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Sample records for qds-supported rna oligonucleotide

  1. A comparative analysis of measles virus RNA by oligonucleotide fingerprinting

    International Nuclear Information System (INIS)

    Stephenson, J.R.; Meulen, V. ter

    1982-01-01

    Isolates from two cases of acute measles, one case of acute measles encephalitis and three patients with subacute sclerosing panencephalitis were compared. This comparison was based upon the electrophoretic analysis of T 1 oligonucleotides from single-stranded, full-length RNA isolated from cytoplasmic nucleocapsids. Although all viruses have oligonucleotides in common, each isolate generated a unique pattern of oligonucleotides. However, no group of oligonucleotides was observed which would allow differentiation between viruses isolated from acute infections and those isolated from CNS diseases; indicating that probably all measles viruses differ in their nucleotide sequence, regardless of origin. (Author)

  2. The Spectral Properties and Photostability of DNA, RNA and Oligonucleotides

    International Nuclear Information System (INIS)

    Kudrya, V.Yu.; Yashchuk, V.M.

    2012-01-01

    The present work discusses the results of comparative investigations of the optical absorption, luminescence, and photostability of the biomacromolecules (DNA, RNA), as well as synthetic poly- and oligonucleotides. The separate nucleotides in DNA and RNA are examined as almost independent absorbing centers. It is confirmed that the main triplet excitons traps responsible for the DNA phosphorescence emission are AT-complexes in DNA. In contrast to DNA, the main triplet excitons traps in RNA are adenosine bases. These bases are the most photostable against UV-irradiation as compared with all other nucleotides in both DNA and RNA. The fact of the photostability of adenosine bases and the AT-complex provides the existence of the DNA/RNA self-protection mechanisms against a damage caused by UV-irradiation. It is found the deoxyribonucleotides are more photostable than the corresponding ribonucleotides. So, the results presented here show that DNA is more photostable than RNA.

  3. Effective Anti-miRNA Oligonucleotides Show High Releasing Rate of MicroRNA from RNA-Induced Silencing Complex.

    Science.gov (United States)

    Ariyoshi, Jumpei; Matsuyama, Yohei; Kobori, Akio; Murakami, Akira; Sugiyama, Hiroshi; Yamayoshi, Asako

    2017-10-01

    MicroRNAs (miRNAs) regulate gene expression by forming RNA-induced silencing complexes (RISCs) and have been considered as promising therapeutic targets. MiRNA is an essential component of RISC for the modulation of gene expression. Therefore, the release of miRNA from RISC is considered as an effective method for the inhibition of miRNA functions. In our previous study, we reported that anti-miRNA oligonucleotides (AMOs), which are composed of the 2'-O-methyl (2'-OMe) RNA, could induce the release of miRNA from RISC. However, the mechanisms underlying the miRNA-releasing effects of chemically modified AMOs, which are conventionally used as anti-cancer drugs, are still unclear. In this study, we investigated the relationship between the miRNA releasing rate from RISC and the inhibitory effect on RISC activity (IC 50 ) using conventional chemically modified AMOs. We demonstrated that the miRNA-releasing effects of AMOs are directly proportional to the IC 50 values, and AMOs, which have an ability to promote the release of miRNA from RISC, can effectively inhibit RISC activity in living cells.

  4. Photodynamic antisense regulation of mRNA having a point mutation with psoralen-conjugated oligonucleotide.

    Science.gov (United States)

    Higuchi, Maiko; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

    2008-01-01

    Nucleic acid-based drugs, such as antisense oligonucleotide, ribozyme, and small interfering RNA, are specific compounds that inhibit gene expression at the post-transcriptional level. To develop more effective nucleic acid-based drugs, we focused on photo-reactive antisense oligonucleotides. We have optimized the structure of psoralen-conjugated oligonucleotide to improve their sequence selectivity and photo-crosslinking efficiency. Previously, we reported that photo reactive oligonucleotides containing 2'-O-psoralenyl-methoxyethyl adenosine (2'-Ps-eom) showed drastic photo-reactivity with a strictly sequence specific manner in vitro. In this report, we evaluated the binding ability toward intracellular target mRNA. The 2'-Ps-eom selectively photo-cross-linked to the target mRNA extracted from cells. The 2'-Ps-eom also cross-linked to target mRNA in cells. Furthermore, 2'-Ps-eom did not cross-link to mRNA having a mismatch base. These results suggest that 2'-Ps-eom is a powerful antisense molecule to inhibit the expression of mRNA having a point mutation.

  5. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

    Science.gov (United States)

    Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O

    1994-09-01

    Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.

  6. Assignment methodology for larger RNA oligonucleotides: Application to an ATP-binding RNA aptamer

    International Nuclear Information System (INIS)

    Dieckmann, Thorsten; Feigon, Juli

    1997-01-01

    The use of uniform 13C, 15N labeling in the NMR spectroscopic study of RNA structures has greatly facilitated the assignment process in small RNA oligonucleotides. For ribose spinsystem assignments, exploitation of these labels has followed previously developed methods for the study of proteins. However, for sequential assignment of the exchangeable and nonexchangeable protons of the nucleotides, it has been necessary to develop a variety of new NMR experiments. Even these are of limited utility in the unambiguous assignment of larger RNAs due to the short carbon relaxation times and extensive spectral overlap for all nuclei.These problems can largely be overcome by the additional use of base-type selectively 13C, 15N-labeled RNA in combination with a judicious use of related RNAs with base substitutions. We report the application of this approach to a 36-nucleotide ATP-binding RNA aptamer in complex with AMP. Complete sequential 1H assignments, as well as the majority of 13C and 15N assignments, were obtained

  7. LNA-modified oligonucleotides mediate specific inhibition of microRNA function

    DEFF Research Database (Denmark)

    Ørom, Ulf Andersson; Kauppinen, Sakari; Lund, Anders H

    2006-01-01

    microRNAs are short, endogenous non-coding RNAs that act as post-transcriptional modulators of gene expression. Important functions for microRNAs have been found in the regulation of development, cellular proliferation and differentiation, while perturbed miRNA expression patterns have been...... observed in many human cancers. Here we present a method for specific inhibition of miRNA function through interaction with LNA-modified antisense oligonucleotides and report the specificity of this application. We show that LNA-modified oligonucleotides can inhibit exogenously introduced miRNAs with high...... specificity using a heterologous reporter assay, and furthermore demonstrate their ability to inhibit an endogenous miRNA in Drosophila melanogaster cells, leading to up-regulation of the cognate target protein. The method shows stoichiometric and reliable inhibition of the targeted miRNA and can thus...

  8. Functional regulation of RNA-induced silencing complex by photoreactive oligonucleotides.

    Science.gov (United States)

    Matsuyama, Yohei; Yamayoshi, Asako; Kobori, Akio; Murakami, Akira

    2014-02-01

    We developed a novel method for regulation of RISC function by photoreactive oligonucleotides (Ps-Oligo) containing 2'-O-psoralenylmethoxyethyl adenosine (Aps). We observed that inhibitory effects of Ps-Oligos on RISC function were enhanced by UV-irradiation compared with 2'-O-methyl-oligonucleotide without Aps. These results suggest Ps-Oligo inhibited RISC function by cross-linking effect, and we propose that the concept described in this report may be promising and applicable one to regulate the small RNA-mediated post-transcriptional regulation. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

  9. Synthesis and Structural Characterization of 2'-Fluoro-α-L-RNA-Modified Oligonucleotides

    DEFF Research Database (Denmark)

    Bundgaard Jensen, Troels; Pasternak, Anna; Stahl Madsen, Andreas

    2011-01-01

    with the smallest destabilization towards RNA. Thermodynamic data show that the duplex formation with 2'-fluoro-α-L-RNA nucleotides is enthalpically disfavored but entropically favored. 2'-Fluoro-α-L-RNA nucleotides exhibit very good base pairing specificity following Watson-Crick rules. The 2'-fluoro......-α-L-RNA monomer was designed as a monocyclic mimic of the bicyclic α-L-LNA, and molecular modeling showed that this indeed is the case as the 2'-fluoro monomer adopts a C3'-endo/C2'-exo sugar pucker. Molecular modeling of modified duplexes show that the 2'-fluoro-α-L-RNA nucleotides partake in Watson-Crick base......We describe the synthesis and binding properties of oligonucleotides that contain one or more 2'-fluoro-α-L-RNA thymine monomer(s). Incorporation of 2'-fluoro-α-L-RNA thymine into oligodeoxynucleotides decreased thermal binding stability slightly upon hybridization with complementary DNA and RNA...

  10. Stability of non-Watson-Crick G-A/A-G base pair in synthetic DNA and RNA oligonucleotides.

    Science.gov (United States)

    Ito, Yuko; Sone, Yumiko; Mizutani, Takaharu

    2004-03-01

    A non-Watson-Crick G-A/A-G base pair is found in SECIS (selenocysteine-insertion sequence) element in the 3'-untranslated region of Se-protein mRNAs and in the functional site of the hammerhead ribozyme. We studied the stability of G-A/A-G base pair (bold) in 17mer GT(U)GACGGAAACCGGAAC synthetic DNA and RNA oligonucleotides by thermal melting experiments and gel electrophoresis. The measured Tm value of DNA oligonucleotide having G-A/A-G pair showed an intermediate value (58 degrees C) between that of Watson-Crick G-C/C-G base pair (75 degrees C) and that of G-G/A-A of non-base-pair (40 degrees C). Similar thermal melting patterns were obtained with RNA oligonucleotides. This result indicates that the secondary structure of oligonucleotide having G-A/A-G base pair is looser than that of the G-C type Watson-Crick base pair. In the comparison between RNA and DNA having G-A/A-G base pair, the Tm value of the RNA oligonucleotide was 11 degrees C lower than that of DNA, indicating that DNA has a more rigid structure than RNA. The stained pattern of oligonucleotide on polyacrylamide gel clarified that the mobility of the DNA oligonucleotide G-A/A-G base pair changed according to the urea concentration from the rigid state (near the mobility of G-C/C-G oligonucleotide) in the absence of urea to the random state (near the mobility of G-G/A-A oligonucleotide) in 7 M urea. However, the RNA oligonucleotide with G-A/A-G pair moved at an intermediate mobility between that of oligonucleotide with G-C/C-G and of the oligonucleotide with G-G/A-A, and the mobility pattern did not depend on urea concentration. Thus, DNA and RNA oligonucleotides with the G-A/A-G base pair showed a pattern indicating an intermediate structure between the rigid Watson-Crick base pair and the random structure of non-base pair. RNA with G-A/A-G base pair has the intermediate structure not influenced by urea concentration. Finally, this study indicated that the intermediate rigidity imparted by Non

  11. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

    OpenAIRE

    Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O

    1994-01-01

    Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed prelim...

  12. Detection of tmRNA molecules on microarrays at low temperatures using helper oligonucleotides

    Directory of Open Access Journals (Sweden)

    Palta Priit

    2010-04-01

    Full Text Available Abstract Background The hybridization of synthetic Streptococcus pneumoniae tmRNA on a detection microarray is slow at 34°C resulting in low signal intensities. Results We demonstrate that adding specific DNA helper oligonucleotides (chaperones to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of Streptococcus pneumoniae tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46°C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule. Conclusions The sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.

  13. Multi-gene detection and identification of mosquito-borne RNA viruses using an oligonucleotide microarray.

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    Nathan D Grubaugh

    Full Text Available BACKGROUND: Arthropod-borne viruses are important emerging pathogens world-wide. Viruses transmitted by mosquitoes, such as dengue, yellow fever, and Japanese encephalitis viruses, infect hundreds of millions of people and animals each year. Global surveillance of these viruses in mosquito vectors using molecular based assays is critical for prevention and control of the associated diseases. Here, we report an oligonucleotide DNA microarray design, termed ArboChip5.1, for multi-gene detection and identification of mosquito-borne RNA viruses from the genera Flavivirus (family Flaviviridae, Alphavirus (Togaviridae, Orthobunyavirus (Bunyaviridae, and Phlebovirus (Bunyaviridae. METHODOLOGY/PRINCIPAL FINDINGS: The assay utilizes targeted PCR amplification of three genes from each virus genus for electrochemical detection on a portable, field-tested microarray platform. Fifty-two viruses propagated in cell-culture were used to evaluate the specificity of the PCR primer sets and the ArboChip5.1 microarray capture probes. The microarray detected all of the tested viruses and differentiated between many closely related viruses such as members of the dengue, Japanese encephalitis, and Semliki Forest virus clades. Laboratory infected mosquitoes were used to simulate field samples and to determine the limits of detection. Additionally, we identified dengue virus type 3, Japanese encephalitis virus, Tembusu virus, Culex flavivirus, and a Quang Binh-like virus from mosquitoes collected in Thailand in 2011 and 2012. CONCLUSIONS/SIGNIFICANCE: We demonstrated that the described assay can be utilized in a comprehensive field surveillance program by the broad-range amplification and specific identification of arboviruses from infected mosquitoes. Furthermore, the microarray platform can be deployed in the field and viral RNA extraction to data analysis can occur in as little as 12 h. The information derived from the ArboChip5.1 microarray can help to establish

  14. Assessing Specific Oligonucleotides and Small Molecule Antibiotics for the Ability to Inhibit the CRD-BP-CD44 RNA Interaction

    Science.gov (United States)

    Thomsen, Dana; Lee, Chow H.

    2014-01-01

    Studies on Coding Region Determinant-Binding Protein (CRD-BP) and its orthologs have confirmed their functional role in mRNA stability and localization. CRD-BP is present in extremely low levels in normal adult tissues, but it is over-expressed in many types of aggressive human cancers and in neonatal tissues. Although the exact role of CRD-BP in tumour progression is unclear, cumulative evidence suggests that its ability to physically associate with target mRNAs is an important criterion for its oncogenic role. CRD-BP has high affinity for the 3′UTR of the oncogenic CD44 mRNA and depletion of CRD-BP in cells led to destabilization of CD44 mRNA, decreased CD44 expression, reduced adhesion and disruption of invadopodia formation. Here, we further characterize the CRD-BP-CD44 RNA interaction and assess specific antisense oligonucleotides and small molecule antibiotics for their ability to inhibit the CRD-BP-CD44 RNA interaction. CRD-BP has a high affinity for binding to CD44 RNA nts 2862–3055 with a Kd of 645 nM. Out of ten antisense oligonucleotides spanning nts 2862–3055, only three antisense oligonucleotides (DD4, DD7 and DD10) were effective in competing with CRD-BP for binding to 32P-labeled CD44 RNA. The potency of DD4, DD7 and DD10 in inhibiting the CRD-BP-CD44 RNA interaction in vitro correlated with their ability to specifically reduce the steady-state level of CD44 mRNA in cells. The aminoglycoside antibiotics neomycin, paramomycin, kanamycin and streptomycin effectively inhibited the CRD-BP-CD44 RNA interaction in vitro. Assessing the potential inhibitory effect of aminoglycoside antibiotics including neomycin on the CRD-BP-CD44 mRNA interaction in cells proved difficult, likely due to their propensity to non-specifically bind nucleic acids. Our results have important implications for future studies in finding small molecules and nucleic acid-based inhibitors that interfere with protein-RNA interactions. PMID:24622399

  15. Inhibition of Xenograft tumor growth by gold nanoparticle-DNA oligonucleotide conjugates-assisted delivery of BAX mRNA.

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    Ji-Hyun Yeom

    Full Text Available Use of non-biological agents for mRNA delivery into living systems in order to induce heterologous expression of functional proteins may provide more advantages than the use of DNA and/or biological vectors for delivery. However, the low efficiency of mRNA delivery into live animals, using non-biological systems, has hampered the use of mRNA as a therapeutic molecule. Here, we show that gold nanoparticle-DNA oligonucleotide (AuNP-DNA conjugates can serve as universal vehicles for more efficient delivery of mRNA into human cells, as well as into xenograft tumors generated in mice. Injections of BAX mRNA loaded on AuNP-DNA conjugates into xenograft tumors resulted in highly efficient mRNA delivery. The delivered mRNA directed the efficient production of biologically functional BAX protein, a pro-apoptotic factor, consequently inhibiting tumor growth. These results demonstrate that mRNA delivery by AuNP-DNA conjugates can serve as a new platform for the development of safe and efficient gene therapy.

  16. Antiresistin RNA Oligonucleotide Ameliorates Diet-Induced Nonalcoholic Fatty Liver Disease in Mice through Attenuating Proinflammatory Cytokines

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    Yi Tan

    2015-01-01

    Full Text Available The aim of this study was to determine whether inhibition of resistin by a synthetic antiresistin RNA (oligonucleotide oligo ameliorates metabolic and histological abnormalities in nonalcoholic fatty liver disease (NAFLD induced by high-fat diet (HFD in mice. The antiresistin RNA oligo and a scrambled control oligo (25 mg/kg of body weight were i.p. injected to HFD mice. Serum metabolic parameters and hepatic enzymes were measured after 4-week treatment. The treatment significantly reduced epididymal fat and attenuated the elevated serum resistin, cholesterol, triglycerides, glucose, and insulin with an improved glucose tolerance test. Antiresistin RNA oligo also normalized serum AST and ALT levels with improved pathohistology of NAFLD. Immunoblotting and qRT-PCR revealed that decreased protein and mRNA expression of resistin in fat and liver tissues of the treated mice were associated with reduction of adipose TNF-α and IL-6 expression and secretion into circulation. mRNA and protein expression of hepatic phosphoenolpyruvate carboxykinase (PEPCK and sterol regulatory element-binding protein-1c (SREBP-1c were also significantly decreased in the treated mice. Our results suggest that resistin may exacerbate NAFLD in metabolic syndrome through upregulating inflammatory cytokines and hepatic PEPCK and SREBP-1c. Antiresistin RNA oligo ameliorated metabolic abnormalities and histopathology of NAFLD through attenuating proinflammatory cytokines.

  17. Characterization of rat brain NCAM mRNA using DNA oligonucleotide probes

    DEFF Research Database (Denmark)

    Andersson, A M; Gaardsvoll, H; Giladi, E

    1990-01-01

    A number of different isoforms of the neural cell adhesion molecule (NCAM) have been identified. The difference between these is due to alternative splicing of a single NCAM gene. In rat brain NCAM mRNAs with sizes of 7.4, 6.7, 5.2, 4.3 and 2.9 kb have been reported. We have synthesized six DNA...... oligonucleotides, that hybridize to different exons in the NCAM gene. Furthermore we have constructed three oligonucleotides, that exclusively hybridize to mRNAs lacking certain exons, by letting them consist of sequences adjacent to both sides of the splice sites. By means of these probes we have characterized...... the five NCAM mRNAs in rat brain....

  18. Development of Novel Antisense Oligonucleotides for the Functional Regulation of RNA-Induced Silencing Complex (RISC) by Promoting the Release of microRNA from RISC.

    Science.gov (United States)

    Ariyoshi, Jumpei; Momokawa, Daiki; Eimori, Nao; Kobori, Akio; Murakami, Akira; Yamayoshi, Asako

    2015-12-16

    MicroRNAs (miRNAs) are known to be important post-transcription regulators of gene expression. Aberrant miRNA expression is associated with pathological disease processes, including carcinogenesis. Therefore, miRNAs are considered significant therapeutic targets for cancer therapy. MiRNAs do not act alone, but exhibit their functions by forming RNA-induced silencing complex (RISC). Thus, the regulation of RISC activity is a promising approach for cancer therapy. MiRNA is a core component of RISC and is an essential to RISC for recognizing target mRNA. Thereby, it is expected that development of the method to promote the release of miRNA from RISC would be an effective approach for inhibition of RISC activity. In this study, we synthesized novel peptide-conjugated oligonucleotides (RINDA-as) to promote the release of miRNA from RISC. RINDA-as showed a high rate of miRNA release from RISC and high level of inhibitory effect on RISC activity.

  19. Family- and genus-level 16S rRNA-targeted oligonucleotide probes for ecological studies of methanotrophic bacteria.

    Science.gov (United States)

    Gulledge, J; Ahmad, A; Steudler, P A; Pomerantz, W J; Cavanaugh, C M

    2001-10-01

    Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signatures that distinguish between the two genera in this family, Methylocystis and Methylosinus, were identified. Two other probes target, as a single group, a majority of the known methanotrophs belonging to the family Methylococcaceae (type I/X methanotrophs). The remaining probes target members of individual genera of the Methylococcaceae, including Methylobacter, Methylomonas, Methylomicrobium, Methylococcus, and Methylocaldum. One of the family-level probes also covers all methanotrophic endosymbionts of marine mollusks for which 16S rRNA sequences have been published. The two known species of the newly described genus Methylosarcina gen. nov. are covered by a probe that otherwise targets only members of the closely related genus Methylomicrobium. None of the probes covers strains of the newly proposed genera Methylocella and "Methylothermus," which are polyphyletic with respect to the recognized methanotrophic families. Empirically determined midpoint dissociation temperatures were 49 to 57 degrees C for all probes. In dot blot screening against RNA from positive- and negative-control strains, the probes were specific to their intended targets. The broad coverage and high degree of specificity of this new suite of probes will provide more detailed, quantitative information about the community structure of methanotrophs in environmental samples than was previously available.

  20. Oligonucleotides targeting TCF4 triplet repeat expansion inhibit RNA foci and mis-splicing in Fuchs' dystrophy.

    Science.gov (United States)

    Hu, Jiaxin; Rong, Ziye; Gong, Xin; Zhou, Zhengyang; Sharma, Vivek K; Xing, Chao; Watts, Jonathan K; Corey, David R; Mootha, V Vinod

    2018-03-15

    Fuchs' endothelial corneal dystrophy (FECD) is the most common repeat expansion disorder. FECD impacts 4% of U.S. population and is the leading indication for corneal transplantation. Most cases are caused by an expanded intronic CUG tract in the TCF4 gene that forms nuclear foci, sequesters splicing factors and impairs splicing. We investigated the sense and antisense RNA landscape at the FECD gene and find that the sense-expanded repeat transcript is the predominant species in patient corneas. In patient tissue, sense foci number were negatively correlated with age and showed no correlation with sex. Each endothelial cell has ∼2 sense foci and each foci is single RNA molecule. We designed antisense oligonucleotides (ASOs) to target the mutant-repetitive RNA and demonstrated potent inhibition of foci in patient-derived cells. Ex vivo treatment of FECD human corneas effectively inhibits foci and reverses pathological changes in splicing. FECD has the potential to be a model for treating many trinucleotide repeat diseases and targeting the TCF4 expansion with ASOs represents a promising therapeutic strategy to prevent and treat FECD.

  1. Fluorescence detection of natural RNA using rationally designed "clickable" oligonucleotide probes

    DEFF Research Database (Denmark)

    Okholm, Anders; Kjems, Jørgen; Astakhova, Kira

    2014-01-01

    Herein a reliable approach to the design of effective fluorescent probes for RNA detection is described. The fluorescence signalling of hybridization by internally positioned polyaromatic hydrocarbons and rhodamine dyes was achieved with a low fluorescence background signal, high fluorescence qua...... quantum yields at ambient and elevated temperature, high selectivity and signal specificity of the probes when binding to miR-7 and circRNA targets....

  2. Inefficient cationic lipid-mediated siRNA and antisense oligonucleotide transfer to airway epithelial cells in vivo

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    Hu Jim

    2006-02-01

    Full Text Available Abstract Background The cationic lipid Genzyme lipid (GL 67 is the current "gold-standard" for in vivo lung gene transfer. Here, we assessed, if GL67 mediated uptake of siRNAs and asODNs into airway epithelium in vivo. Methods Anti-lacZ and ENaC (epithelial sodium channel siRNA and asODN were complexed to GL67 and administered to the mouse airway epithelium in vivo Transfection efficiency and efficacy were assessed using real-time RT-PCR as well as through protein expression and functional studies. In parallel in vitro experiments were carried out to select the most efficient oligonucleotides. Results In vitro, GL67 efficiently complexed asODNs and siRNAs, and both were stable in exhaled breath condensate. Importantly, during in vitro selection of functional siRNA and asODN we noted that asODNs accumulated rapidly in the nuclei of transfected cells, whereas siRNAs remained in the cytoplasm, a pattern consistent with their presumed site of action. Following in vivo lung transfection siRNAs were only visible in alveolar macrophages, whereas asODN also transfected alveolar epithelial cells, but no significant uptake into conducting airway epithelial cells was seen. SiRNAs and asODNs targeted to β-galactosidase reduced βgal mRNA levels in the airway epithelium of K18-lacZ mice by 30% and 60%, respectively. However, this was insufficient to reduce protein expression. In an attempt to increase transfection efficiency of the airway epithelium, we increased contact time of siRNA and asODN using the in vivo mouse nose model. Although highly variable and inefficient, transfection of airway epithelium with asODN, but not siRNA, was now seen. As asODNs more effectively transfected nasal airway epithelial cells, we assessed the effect of asODN against ENaC, a potential therapeutic target in cystic fibrosis; no decrease in ENaC mRNA levels or function was detected. Conclusion This study suggests that although siRNAs and asODNs can be developed to inhibit

  3. Dynamics of co-transcriptional pre-mRNA folding influences the induction of dystrophin exon skipping by antisense oligonucleotides.

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    Keng Boon Wee

    Full Text Available Antisense oligonucleotides (AONs mediated exon skipping offers potential therapy for Duchenne muscular dystrophy. However, the identification of effective AON target sites remains unsatisfactory for lack of a precise method to predict their binding accessibility. This study demonstrates the importance of co-transcriptional pre-mRNA folding in determining the accessibility of AON target sites for AON induction of selective exon skipping in DMD. Because transcription and splicing occur in tandem, AONs must bind to their target sites before splicing factors. Furthermore, co-transcriptional pre-mRNA folding forms transient secondary structures, which redistributes accessible binding sites. In our analysis, to approximate transcription elongation, a "window of analysis" that included the entire targeted exon was shifted one nucleotide at a time along the pre-mRNA. Possible co-transcriptional secondary structures were predicted using the sequence in each step of transcriptional analysis. A nucleotide was considered "engaged" if it formed a complementary base pairing in all predicted secondary structures of a particular step. Correlation of frequency and localisation of engaged nucleotides in AON target sites accounted for the performance (efficacy and efficiency of 94% of 176 previously reported AONs. Four novel insights are inferred: (1 the lowest frequencies of engaged nucleotides are associated with the most efficient AONs; (2 engaged nucleotides at 3' or 5' ends of the target site attenuate AON performance more than at other sites; (3 the performance of longer AONs is less attenuated by engaged nucleotides at 3' or 5' ends of the target site compared to shorter AONs; (4 engaged nucleotides at 3' end of a short target site attenuates AON efficiency more than at 5' end.

  4. In vitro detection of mdr1 mRNA in murine leukemia cells with 111In-labeled oligonucleotide

    International Nuclear Information System (INIS)

    Bai Jingming; Yokoyama, Kunihiko; Kinuya, Seigo; Michigishi, Takatoshi; Tonami, Norihisa; Shiba, Kazuhiro; Matsushita, Ryo; Nomura, Masaaki

    2004-01-01

    The feasibility of intracellular mdr1 mRNA expression detection with radiolabeled antisense oligonucleotide (ODN) was investigated in the murine leukemia cell line, P388/S, and its subclonal, adriamycin-resistant cell line, P388/R. The expression level of mdr1 mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). Existence of the multidrug resistance (MDR) phenomenon was assessed via cellular uptake of 99m Tc-sestamibi (MIBI), a known substrate for P-glycoprotein. A 15-mer phosphorothioate antisense ODN complementary to the sequences located at -1 to 14 of mdr1 mRNA and its corresponding sense ODN were conjugated with the cyclic anhydride of diethylene triamine penta-acetic acid (cDTPA) via an amino group linked to the terminal phosphate at the 5' end at pH 8-9. The DTPA-ODN complexes at concentrations of 0.1-17.4 μMwere reacted with 111 InCl 3 at pH 5 for 1 h. The hybridization affinity of labeled ODN was evaluated with size-exclusion high-performance liquid chromatography following incubation with the complementary sequence. Cellular uptake of labeled ODN was examined in vitro. Furthermore, enhancing effects of synthetic lipid carriers (Transfast) on transmembrane delivery of ODN were assessed. P388/R cells displayed intense mdr1 mRNA expression in comparison with P388/S cells. 99m Tc-MIBI uptake in P388/S cells was higher than that in P388/R cells. Specific radioactivity up to 1,634 MBq/nmol was achieved via elevation of added radioactivity relative to ODN molar amount. The hybridization affinity of antisense 111 In-ODN was preserved at approximately 85% irrespective of specific activity. Cellular uptake of antisense 111 In-ODN did not differ from that of sense 111 In-ODN in either P388/S cells or P388/R cells. However, lipid carrier incorporation significantly increased transmembrane delivery of 111 In-ODN; moreover, specific uptake of antisense 111 In-ODN was demonstrated in P388/R cells. Radiolabeling of ODN at high specific

  5. In Vitro Antiviral Activity of Circular Triple Helix Forming Oligonucleotide RNA towards Feline Infectious Peritonitis Virus Replication

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    Oi Kuan Choong

    2014-01-01

    Full Text Available Feline Infectious Peritonitis (FIP is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV, a virulent mutant strain of Feline Enteric Coronavirus (FECV. Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO RNAs (TFO1 to TFO5, which target the different regions of virulent feline coronavirus (FCoV strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10 from 1014 in the virus-inoculated cells to 109 in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.

  6. In vitro antiviral activity of circular triple helix forming oligonucleotide RNA towards Feline Infectious Peritonitis virus replication.

    Science.gov (United States)

    Choong, Oi Kuan; Mehrbod, Parvaneh; Tejo, Bimo Ario; Omar, Abdul Rahman

    2014-01-01

    Feline Infectious Peritonitis (FIP) is a severe fatal immune-augmented disease in cat population. It is caused by FIP virus (FIPV), a virulent mutant strain of Feline Enteric Coronavirus (FECV). Current treatments and prophylactics are not effective. The in vitro antiviral properties of five circular Triple-Helix Forming Oligonucleotide (TFO) RNAs (TFO1 to TFO5), which target the different regions of virulent feline coronavirus (FCoV) strain FIPV WSU 79-1146 genome, were tested in FIPV-infected Crandell-Rees Feline Kidney (CRFK) cells. RT-qPCR results showed that the circular TFO RNAs, except TFO2, inhibit FIPV replication, where the viral genome copy numbers decreased significantly by 5-fold log10 from 10(14) in the virus-inoculated cells to 10(9) in the circular TFO RNAs-transfected cells. Furthermore, the binding of the circular TFO RNA with the targeted viral genome segment was also confirmed using electrophoretic mobility shift assay. The strength of binding kinetics between the TFO RNAs and their target regions was demonstrated by NanoITC assay. In conclusion, the circular TFOs have the potential to be further developed as antiviral agents against FIPV infection.

  7. Optimization of oligonucleotide arrays and RNA amplification protocols for analysis of transcript structure and alternative splicing.

    Science.gov (United States)

    Castle, John; Garrett-Engele, Phil; Armour, Christopher D; Duenwald, Sven J; Loerch, Patrick M; Meyer, Michael R; Schadt, Eric E; Stoughton, Roland; Parrish, Mark L; Shoemaker, Daniel D; Johnson, Jason M

    2003-01-01

    Microarrays offer a high-resolution means for monitoring pre-mRNA splicing on a genomic scale. We have developed a novel, unbiased amplification protocol that permits labeling of entire transcripts. Also, hybridization conditions, probe characteristics, and analysis algorithms were optimized for detection of exons, exon-intron edges, and exon junctions. These optimized protocols can be used to detect small variations and isoform mixtures, map the tissue specificity of known human alternative isoforms, and provide a robust, scalable platform for high-throughput discovery of alternative splicing.

  8. The cellular uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite nanoparticles

    OpenAIRE

    Negin Saffarzadeh; Seyed Mehdi Kalantar; Ali Jebali; Seyed Hossein Hekmatimoghaddam; Mohammad Hassan Sheikhha; Ehsan Farashahi

    2014-01-01

    Objective(s): Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs) and 1,2-dioleoyl-sn-glycero-3-phosphoethanol​amine (DOPE)-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Materials and Methods: Calcium...

  9. Quantification of Different Eubacterium spp. in Human Fecal Samples with Species-Specific 16S rRNA-Targeted Oligonucleotide Probes

    OpenAIRE

    Schwiertz, Andreas; Le Blay, Gwenaelle; Blaut, Michael

    2000-01-01

    Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none...

  10. miRNA oligonucleotide and sponge for miRNA-21 inhibition mediated by PEI-PLL in breast cancer therapy.

    Science.gov (United States)

    Gao, Shiqian; Tian, Huayu; Guo, Ye; Li, Yuce; Guo, Zhaopei; Zhu, Xiaojuan; Chen, Xuesi

    2015-10-01

    MicroRNA-21 (miR-21) inhibition is a promising biological strategy for breast cancer therapy. However its application is limited by the lack of efficient miRNA inhibitor delivery systems. As a cationic polymer transfection material for nucleic acids, the poly (l-lysine)-modified polyethylenimine (PEI-PLL) copolymer combines the high transfection efficiency of polyethylenimine (PEI) and the good biodegradability of polyllysine (PLL). In this work, PEI-PLL was successfully synthesized and confirmed to transfect plasmid and oligonucleotide more effectively than PEI in MCF-7 cells (human breast cancer cells). In this regard, two kinds of miR-21 inhibitors, miR-21 sponge plasmid DNA (Sponge) and anti-miR-21 oligonucleotide (AMO), were transported into MCF-7 cells by PEI-PLL respectively. The miR-21 expression and the cellular physiology were determined post transfection. Compared with the negative control, PEI-PLL/Sponge or PEI-PLL/AMO groups exhibited lower miR-21 expression and cell viability. The anti-tumor mechanism of PEI-PLL/miR-21 inhibitors was further studied by cell cycle and western blot analyses. The results indicated that the miR-21 inhibition could induce the cell cycle arrest in G1 phase, upregulate the expression of Programmed Cell Death Protein 4 (PDCD4) and thus active the caspase-3 apoptosis pathway. Interestingly, the PEI-PLL/Sponge and PEI-PLL/AMO also sensitized the MCF-7 cells to anti-tumor drugs, doxorubicin (DOX) and cisplatin (CDDP). These results demonstrated that PEI-PLL/Sponge and PEI-PLL/AMO complexes would be two novel and promising gene delivery systems for breast cancer gene therapy based on miR-21 inhibition. This work was a combination of the high transfection efficiency of polyethylenimine (PEI), the good biodegradability of polyllysine (PLL) and the breast cancer-killing effect of miR-21 inhibitors. The poly (l-lysine)-modified polyethylenimine (PEI-PLL) copolymer was employed as the vector of miR-21 sponge plasmid DNA (Sponge) or

  11. Resolving prokaryotic taxonomy without rRNA: longer oligonucleotide word lengths improve genome and metagenome taxonomic classification.

    Science.gov (United States)

    Alsop, Eric B; Raymond, Jason

    2013-01-01

    Oligonucleotide signatures, especially tetranucleotide signatures, have been used as method for homology binning by exploiting an organism's inherent biases towards the use of specific oligonucleotide words. Tetranucleotide signatures have been especially useful in environmental metagenomics samples as many of these samples contain organisms from poorly classified phyla which cannot be easily identified using traditional homology methods, including NCBI BLAST. This study examines oligonucleotide signatures across 1,424 completed genomes from across the tree of life, substantially expanding upon previous work. A comprehensive analysis of mononucleotide through nonanucleotide word lengths suggests that longer word lengths substantially improve the classification of DNA fragments across a range of sizes of relevance to high throughput sequencing. We find that, at present, heptanucleotide signatures represent an optimal balance between prediction accuracy and computational time for resolving taxonomy using both genomic and metagenomic fragments. We directly compare the ability of tetranucleotide and heptanucleotide world lengths (tetranucleotide signatures are the current standard for oligonucleotide word usage analyses) for taxonomic binning of metagenome reads. We present evidence that heptanucleotide word lengths consistently provide more taxonomic resolving power, particularly in distinguishing between closely related organisms that are often present in metagenomic samples. This implies that longer oligonucleotide word lengths should replace tetranucleotide signatures for most analyses. Finally, we show that the application of longer word lengths to metagenomic datasets leads to more accurate taxonomic binning of DNA scaffolds and have the potential to substantially improve taxonomic assignment and assembly of metagenomic data.

  12. Resolving prokaryotic taxonomy without rRNA: longer oligonucleotide word lengths improve genome and metagenome taxonomic classification.

    Directory of Open Access Journals (Sweden)

    Eric B Alsop

    Full Text Available Oligonucleotide signatures, especially tetranucleotide signatures, have been used as method for homology binning by exploiting an organism's inherent biases towards the use of specific oligonucleotide words. Tetranucleotide signatures have been especially useful in environmental metagenomics samples as many of these samples contain organisms from poorly classified phyla which cannot be easily identified using traditional homology methods, including NCBI BLAST. This study examines oligonucleotide signatures across 1,424 completed genomes from across the tree of life, substantially expanding upon previous work. A comprehensive analysis of mononucleotide through nonanucleotide word lengths suggests that longer word lengths substantially improve the classification of DNA fragments across a range of sizes of relevance to high throughput sequencing. We find that, at present, heptanucleotide signatures represent an optimal balance between prediction accuracy and computational time for resolving taxonomy using both genomic and metagenomic fragments. We directly compare the ability of tetranucleotide and heptanucleotide world lengths (tetranucleotide signatures are the current standard for oligonucleotide word usage analyses for taxonomic binning of metagenome reads. We present evidence that heptanucleotide word lengths consistently provide more taxonomic resolving power, particularly in distinguishing between closely related organisms that are often present in metagenomic samples. This implies that longer oligonucleotide word lengths should replace tetranucleotide signatures for most analyses. Finally, we show that the application of longer word lengths to metagenomic datasets leads to more accurate taxonomic binning of DNA scaffolds and have the potential to substantially improve taxonomic assignment and assembly of metagenomic data.

  13. Isolation and characterization of human glycophorin A cDNA clones by a synthetic oligonucleotide approach: nucleotide sequence and mRNA structure

    International Nuclear Information System (INIS)

    Siebert, P.D.; Fukuda, M.

    1986-01-01

    In an effort to understand the relationships among and the regulation of human glycophorins, the authors have isolated and characterized several glycophorin A-specific cDNA clones obtained from a human erythroleukemic K562 cell cDNA library. This was accomplished by using mixed synthetic oligonucleotides, corresponding to various regions of the known amino acid sequence, to prime the synthesis of the cDNA as well as to screen the cDNA library. They also used synthetic oligonucleotides to sequence the largest of the glycophorin cDNAs. The nucleotide sequence obtained suggests the presence of a potential leader peptide, consistent with the membrane localization of this glycoprotein. Examination of the structure of glycophorin mRNA by blot hybridization revealed the existence of several electrophoretically distinct mRNAs numbering three or four, depending on the size of the glycophorin cDNA used as a hybridization probe. The smaller cDNA hybridized to three mRNAs of approximately 2.8, 1.7, and 1.0 kilobases. In contrast, the larger cDNA hybridized to an additional mRNA of approximately 0.6 kilobases. Further examination of the relationships between these multiple mRNAs by blot hybridization was conducted with the use of exact-sequence oligonucleotide probes constructed from various regions of the cDNA representing portions of the amino acid sequence of glycophorin A with or without known homology with glycophorin B. In total, the results obtained are consistent with the hypothesis that the three larger mRNAs represent glycophorin A gene transcripts and that the smallest (0.6 kilobase) mRNA may be specific for glycophorin B

  14. The cellular uptake of antisense oligonucleotid of E6 mRNA into cervical cancer cells by DOPE-modified hydroxyapatite nanoparticles

    Directory of Open Access Journals (Sweden)

    Negin Saffarzadeh

    2014-10-01

    Full Text Available Objective(s: Although several chemical and physical methods for gene delivery have been introduced, their cytotoxicity, non-specific immune responses and the lack of biodegradability remain the main issues. In this study, hydroxyapatite nanoparticles (NPs and 1,2-dioleoyl-sn-glycero-3-phosphoethanol​amine (DOPE-modified hydroxyapatite NPs was coated with antisense oligonucleotide of E6 mRNA, and their uptakes into the cervical cancer cell line were evaluated. Materials and Methods: Calcium nitrate and diammonium phosphate were used for the synthesis of the hydroxyapatite nanoparticle. Thus, they were coated with polyethylene glycol (PEG, DOPE and antisense oligonucleotide of E6 mRNA using a cross-linker. Then, hydroxyapatite NPs and DOPE-modified hydroxyapatite NPs were incubated 48 hours with cervical cancer cells and their uptakes were evaluated by fluorescent microscopy. Results: The hydroxyapatite NPs had different shapes and some agglomeration with average size of 100 nm. The results showed DOPE-modified hydroxyapatite NPs had higher uptake than hydroxyapatite NPs (P

  15. Synthesis and conformational properties of oligonucleotides incorporating 2'-O-phosphorylated ribonucleotides as structural motifs of pre-tRNA splicing intermediates.

    Science.gov (United States)

    Tsuruoka, H; Shohda, K; Wada, T; Sekine, M

    2000-11-03

    To synthesize oligonucleotides containing 2'-O-phosphate groups, four kinds of ribonucleoside 3'-phosphoramidite building blocks 6a-d having the bis(2-cyano-1,1-dimethylethoxy)thiophosphoryl (BCMETP) group were prepared according to our previous phosphorylation procedure. These phosphoramidite units 6a-d were not contaminated with 3'-regioisomers and were successfully applied to solid-phase synthesis to give oligodeoxyuridylates 15, 16 and oligouridylates 21, 22. Self-complementary Drew-Dickerson DNA 12mers 24-28 replaced by a 2'-O-phosphorylated ribonucleotide at various positions were similarly synthesized. In these syntheses, it turned out that KI(3) was the most effective reagent for oxidative desulfurization of the initially generated thiophosphate group to the phosphate group on polymer supports. Without using this conversion step, a tridecadeoxyuridylate 17 incorporating a 2'-O-thiophosphorylated uridine derivative was also synthesized. To investigate the effect of the 2'-phosphate group on the thermal stability and 3D-structure of DNA(RNA) duplexes, T(m) measurement of the self-complementary oligonucleotides obtained and MD simulation of heptamer duplexes 33-36 were carried out. According to these analyses, it was suggested that the nucleoside ribose moiety phosphorylated at the 2'-hydroxyl function predominantly preferred C2'-endo to C3'-endo conformation in DNA duplexes so that it did not significantly affect the stability of the DNA duplex. On the other hand, the 2'-modified ribose moiety was expelled to give a C3'-endo conformation in RNA duplexes so that the RNA duplexes were extremely destabilized.

  16. High affinity RNA targeting by oligonucleotides displaying aromatic stacking and amino groups in the major groove. Comparison of triazoles and phenylsubstituents

    DEFF Research Database (Denmark)

    Kumar, Pawan; Hornum, Mick; Nielsen, Lise Junker

    2014-01-01

    Three 5-modified 2'-deoxyuridine nucleosides were synthesized and incorporated into oligonucleotides and compared with the previously published 5-(1-phenyl-1,2,3-triazol-4-yl)-2'-deoxyuridine monomer W. The introduction of an aminomethyl group on the phenyl group led to monomer X, which was found...... to thermally stabilize a 9-mer DNA:RNA duplex, presumably through the partial neutralization of the negative charge of the backbone. By also taking advantage of the stacking interactions in the major groove of two or more of the monomer X, an extremely high thermal stability was obtained. A regioisomer...... monomer Z was incorporated for comparison, and it was found to give a more neutral influence on duplex stability indicating less efficient stacking interactions. The duplexes were investigated by CD spectroscopy and MD simulations....

  17. Differential detection of type II methanotrophic bacteria in acidic peatlands using newly developed 16S rRNA-targeted fluorescent oligonucleotide probes.

    Science.gov (United States)

    Dedysh, Svetlana N; Dunfield, Peter F; Derakshani, Manigee; Stubner, Stephan; Heyer, Jürgen; Liesack, Werner

    2003-04-01

    Abstract Based on an extensive 16S rRNA sequence database for type II methanotrophic bacteria, a set of 16S rRNA-targeted oligonucleotide probes was developed for differential detection of specific phylogenetic groups of these bacteria by fluorescence in situ hybridisation (FISH). This set of oligonucleotides included a genus-specific probe for Methylocystis (Mcyst-1432) and three species-specific probes for Methylosinus sporium (Msins-647), Methylosinus trichosporium (Msint-1268) and the recently described acidophilic methanotroph Methylocapsa acidiphila (Mcaps-1032). These novel probes were applied to further characterise the type II methanotroph community that was detected in an acidic Sphagnum peat from West Siberia in a previous study (Dedysh et al. (2001) Appl. Environ. Microbiol. 67, 4850-4857). The largest detectable population of indigenous methanotrophs simultaneously hybridised with a group-specific probe targeting all currently known Methylosinus/Methylocystis spp. (M-450), with a genus-specific probe for Methylocystis spp. (Mcyst-1432), and with an additional probe (Mcyst-1261) that had been designed to target a defined phylogenetic subgroup of Methylocystis spp. The same subgroup of Methylocystis was also detected in acidic peat sampled from Sphagnum-dominated wetland in northern Germany. The population size of this peat-inhabiting Methylocystis subgroup was 2.0+/-0.1x10(6) cells g(-1) (wet weight) of peat from Siberia and 5.5+/-0.5x10(6) cells g(-1) of peat from northern Germany. This represented 60 and 95%, respectively, of the total number of methanotroph cells detected by FISH in these two wetland sites. Other major methanotroph populations were M. acidiphila and Methylocella palustris. Type I methanotrophs accounted for not more than 1% of total methanotroph cells. Neither M. trichosporium nor M. sporium were detected in acidic Sphagnum peat.

  18. An oligonucleotide complementary to the SL-B1 domain in the 3'-end of the minus-strand RNA of the hepatitis C virus inhibits in vitro initiation of RNA synthesis by the viral polymerase

    International Nuclear Information System (INIS)

    Reigadas, Sandrine; Ventura, Michel; Andreola, Marie-Line; Michel, Justine; Gryaznov, Sergei; Tarrago-Litvak, Laura; Litvak, Simon; Astier-Gin, Therese

    2003-01-01

    We describe oligonucleotides (ODNs) that inhibit hepatitis C virus (HCV) RNA synthesis in vitro. From a series of 13 ODNs complementary to the 3'-end of the minus-strand HCV RNA, only 4 inhibited RNA synthesis with IC 50 values lower than 1 μM. The inhibition was sequence-specific, since no effect was observed when the ODNs were used with a noncomplementary template. The introduction of a 2'-O-methyl modification increased the inhibitor activity 11-fold (IC 50 = 50 nM) in just 1 (ODN7) of the 4 inhibitory ODNs. ODNs did not inhibit RNA synthesis by interfering with the elongation process as no short RNAs products were detected. We also show that ODN7 did not prevent binding of NS5B to the template or cause polymerase trapping by the duplex RNA/ODN. Our data demonstrate that ODN7 inhibits the initiation process, most probably by modifying structural features present at the 3'-end of the minus-strand RNA

  19. Characterization of the effect of sample quality on high density oligonucleotide microarray data using progressively degraded rat liver RNA

    Directory of Open Access Journals (Sweden)

    Rosenzweig Barry A

    2007-09-01

    Full Text Available Abstract Background The interpretability of microarray data can be affected by sample quality. To systematically explore how RNA quality affects microarray assay performance, a set of rat liver RNA samples with a progressive change in RNA integrity was generated by thawing frozen tissue or by ex vivo incubation of fresh tissue over a time course. Results Incubation of tissue at 37°C for several hours had little effect on RNA integrity, but did induce changes in the transcript levels of stress response genes and immune cell markers. In contrast, thawing of tissue led to a rapid loss of RNA integrity. Probe sets identified as most sensitive to RNA degradation tended to be located more than 1000 nucleotides upstream of their transcription termini, similar to the positioning of control probe sets used to assess sample quality on Affymetrix GeneChip® arrays. Samples with RNA integrity numbers less than or equal to 7 showed a significant increase in false positives relative to undegraded liver RNA and a reduction in the detection of true positives among probe sets most sensitive to sample integrity for in silico modeled changes of 1.5-, 2-, and 4-fold. Conclusion Although moderate levels of RNA degradation are tolerated by microarrays with 3'-biased probe selection designs, in this study we identify a threshold beyond which decreased specificity and sensitivity can be observed that closely correlates with average target length. These results highlight the value of annotating microarray data with metrics that capture important aspects of sample quality.

  20. Synthesis, Improved Antisense Activity and Structural Rationale for the Divergent RNA Affinities of 3;#8242;-Fluoro Hexitol Nucleic Acid (FHNA and Ara-FHNA) Modified Oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Egli, Martin; Pallan, Pradeep S.; Allerson, Charles R.; Prakash, Thazha P.; Berdeja, Andres; Yu, Jinghua; Lee, Sam; Watt, Andrew; Gaus, Hans; Bhat, Balkrishen; Swayze, Eric E.; Seth, Punit P. (Isis Pharm.); (Vanderbilt)

    2012-03-16

    The synthesis, biophysical, structural, and biological properties of both isomers of 3'-fluoro hexitol nucleic acid (FHNA and Ara-FHNA) modified oligonucleotides are reported. Synthesis of the FHNA and Ara-FHNA thymine phosphoramidites was efficiently accomplished starting from known sugar precursors. Optimal RNA affinities were observed with a 3'-fluorine atom and nucleobase in a trans-diaxial orientation. The Ara-FHNA analog with an equatorial fluorine was found to be destabilizing. However, the magnitude of destabilization was sequence-dependent. Thus, the loss of stability is sharply reduced when Ara-FHNA residues were inserted at pyrimidine-purine (Py-Pu) steps compared to placement within a stretch of pyrimidines (Py-Py). Crystal structures of A-type DNA duplexes modified with either monomer provide a rationalization for the opposing stability effects and point to a steric origin of the destabilization caused by the Ara-FHNA analog. The sequence dependent effect can be explained by the formation of an internucleotide C-F {hor_ellipsis} H-C pseudo hydrogen bond between F3' of Ara-FHNA and C8-H of the nucleobase from the 3'-adjacent adenosine that is absent at Py-Py steps. In animal experiments, FHNA-modified antisense oligonucleotides formulated in saline showed a potent downregulation of gene expression in liver tissue without producing hepatotoxicity. Our data establish FHNA as a useful modification for antisense therapeutics and also confirm the stabilizing influence of F(Py) {hor_ellipsis} H-C(Pu) pseudo hydrogen bonds in nucleic acid structures.

  1. Short locked nucleic acid antisense oligonucleotides potently reduce apolipoprotein B mRNA and serum cholesterol in mice and non-human primates

    DEFF Research Database (Denmark)

    Straarup, Ellen Marie; Fisker, Niels; Hedtjärn, Maj

    2010-01-01

    -life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1-2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non...... using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates....

  2. The Medicinal Chemistry of Therapeutic Oligonucleotides.

    Science.gov (United States)

    Wan, W Brad; Seth, Punit P

    2016-11-10

    Oligonucleotide-based therapeutics have made rapid progress in the clinic for treatment of a variety of disease indications. Unmodified oligonucleotides are polyanionic macromolecules with poor drug-like properties. Over the past two decades, medicinal chemists have identified a number of chemical modification and conjugation strategies which can improve the nuclease stability, RNA-binding affinity, and pharmacokinetic properties of oligonucleotides for therapeutic applications. In this perspective, we present a summary of the most commonly used nucleobase, sugar and backbone modification, and conjugation strategies used in oligonucleotide medicinal chemistry.

  3. Gene editing by co-transformation of TALEN and chimeric RNA/DNA oligonucleotides on the rice OsEPSPS gene and the inheritance of mutations.

    Directory of Open Access Journals (Sweden)

    Mugui Wang

    Full Text Available Although several site-specific nucleases (SSNs, such as zinc-finger nucleases (ZFNs, transcription activator-like effector nucleases (TALENs, and the clustered regularly interspaced short palindromic repeat (CRISPR/Cas, have emerged as powerful tools for targeted gene editing in many organisms, to date, gene targeting (GT in plants remains a formidable challenge. In the present study, we attempted to substitute a single base in situ on the rice OsEPSPS gene by co-transformation of TALEN with chimeric RNA/DNA oligonucleotides (COs, including different strand composition such as RNA/DNA (C1 or DNA/RNA (C2 but contained the same target base to be substituted. In contrast to zero GT event obtained by the co-transformation of TALEN with homologous recombination plasmid (HRP, we obtained one mutant showing target base substitution although accompanied by undesired deletion of 12 bases downstream the target site from the co-transformation of TALEN and C1. In addition to this typical event, we also obtained 16 mutants with different length of base deletions around the target site among 105 calli lines derived from transformation of TALEN alone (4/19 as well as co-transformation of TELAN with either HRP (5/30 or C1 (2/25 or C2 (5/31. Further analysis demonstrated that the homozygous gene-edited mutants without foreign gene insertion could be obtained in one generation. The induced mutations in transgenic generation were also capable to pass to the next generation stably. However, the genotypes of mutants did not segregate normally in T1 population, probably due to lethal mutations. Phenotypic assessments in T1 generation showed that the heterozygous plants with either one or three bases deletion on target sequence, called d1 and d3, were more sensitive to glyphosate and the heterozygous d1 plants had significantly lower seed-setting rate than wild-type.

  4. Comparison of three techniques for generation of tolerogenic dendritic cells: siRNA, oligonucleotide antisense, and antibody blocking.

    Science.gov (United States)

    Karimi, Mohammad Hossein; Ebadi, Padideh; Pourfathollah, Ali Akbar; Moazzeni, Mohammad; Soheili, Zahra Soheila; Samiee, Shahram

    2010-12-01

    In recent years, a new view of dendritic cells (DCs) as a main regulator of immunity to induce and maintain tolerance has been established. In vitro manipulation of their development and maturation is a topic of DC therapeutic application, which utilizes their inherent tolerogenicity. In this field, the therapeutic potential of antisense, siRNA, and blocking antibody are an interesting goal. In the present study, the efficiency of these three methods--siRNA, antisense, and blocking antibody--against CD40 molecule and its function in DCs and BCL1 cell line are compared. DCs were separated from mouse spleen and then cultured in vitro using Lipofectamine 2000 to deliver both silencers; the efficacy of transfection was estimated by flow cytometry. mRNA expression and protein synthesis were assessed by real time-PCR and flow cytometry, respectively. By Annexin V and propidium iodine staining, we could evaluate the viability of transfected cells. Knocking down the CD40 gene into separate groups of DCs by siRNA, antisense, and blocking antibody treated DCs can cause an increase in IL-4, decrease in IL-12, IFN-γ production, and allostimulation activity. Our results indicated that, in comparison to antisense and blocking antibody, siRNAs appear to be quantitatively more efficient in CD40 downregulation and their differences are significant.

  5. Use of 16S rRNA-targeted oligonucleotide probes to investigate the distribution of sulphate-reducing bacteria in estuarine sediments.

    Science.gov (United States)

    Purdy, K J.; Nedwell, D B.; Embley, T M.; Takii, S

    2001-07-01

    The distribution of sulphate-reducing bacteria (SRBs) in three anaerobic sediments, one predominantly freshwater and low sulphate and two predominantly marine and high sulphate, on the River Tama, Tokyo, Japan, was investigated using 16S rRNA-targeted oligonucleotide probes. Hybridisation results and sulphate reduction measurements indicated that SRBs are a minor part of the bacterial population in the freshwater sediments. Only Desulfobulbus and Desulfobacterium were detected, representing 1.6% of the general bacterial probe signal. In contrast, the SRB community detected at the two marine-dominated sites was larger and more diverse, representing 10-11.4% of the bacterial signal and with Desulfobacter, Desulfovibrio, Desulfobulbus and Desulfobacterium detected. In contrast to previous reports our results suggest that Desulfovibrio may not always be the most abundant SRB in anaerobic sediments. Acetate-utilising Desulfobacter were the dominant SRB in the marine-dominated sediments, and Desulfobulbus and Desulfobacterium were active in low-sulphate sediments, where they may utilise electron acceptors other than sulphate.

  6. Quantification of different Eubacterium spp. in human fecal samples with species-specific 16S rRNA-targeted oligonucleotide probes.

    Science.gov (United States)

    Schwiertz, A; Le Blay, G; Blaut, M

    2000-01-01

    Species-specific 16S rRNA-targeted, Cy3 (indocarbocyanine)-labeled oligonucleotide probes were designed and validated to quantify different Eubacterium species in human fecal samples. Probes were directed at Eubacterium barkeri, E. biforme, E. contortum, E. cylindroides (two probes), E. dolichum, E. hadrum, E. lentum, E. limosum, E. moniliforme, and E. ventriosum. The specificity of the probes was tested with the type strains and a range of common intestinal bacteria. With one exception, none of the probes showed cross-hybridization under stringent conditions. The species-specific probes were applied to fecal samples obtained from 12 healthy volunteers. E. biforme, E. cylindroides, E. hadrum, E. lentum, and E. ventriosum could be determined. All other Eubacterium species for which probes had been designed were under the detection limit of 10(7) cells g (dry weight) of feces(-1). The cell counts obtained are essentially in accordance with the literature data, which are based on colony counts. This shows that whole-cell in situ hybridization with species-specific probes is a valuable tool for the enumeration of Eubacterium species in feces.

  7. Nucleic acid helices: I. Structure of M1 RNA from E. coli as determined bypsoralen crosslinking. II. Thermodynamics of the helix-coil transition of DNA oligonucleotides in solutions containing 3.0 M tetramethylammonium chloride

    International Nuclear Information System (INIS)

    Lipson, S.E.

    1987-01-01

    This work includes two different investigations examining nucleic acid helices. The first study discusses secondary and tertiary interactions in the RNA moiety of ribonuclease P from Escherichia coli. The second study discusses the thermodynamics of the helix-coil transition of DNA oligonucleotides in solutions containing tetramethylammonium chloride. The RNA moiety of ribonuclease P from Escherichia coli (M1 RNA) has been photoreacted with 4'-hydroxymethyl-4,5'8-trimethylpsoralen and long wave UV light (320-380 nm) in a buffer in which the M1 RNA alone acts as a true catalyst of tRNA processing. Limited specific digestion followed by two dimensional gel electrophoresis yields fragments crosslinked by HMT. The positions of the crosslinks have been determined to within ±15 nucleotides by photoreversal of the isolated crosslinked fragments and enzymatic sequencing of the resulting RNA. Further assignments of the exact locations of the crosslinks have been made on the known photoreactivity of the psoralen with different bases

  8. Patterns of oligonucleotide sequences in viral and host cell RNA identify mediators of the host innate immune system.

    Directory of Open Access Journals (Sweden)

    Benjamin D Greenbaum

    Full Text Available The innate immune response provides a first line of defense against pathogens by targeting generic differential features that are present in foreign organisms but not in the host. These innate responses generate selection forces acting both in pathogens and hosts that further determine their co-evolution. Here we analyze the nucleic acid sequence fingerprints of these selection forces acting in parallel on both host innate immune genes and ssRNA viral genomes. We do this by identifying dinucleotide biases in the coding regions of innate immune response genes in plasmacytoid dendritic cells, and then use this signal to identify other significant host innate immune genes. The persistence of these biases in the orthologous groups of genes in humans and chickens is also examined. We then compare the significant motifs in highly expressed genes of the innate immune system to those in ssRNA viruses and study the evolution of these motifs in the H1N1 influenza genome. We argue that the significant under-represented motif pattern of CpG in an AU context--which is found in both the ssRNA viruses and innate genes, and has decreased throughout the history of H1N1 influenza replication in humans--is immunostimulatory and has been selected against during the co-evolution of viruses and host innate immune genes. This shows how differences in host immune biology can drive the evolution of viruses that jump into species with different immune priorities than the original host.

  9. Discriminating a Single Nucleotide Difference for Enhanced miRNA Detection Using Tunable Graphene and Oligonucleotide Nanodevices.

    Science.gov (United States)

    Robertson, Neil M; Hizir, Mustafa Salih; Balcioglu, Mustafa; Wang, Rui; Yavuz, Mustafa Selman; Yumak, Hasan; Ozturk, Birol; Sheng, Jia; Yigit, Mehmet V

    2015-09-15

    In this study we have reported our efforts to address some of the challenges in the detection of miRNAs using water-soluble graphene oxide and DNA nanoassemblies. Purposefully inserting mismatches at specific positions in our DNA (probe) strands shows increasing specificity against our target miRNA, miR-10b, over miR-10a which varies by only a single nucleotide. This increased specificity came at a loss of signal intensity within the system, but we demonstrated that this could be addressed with the use of DNase I, an endonuclease capable of cleaving the DNA strands of the RNA/DNA heteroduplex and recycling the RNA target to hybridize to another probe strand. As we previously demonstrated, this enzymatic signal also comes with an inherent activity of the enzyme on the surface-adsorbed probe strands. To remove this activity of DNase I and the steady nonspecific increase in the fluorescence signal without compromising the recovered signal, we attached a thermoresponsive PEGMA polymer (poly(ethylene glycol) methyl ether methacrylate) to nGO. This smart polymer is able to shield the probes adsorbed on the nGO surface from the DNase I activity and is capable of tuning the detection capacity of the nGO nanoassembly with a thermoswitch at 39 °C. By utilizing probes with multiple mismatches, DNase I cleavage of the DNA probe strands, and the attachment of PEGMA polymers to graphene oxide to block undesired DNase I activity, we were able to detect miR-10b from liquid biopsy mimics and breast cancer cell lines. Overall we have reported our efforts to improve the specificity, increase the sensitivity, and eliminate the undesired enzymatic activity of DNase I on surface-adsorbed probes for miR-10b detection using water-soluble graphene nanodevices. Even though we have demonstrated only the discrimination of miR-10b from miR-10a, our approach can be extended to other short RNA molecules which differ by a single nucleotide.

  10. Peptide-LNA oligonucleotide conjugates

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Hansen, Lykke Haastrup; Vester, Birte

    2013-01-01

    properties, peptides were introduced into oligonucleotides via a 2'-alkyne-2'-amino-LNA scaffold. Derivatives of methionine- and leucine-enkephalins were chosen as model peptides of mixed amino acid content, which were singly and doubly incorporated into LNA/DNA strands using highly efficient copper......(i)-catalyzed azide-alkyne cycloaddition (CuAAC) "click" chemistry. DNA/RNA target binding affinity and selectivity of the resulting POCs were improved in comparison to LNA/DNA mixmers and unmodified DNA controls. This clearly demonstrates that internal attachment of peptides to oligonucleotides can significantly...

  11. Therapeutic Oligonucleotides Targeting Liver Disease: TTR Amyloidosis

    Directory of Open Access Journals (Sweden)

    Christoph Niemietz

    2015-09-01

    Full Text Available The liver has become an increasingly interesting target for oligonucleotide therapy. Mutations of the gene encoding transthyretin (TTR, expressed in vast amounts by the liver, result in a complex degenerative disease, termed familial amyloid polyneuropathy (FAP. Misfolded variants of TTR are linked to the establishment of extracellular protein deposition in various tissues, including the heart and the peripheral nervous system. Recent progress in the chemistry and formulation of antisense (ASO and small interfering RNA (siRNA designed for a knockdown of TTR mRNA in the liver has allowed to address the issue of gene-specific molecular therapy in a clinical setting of FAP. The two therapeutic oligonucleotides bind to RNA in a sequence specific manner but exploit different mechanisms. Here we describe major developments that have led to the advent of therapeutic oligonucleotides for treatment of TTR-related disease.

  12. Visualization of mcr mRNA in a methanogen by fluorescence in situ hybridization with an oligonucleotide probe and two-pass tyramide signal amplification (two-pass TSA-FISH).

    Science.gov (United States)

    Kubota, Kengo; Ohashi, Akiyoshi; Imachi, Hiroyuki; Harada, Hideki

    2006-09-01

    Two-pass tyramide signal amplification-fluorescence in situ hybridization (two-pass TSA-FISH) with a horseradish peroxidase (HRP)-labeled oligonucleotide probe was applied to detect prokaryotic mRNA. In this study, mRNA of a key enzyme for methanogenesis, methyl coenzyme M reductase (mcr), in Methanococcus vannielii was targeted. Applicability of mRNA-targeted probes to in situ hybridization was verified by Clone-FISH. It was observed that sensitivity of two-pass TSA-FISH was significantly higher than that of TSA-FISH, which was further increased by the addition of dextran sulphate in TSA working solution. Signals from two-pass TSA-FISH were more reliable compared to the weak, spotty signals yielded by TSA-FISH.

  13. Detection and enumeration of methanotrophs in acidic Sphagnum peat by 16S rRNA fluorescence in situ hybridization, including the use of newly developed oligonucleotide probes for Methylocella palustris.

    Science.gov (United States)

    Dedysh, S N; Derakshani, M; Liesack, W

    2001-10-01

    Two 16S rRNA-targeted oligonucleotide probes, Mcell-1026 and Mcell-181, were developed for specific detection of the acidophilic methanotroph Methylocella palustris using fluorescence in situ hybridization (FISH). The fluorescence signal of probe Mcell-181 was enhanced by its combined application with the oligonucleotide helper probe H158. Mcell-1026 and Mcell-181, as well as 16S rRNA oligonucleotide probes with reported group specificity for either type I methanotrophs (probes M-84 and M-705) or the Methylosinus/Methylocystis group of type II methanotrophs (probes MA-221 and M-450), were used in FISH to determine the abundance of distinct methanotroph groups in a Sphagnum peat sample of pH 4.2. M. palustris was enumerated at greater than 10(6) cells per g of peat (wet weight), while the detectable population size of type I methanotrophs was three orders of magnitude below the population level of M. palustris. The cell counts with probe MA-221 suggested that only 10(4) type II methanotrophs per g of peat (wet weight) were present, while the use of probe M-450 revealed more than 10(6) type II methanotroph cells per g of the same samples. This discrepancy was due to the fact that probe M-450 targets almost all currently known strains of Methylosinus and Methylocystis, whereas probe MA-221, originally described as group specific, does not detect a large proportion of Methylocystis strains. The total number of methanotrophic bacteria detected by FISH was 3.0 (+/-0.2) x 10(6) cells per g (wet weight) of peat. This was about 0.8% of the total bacterial cell number. Thus, our study clearly suggests that M. palustris and a defined population of Methylocystis spp. were the predominant methanotrophs detectable by FISH in an acidic Sphagnum peat bog.

  14. Nucleic acid helices: I. Structure of M1 RNA from E. coli as determined bypsoralen crosslinking. II. Thermodynamics of the helix-coil transition of DNA oligonucleotides in solutions containing 3. 0 M tetramethylammonium chloride

    Energy Technology Data Exchange (ETDEWEB)

    Lipson, S.E.

    1987-01-01

    This work includes two different investigations examining nucleic acid helices. The first study discusses secondary and tertiary interactions in the RNA moiety of ribonuclease P from Escherichia coli. The second study discusses the thermodynamics of the helix-coil transition of DNA oligonucleotides in solutions containing tetramethylammonium chloride. The RNA moiety of ribonuclease P from Escherichia coli (M1 RNA) has been photoreacted with 4{prime}-hydroxymethyl-4,5{prime}8-trimethylpsoralen and long wave UV light (320-380 nm) in a buffer in which the M1 RNA alone acts as a true catalyst of tRNA processing. Limited specific digestion followed by two dimensional gel electrophoresis yields fragments crosslinked by HMT. The positions of the crosslinks have been determined to within {plus minus}15 nucleotides by photoreversal of the isolated crosslinked fragments and enzymatic sequencing of the resulting RNA. Further assignments of the exact locations of the crosslinks have been made on the known photoreactivity of the psoralen with different bases.

  15. Isolation and characterization of human glycophorin A cDNAs using a synthetic oligonucleotide approach: nucleotide sequence, mRNA structure and regulation by 12-O-tetradecanoylphorbol 13-acetate (TPA)

    International Nuclear Information System (INIS)

    Siebert, P.D.; Fukuda, M.

    1986-01-01

    The authors have previously shown that treatment of human erythroleukemic K562 cells with the tumor-promoting phorbol ester, TPA, results in a diminished expression of glycophorin A at the level of protein biosynthesis and in vitro mRNA translation activity. To further examine the structure, relationships and expression of human glycophorins they have successfully isolated and sequenced several glycophorin A specific cDNA clones derived from K562 cells, by making extensive use of mixed and exact synthetic oligonucleotides as primers and radioactively labeled probes. The nucleotide sequence obtained from the largest glycophorin A cDNA suggests the presence of a hydrophobic leader-like peptide of at least 19 amino acids. Northern gel analysis using both whole cDNA-plasmid and synthetic oligonucleotide probes revealed the existence of multiple mRNAs, three of which they believe to be glycophorin A-specific, whereas a fourth and smaller mRNA appears to be glycophorin B-specific. Furthermore, the abundance of all four glycophorin mRNAs were found to be extensively reduced following treatment of K562 cells with TPA suggesting coordinate regulation, possibly at the level of gene transcription

  16. Problems connected with the use of oligonucleotide probes with a high degree of degeneracy. Identification of mRNA and of cDNA clones corresponding to the gene of the. cap alpha. -subunit of Na/sup +/, K/sup +/-ATPase

    Energy Technology Data Exchange (ETDEWEB)

    Petrukhin, K.E.; Grishin, A.V.; Arsenyan, S.G.; Broude, N.E.; Grinkevich, V.A.; Filippova, L.Yu.; Severtsova, I.V.; Modyanov, N.N.

    1986-10-01

    To identify and search for nucleotide sequences containing the structural part of the gene of the ..cap alpha..-subunit of Na/sup +/, K/sup +/-ATPase, 17-membered oligonucleotide probes corresponding to the peptide Lys-Asp-Ala-Phe-Gln-Asn have been synthesized. It has been shown that, with a 64-fold degeneracyd, the 17-membered probe is suitable only for the identification of a specific sequence in mRNA. To search for clones containing cDNA fragments, preliminary fractionation of the probes with the aid of HPLC or the resynthesis of groups of oligonucleotides with a lower degeneracy is necessary.

  17. Oligonucleotide-based theranostic nanoparticles in cancer therapy

    Science.gov (United States)

    Shahbazi, Reza; Ozpolat, Bulent; Ulubayram, Kezban

    2016-01-01

    Theranostic approaches, combining the functionality of both therapy and imaging, have shown potential in cancer nanomedicine. Oligonucleotides such as small interfering RNA and microRNA, which are powerful therapeutic agents, have been effectively employed in theranostic systems against various cancers. Nanoparticles are used to deliver oligonucleotides into tumors by passive or active targeting while protecting the oligonucleotides from nucleases in the extracellular environment. The use of quantum dots, iron oxide nanoparticles and gold nanoparticles and tagging with contrast agents, like fluorescent dyes, optical or magnetic agents and various radioisotopes, has facilitated early detection of tumors and evaluation of therapeutic efficacy. In this article, we review the advantages of theranostic applications in cancer therapy and imaging, with special attention to oligonucleotide-based therapeutics. PMID:27102380

  18. Attenuation of alpha2A-adrenergic receptor expression in neonatal rat brain by RNA interference or antisense oligonucleotide reduced anxiety in adulthood.

    Science.gov (United States)

    Shishkina, G T; Kalinina, T S; Dygalo, N N

    2004-01-01

    Brain alpha2-adrenergic receptors (alpha2-ARs) have been implicated in the regulation of anxiety, which is associated with stress. Environmental treatments during neonatal development could modulate the level of brain alpha2-AR expression and alter anxiety in adults, suggesting possible involvement of these receptors in early-life programming of anxiety state. The present study was undertaken to determine whether the reduction of the expression of A subtype of these receptors most abundant in the neonatal brain affects anxiety-related behavior in adulthood. We attenuated the expression of alpha2A-ARs during neonatal life by two different sequence specific approaches, antisense technology and RNA interference. Treatment of rats with the antisense oligodeoxynucleotide or short interfering RNA (siRNA) against alpha2A-ARs on the days 2-4 of their life, produced a marked acute decrease in the levels of both alpha2A-AR mRNA and [3H]RX821002 binding sites in the brainstem into which drugs were injected. The decrease of alpha2A-AR expression in the neonatal brainstem influenced the development of this receptor system in the brain regions as evidenced by the increased number of [3H]RX821002 binding sites in the hypothalamus of adult animals with both neonatal alpha2A-AR knockdown treatments; also in the frontal cortex of antisense-treated, and in the hippocampus of siRNA-treated adult rats. These adult animals also demonstrated a decreased anxiety in the elevated plus-maze as evidenced by an increased number of the open arm entries, greater proportion of time spent in the open arms, and more than a two-fold increase in the number of exploratory head dips. The results provide the first evidence that the reduction in the brain expression of a gene encoding for alpha2A-AR during neonatal life led to the long-term neurochemical and behavioral alterations. The data suggests that alterations in the expression of the receptor-specific gene during critical periods of brain

  19. Oligonucleotide Therapy for Obstructive and Restrictive Respiratory Diseases

    Directory of Open Access Journals (Sweden)

    Wupeng Liao

    2017-01-01

    Full Text Available Inhaled oligonucleotide is an emerging therapeutic modality for various common respiratory diseases, including obstructive airway diseases like asthma and chronic obstructive pulmonary disease (COPD and restrictive airway diseases like idiopathic pulmonary fibrosis (IPF. The advantage of direct accessibility for oligonucleotide molecules to the lung target sites, bypassing systemic administration, makes this therapeutic approach promising with minimized potential systemic side effects. Asthma, COPD, and IPF are common chronic respiratory diseases, characterized by persistent airway inflammation and dysregulated tissue repair and remodeling, although each individual disease has its unique etiology. Corticosteroids have been widely prescribed for the treatment of asthma, COPD, and IPF. However, the effectiveness of corticosteroids as an anti-inflammatory drug is limited by steroid resistance in severe asthma, the majority of COPD cases, and pulmonary fibrosis. There is an urgent medical need to develop target-specific drugs for the treatment of these respiratory conditions. Oligonucleotide therapies, including antisense oligonucleotide (ASO, small interfering RNA (siRNA, and microRNA (miRNA are now being evaluated both pre-clinically and clinically as potential therapeutics. The mechanisms of action of ASO and siRNA are highly target mRNA specific, ultimately leading to target protein knockdown. miRNA has both biomarker and therapeutic values, and its knockdown by a miRNA antagonist (antagomir has a broader but potentially more non-specific biological outcome. This review will compile the current findings of oligonucleotide therapeutic targets, verified in various respiratory disease models and in clinical trials, and evaluate different chemical modification approaches to improve the stability and potency of oligonucleotides for the treatment of respiratory diseases.

  20. Stereospecificity of oligonucleotide interactions revisited: no evidence for heterochiral hybridization and ribozyme/DNAzyme activity.

    Directory of Open Access Journals (Sweden)

    Kai Hoehlig

    Full Text Available A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications.

  1. Inhibition of iridovirus protein synthesis and virus replication by antisense morpholino oligonucleotides targeted to the major capsid protein, the 18 kDa immediate-early protein, and a viral homolog of RNA polymerase II

    International Nuclear Information System (INIS)

    Sample, Robert; Bryan, Locke; Long, Scott; Majji, Sai; Hoskins, Glenn; Sinning, Allan; Olivier, Jake; Chinchar, V. Gregory

    2007-01-01

    Frog virus 3 (FV3) is a large DNA virus that encodes ∼ 100 proteins. Although the general features of FV3 replication are known, the specific roles that most viral proteins play in the virus life cycle have not yet been elucidated. To address the question of viral gene function, antisense morpholino oligonucleotides (asMOs) were used to transiently knock-down expression of specific viral genes and thus infer their role in virus replication. We designed asMOs directed against the major capsid protein (MCP), an 18 kDa immediate-early protein (18K) that was thought to be a viral regulatory protein, and the viral homologue of the largest subunit of RNA polymerase II (vPol-IIα). All three asMOs successfully inhibited translation of the targeted protein, and two of the three asMOs resulted in marked phenotypic changes. Knock-down of the MCP resulted in a marked reduction in viral titer without a corresponding drop in the synthesis of other late viral proteins. Transmission electron microscopy (TEM) showed that in cells treated with the anti-MCP MO assembly sites were devoid of viral particles and contained numerous aberrant structures. In contrast, inhibition of 18K synthesis did not block virion formation, suggesting that the 18K protein was not essential for replication of FV3 in fathead minnow (FHM) cells. Finally, consistent with the view that late viral gene expression is catalyzed by a virus-encoded or virus-modified Pol-II-like protein, knock-down of vPol-IIα triggered a global decline in late gene expression and virus yields without affecting the synthesis of early viral genes. Collectively, these results demonstrate the utility of using asMOs to elucidate the function of FV3 proteins

  2. Tandem Oligonucleotide Probe Annealing and Elongation To Discriminate Viral Sequence

    DEFF Research Database (Denmark)

    Taskova, Maria; Uhd, Jesper; Miotke, Laura

    2017-01-01

    opportunities in transcriptome analysis, virology, and other fields. Herein, we report for the first time a "click" chemistry approach to oligonucleotide probe elongation as a novel approach to specifically detect a viral sequence. We hybridized a library of short, terminally labeled probes to Ebola virus RNA...

  3. Fully automated parallel oligonucleotide synthesizer

    Czech Academy of Sciences Publication Activity Database

    Lebl, M.; Burger, Ch.; Ellman, B.; Heiner, D.; Ibrahim, G.; Jones, A.; Nibbe, M.; Thompson, J.; Mudra, Petr; Pokorný, Vít; Poncar, Pavel; Ženíšek, Karel

    2001-01-01

    Roč. 66, č. 8 (2001), s. 1299-1314 ISSN 0010-0765 Institutional research plan: CEZ:AV0Z4055905 Keywords : automated oligonucleotide synthesizer Subject RIV: CC - Organic Chemistry Impact factor: 0.778, year: 2001

  4. Chemically modified oligonucleotides with efficient RNase H response

    DEFF Research Database (Denmark)

    Vester, Birte; Boel, Anne Marie; Lobedanz, Sune

    2008-01-01

    Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly...... in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage....

  5. A Kinetic Model Explains Why Shorter and Less Affine Enzyme-recruiting Oligonucleotides Can Be More Potent

    Directory of Open Access Journals (Sweden)

    Lykke Pedersen

    2014-01-01

    Full Text Available Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reasoned to confer higher potency, i.e., modified oligonucleotides can be dosed at lower concentrations to achieve the same effect. Surprisingly, shorter and less affine oligonucleotides sometimes display increased potency. To explain this apparent contradiction, increased uptake or decreased propensity to form structures have been suggested as possible mechanisms. Here, we provide an alternative explanation that invokes only the kinetics behind oligonucleotide-mediated cleavage of RNA targets. A model based on the law of mass action predicts, and experiments support, the existence of an optimal binding affinity. Exaggerated affinity, and not length per se, is detrimental to potency. This finding clarifies how to optimally apply high-affinity modifications in the discovery of potent antisense oligonucleotide drugs.

  6. RNA.

    Science.gov (United States)

    Darnell, James E., Jr.

    1985-01-01

    Ribonucleic acid (RNA) converts genetic information into protein and usually must be processed to serve its function. RNA types, chemical structure, protein synthesis, translation, manufacture, and processing are discussed. Concludes that the first genes might have been spliced RNA and that humans might be closer than bacteria to primitive…

  7. Electronic Structures of LNA Phosphorothioate Oligonucleotides

    DEFF Research Database (Denmark)

    Bohr, Henrik G.; Shim, Irene; Stein, Cy

    2017-01-01

    Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM) calculations and chromatography experiments on locked nucleic acid (LNA) phosphorothioate (PS) oligonucleotides. iso-potential electrostatic surfaces...

  8. Thermodynamics of Oligonucleotide Duplex Melting

    Science.gov (United States)

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-01-01

    Melting temperatures of oligonucleotides are useful for a number of molecular biology applications, such as the polymerase chain reaction (PCR). Although melting temperatures are often calculated with simplistic empirical equations, application of thermodynamics provides more accurate melting temperatures and an opportunity for students to apply…

  9. Identification of clinically relevant viridans streptococci by an oligonucleotide array.

    Science.gov (United States)

    Chen, Chao Chien; Teng, Lee Jene; Kaiung, Seng; Chang, Tsung Chain

    2005-04-01

    Viridans streptococci (VS) are common etiologic agents of subacute infective endocarditis and are capable of causing a variety of pyogenic infections. Many species of VS are difficult to differentiate by phenotypic traits. An oligonucleotide array based on 16S-23S rRNA gene intergenic spacer (ITS) sequences was developed to identify 11 clinically relevant VS. These 11 species were Streptococcus anginosus, S. constellatus, S. gordonii, S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguinis, S. salivarius, S. sanguinis, and S. uberis. The method consisted of PCR amplification of the ITS regions by using a pair of universal primers, followed by hybridization of the digoxigenin-labeled PCR products to a panel of species-specific oligonucleotides immobilized on a nylon membrane. After 120 strains of the 11 species of VG and 91 strains of other bacteria were tested, the sensitivity and specificity of the oligonucleotide array were found to be 100% (120 of 120 strains) and 95.6% (87 of 91 strains), respectively. S. pneumoniae cross-hybridized to the probes used for the identification of S. mitis, and simple biochemical tests such as optochin susceptibility or bile solubility should be used to differentiate S. pneumoniae from S. mitis. In conclusion, identification of species of VS by use of the present oligonucleotide array is accurate and could be used as an alternative reliable method for species identification of strains of VS.

  10. Recommendations of the Oligonucleotide Safety Working Group's Formulated Oligonucleotide Subcommittee for the Safety Assessment of Formulated Oligonucleotide-Based Therapeutics.

    Science.gov (United States)

    Marlowe, Jennifer L; Akopian, Violetta; Karmali, Priya; Kornbrust, Douglas; Lockridge, Jennifer; Semple, Sean

    2017-08-01

    The use of lipid formulations has greatly improved the ability to effectively deliver oligonucleotides and has been instrumental in the rapid expansion of therapeutic development programs using oligonucleotide drugs. However, the development of such complex multicomponent therapeutics requires the implementation of unique, scientifically sound approaches to the nonclinical development of these drugs, based upon a hybrid of knowledge and experiences drawn from small molecule, protein, and oligonucleotide therapeutic drug development. The relative paucity of directly applicable regulatory guidance documents for oligonucleotide therapeutics in general has resulted in the generation of multiple white papers from oligonucleotide drug development experts and members of the Oligonucleotide Safety Working Group (OSWG). The members of the Formulated Oligonucleotide Subcommittee of the OSWG have utilized their collective experience working with a variety of formulations and their associated oligonucleotide payloads, as well as their insights into regulatory considerations and expectations, to generate a series of consensus recommendations for the pharmacokinetic characterization and nonclinical safety assessment of this unique class of therapeutics. It should be noted that the focus of Subcommittee discussions was on lipid nanoparticle and other types of particulate formulations of therapeutic oligonucleotides and not on conjugates or other types of modifications of oligonucleotide structure intended to facilitate delivery.

  11. Polymerase-endonuclease amplification reaction (PEAR for large-scale enzymatic production of antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Xiaolong Wang

    Full Text Available Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR, for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs.

  12. Chemical Ligation Reactions of Oligonucleotides for Biological and Medicinal Applications.

    Science.gov (United States)

    Abe, Hiroshi; Kimura, Yasuaki

    2018-01-01

    Chemical ligation of oligonucleotides (ONs) is the key reaction for various ON-based technologies. We have tried to solve the problems of RNA interference (RNAi) technology by applying ON chemical ligation to RNAi. We designed a new RNAi system, called intracellular buildup RNAi (IBR-RNAi), where the RNA fragments are built up into active small-interference RNA (siRNA) in cells through a chemical ligation reaction. Using the phosphorothioate and iodoacetyl groups as reactive functional groups for the ligation, we achieved RNAi effects without inducing immune responses. Additionally, we developed a new chemical ligation for IBR-RNAi, which affords a more native-like structure in the ligated product. The new ligation method should be useful not only for IBR-RNAi but also for the chemical synthesis of biofunctional ONs.

  13. Binding of Dumbbell Oligonucleotides to MoMuLV Reverse Transcriptase: Inhibitory Properties of RNase H Activity

    Directory of Open Access Journals (Sweden)

    Ajay Kumar

    2010-01-01

    Full Text Available Dumbbell oligonucleotides with loops of various chemistry were synthesized. Incubation of dumbbell oligonucleotides containing phosphorothioate bonds or trimethylene phosphate linkages in loops with S1 nuclease did not result in significant cleavage under conditions which led to the degradation of dumbbell oligonucleotide containing phophodiester bonds in the loops. The binding of reverse transcriptase of Moloney Murine Leukemia Virus (MoMuLV was evaluated with all the five oligonucleotides. The protein binds to all the dumbbell oligonucleotides with similar affinity. The dissociation constants evaluated using PAGE band mobility shift assays were of the order of 10-7. The inhibitory properties of the retroviral RNase H activity was evaluated using 3H –UTP-labeled RNA:RNA-DNA hybrid. It was found that the best dumbbell oligonucleotide, inhibitor contained phosphorothioate residues in both the loops. Our value studies demonstrated that this particularly designed oligonucleotide displays an IC50 of 18 nM in its inhibition on the reverse transcriptase RNase H activity, a magnitude lower than that of first nucleotide reverse transcriptase of HIV-1, tenofovir, introduced by Gilead Science in the market.

  14. A one-step reaction for the rapid identification of Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti using oligonucleotide primers designed from the 16S-23S rRNA intergenic sequences.

    Science.gov (United States)

    Ferchichi, M; Valcheva, R; Prévost, H; Onno, B; Dousset, X

    2008-06-01

    Species-specific primers targeting the 16S-23S ribosomal DNA (rDNA) intergenic spacer region (ISR) were designed to rapidly discriminate between Lactobacillus mindensis, Lactobacillus panis, Lactobacillus paralimentarius, Lactobacillus pontis and Lactobacillus frumenti species recently isolated from French sourdough. The 16S-23S ISRs were amplified using primers 16S/p2 and 23S/p7, which anneal to positions 1388-1406 of the 16S rRNA gene and to positions 207-189 of the 23S rRNA gene respectively, Escherichia coli numbering (GenBank accession number V00331). Clone libraries of the resulting amplicons were constructed using a pCR2.1 TA cloning kit and sequenced. Species-specific primers were designed based on the sequences obtained and were used to amplify the 16S-23S ISR in the Lactobacillus species considered. For all of them, two PCR amplicons, designated as small ISR (S-ISR) and large ISR (L-ISR), were obtained. The L-ISR is composed of the corresponding S-ISR, interrupted by a sequence containing tRNA(Ile) and tRNA(Ala) genes. Based on these sequences, species-specific primers were designed and proved to identify accurately the species considered among 30 reference Lactobacillus species tested. Designed species-specific primers enable a rapid and accurate identification of L. mindensis, L. paralimentarius, L. panis, L. pontis and L. frumenti species among other lactobacilli. The proposed method provides a powerful and convenient means of rapidly identifying some sourdough lactobacilli, which could be of help in large starter culture surveys.

  15. TIA-1 RRM23 binding and recognition of target oligonucleotides.

    Science.gov (United States)

    Waris, Saboora; García-Mauriño, Sofía M; Sivakumaran, Andrew; Beckham, Simone A; Loughlin, Fionna E; Gorospe, Myriam; Díaz-Moreno, Irene; Wilce, Matthew C J; Wilce, Jacqueline A

    2017-05-05

    TIA-1 (T-cell restricted intracellular antigen-1) is an RNA-binding protein involved in splicing and translational repression. It mainly interacts with RNA via its second and third RNA recognition motifs (RRMs), with specificity for U-rich sequences directed by RRM2. It has recently been shown that RRM3 also contributes to binding, with preferential binding for C-rich sequences. Here we designed UC-rich and CU-rich 10-nt sequences for engagement of both RRM2 and RRM3 and demonstrated that the TIA-1 RRM23 construct preferentially binds the UC-rich RNA ligand (5΄-UUUUUACUCC-3΄). Interestingly, this binding depends on the presence of Lys274 that is C-terminal to RRM3 and binding to equivalent DNA sequences occurs with similar affinity. Small-angle X-ray scattering was used to demonstrate that, upon complex formation with target RNA or DNA, TIA-1 RRM23 adopts a compact structure, showing that both RRMs engage with the target 10-nt sequences to form the complex. We also report the crystal structure of TIA-1 RRM2 in complex with DNA to 2.3 Å resolution providing the first atomic resolution structure of any TIA protein RRM in complex with oligonucleotide. Together our data support a specific mode of TIA-1 RRM23 interaction with target oligonucleotides consistent with the role of TIA-1 in binding RNA to regulate gene expression. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  16. Hetero-oligonucleotide Nanoscale Tiles Capable of Two-Dimensional Lattice Formation as Testbeds for a Rapid, Affordable Purification Methodology

    Science.gov (United States)

    2013-01-01

    devices; however, DNA is not the only polymer that can take advantage of the specicity of the Watson – Crick base-pair to achieve these goals. Central to...14. ABSTRACT 16. SECURITY CLASSIFICATION OF: New nanoscale hetero-oligonucleotide tiles are assembled from DNA , RNA and morpholino oligos and...purified using size exclusion filtration. Homo-oligonucleotide tiles assembled from RP-cartridge processed DNA oligos are purified by nondenaturing gel

  17. Ultramild protein-mediated click chemistry creates efficient oligonucleotide probes for targeting and detecting nucleic acids

    DEFF Research Database (Denmark)

    Nåbo, Lina J.; Madsen, Charlotte S.; Jensen, Knud J.

    2015-01-01

    Functionalized synthetic oligonucleotides are finding growing applications in research, clinical studies, and therapy. However, it is not easy to prepare them in a biocompatible and highly efficient manner. We report a new strategy to synthesize oligonucleotides with promising nucleic acid...... targeting and detection properties. We focus in particular on the pH sensitivity of these new probes and their high target specificity. For the first time, human copper(I)-binding chaperon Cox17 was applied to effectively catalyze click labeling of oligonucleotides. This was performed under ultramild...... conditions with fluorophore, peptide, and carbohydrate azide derivatives. In thermal denaturation studies, the modified probes showed specific binding to complementary DNA and RNA targets. Finally, we demonstrated the pH sensitivity of the new rhodamine-based fluorescent probes in vitro and rationalize our...

  18. Advances in Antisense Oligonucleotide Development for Target Identification, Validation, and as Novel Therapeutics

    Directory of Open Access Journals (Sweden)

    Moizza Mansoor

    2008-01-01

    Full Text Available Antisense oligonucleotides (As-ODNs are single stranded, synthetically prepared strands of deoxynucleotide sequences, usually 18–21 nucleotides in length, complementary to the mRNA sequence of the target gene. As-ODNs are able to selectively bind cognate mRNA sequences by sequence-specific hybridization. This results in cleavage or disablement of the mRNA and, thus, inhibits the expression of the target gene. The specificity of the As approach is based on the probability that, in the human genome, any sequence longer than a minimal number of nucleotides (nt, 13 for RNA and 17 for DNA, normally occurs only once. The potential applications of As-ODNs are numerous because mRNA is ubiquitous and is more accessible to manipulation than DNA. With the publication of the human genome sequence, it has become theoretically possible to inhibit mRNA of almost any gene by As-ODNs, in order to get a better understanding of gene function, investigate its role in disease pathology and to study novel therapeutic targets for the diseases caused by dysregulated gene expression. The conceptual simplicity, the availability of gene sequence information from the human genome, the inexpensive availability of synthetic oligonucleotides and the possibility of rational drug design makes As-ODNs powerful tools for target identification, validation and therapeutic intervention. In this review we discuss the latest developments in antisense oligonucleotide design, delivery, pharmacokinetics and potential side effects, as well as its uses in target identification and validation, and finally focus on the current developments of antisense oligonucleotides in therapeutic intervention in various diseases.

  19. Methods for the preparation of large quantities of complex single-stranded oligonucleotide libraries.

    Science.gov (United States)

    Murgha, Yusuf E; Rouillard, Jean-Marie; Gulari, Erdogan

    2014-01-01

    Custom-defined oligonucleotide collections have a broad range of applications in fields of synthetic biology, targeted sequencing, and cytogenetics. Also, they are used to encode information for technologies like RNA interference, protein engineering and DNA-encoded libraries. High-throughput parallel DNA synthesis technologies developed for the manufacture of DNA microarrays can produce libraries of large numbers of different oligonucleotides, but in very limited amounts. Here, we compare three approaches to prepare large quantities of single-stranded oligonucleotide libraries derived from microarray synthesized collections. The first approach, alkaline melting of double-stranded PCR amplified libraries with a biotinylated strand captured on streptavidin coated magnetic beads results in little or no non-biotinylated ssDNA. The second method wherein the phosphorylated strand of PCR amplified libraries is nucleolyticaly hydrolyzed is recommended when small amounts of libraries are needed. The third method combining in vitro transcription of PCR amplified libraries to reverse transcription of the RNA product into single-stranded cDNA is our recommended method to produce large amounts of oligonucleotide libraries. Finally, we propose a method to remove any primer binding sequences introduced during library amplification.

  20. Structure of an RNA dimer of a regulatory element from human thymidylate synthase mRNA

    OpenAIRE

    Dibrov, Sergey; McLean, Jaime; Hermann, Thomas

    2011-01-01

    An oligonucleotide representing a regulatory element of human thymidylate synthase mRNA has been crystallized as a dimer. The structure of the asymmetric dimer has been determined at 1.97 Å resolution.

  1. Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Hongguang Sun

    2014-01-01

    Full Text Available Aptamers are a class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets. Similar to antibodies, aptamers interact with their targets by recognizing a specific three-dimensional structure and are thus termed “chemical antibodies.” In contrast to protein antibodies, aptamers offer unique chemical and biological characteristics based on their oligonucleotide properties. Hence, they are more suitable for the development of novel clinical applications. Aptamer technology has been widely investigated in various biomedical fields for biomarker discovery, in vitro diagnosis, in vivo imaging, and targeted therapy. This review will discuss the potential applications of aptamer technology as a new tool for targeted cancer therapy with emphasis on the development of aptamers that are able to specifically target cell surface biomarkers. Additionally, we will describe several approaches for the use of aptamers in targeted therapeutics, including aptamer-drug conjugation, aptamer-nanoparticle conjugation, aptamer-mediated targeted gene therapy, aptamer-mediated immunotherapy, and aptamer-mediated biotherapy.

  2. Enzymatic Production of Monoclonal Stoichiometric Single-Stranded DNA Oligonucleotides

    Science.gov (United States)

    Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M.; Högberg, Björn

    2013-01-01

    Single-stranded oligonucleotides are important as research tools as probes for diagnostics and gene therapy. Today, production of oligonucleotides is done via solid-phase synthesis. However, the capabilities of current polymer chemistry are limited in comparison to what can be produced in biological systems. The errors in synthetic DNA increases with oligonucleotide length, and sequence diversity can often be a problem. Here, we present the Monoclonal Stoichiometric (MOSIC) method for enzymatic DNA oligonucleotide production. Using this method, we amplify oligonucleotides from clonal templates followed by digestion of a cutter-hairpin, resulting in pools of monoclonal oligonucleotides with precisely controlled relative stoichiometric ratios. We present data where MOSIC oligonucleotides, 14–378 nt long, were prepared either by in vitro rolling-circle amplification, or by amplification in Escherichia coli in the form of phagemid DNA. The formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides. PMID:23727986

  3. Enzymatic production of 'monoclonal stoichiometric' single-stranded DNA oligonucleotides.

    Science.gov (United States)

    Ducani, Cosimo; Kaul, Corinna; Moche, Martin; Shih, William M; Högberg, Björn

    2013-07-01

    Single-stranded oligonucleotides are important as research tools, as diagnostic probes, in gene therapy and in DNA nanotechnology. Oligonucleotides are typically produced via solid-phase synthesis, using polymer chemistries that are limited relative to what biological systems produce. The number of errors in synthetic DNA increases with oligonucleotide length, and the resulting diversity of sequences can be a problem. Here we present the 'monoclonal stoichiometric' (MOSIC) method for enzyme-mediated production of DNA oligonucleotides. We amplified oligonucleotides from clonal templates derived from single bacterial colonies and then digested cutter hairpins in the products, which released pools of oligonucleotides with precisely controlled relative stoichiometric ratios. We prepared 14-378-nucleotide MOSIC oligonucleotides either by in vitro rolling-circle amplification or by amplification of phagemid DNA in Escherichia coli. Analyses of the formation of a DNA crystal and folding of DNA nanostructures confirmed the scalability, purity and stoichiometry of the produced oligonucleotides.

  4. Oligonucleotides with 1,4-dioxane-based nucleotide monomers

    DEFF Research Database (Denmark)

    Madsen, Andreas S; Wengel, Jesper

    2012-01-01

    An epimeric mixture of H-phosphonates 5R and 5S has been synthesized in three steps from known secouridine 1. Separation of the epimers has been accomplished by RP-HPLC, allowing full characterization and incorporation of monomers X and Y into 9-mer oligonucleotides using H-phosphonates building...... blocks 5R and 5S, respectively. A single incorporation of either monomer X or monomer Y in the central position of a DNA 9-mer results in decreased thermal affinity toward both DNA and RNA complements (ΔT(m) = -3.5 °C/-3.5 °C for monomer X and ΔT(m) = -11.0 °C/-6.5 °C for monomer Y). CD measurements do...

  5. Sulfonamide bearing oligonucleotides: Simple synthesis and efficient RNA recognition

    DEFF Research Database (Denmark)

    Kumar, P.; Chandak, N.; Nielsen, P.

    2012-01-01

    Four pyrimidine nucleosides wherein a benzensulfonamide group is linked to the C-5 position of the uracil nucleobase through a triazolyl or an alkynyl linker were prepared by Cu(I)-assisted azide-alkyne cycloadditions (CuAAC) or Sonogashira reactions, respectively, and incorporated into oligonucl...

  6. Radio-marking and in vivo imagery of oligonucleotides

    International Nuclear Information System (INIS)

    Kuehnast, Bertrand

    2000-01-01

    This research thesis is part of activities aimed at the development of new molecules like oligonucleotides. Its first objective was the development and validation of a marking method with fluorine-18 of oligonucleotides for their in-vivo pharmacological assessment with positron emission tomography (PET). Further investigations addressed the use of iodine-125 for oligonucleotide marking purpose. This radio-marking, and in vivo and ex vivo imagery techniques are described, and their potential is highlighted for the pharmacological assessment of different oligonucleotides

  7. Electronic Structures of LNA Phosphorothioate Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Henrik G. Bohr

    2017-09-01

    Full Text Available Important oligonucleotides in anti-sense research have been investigated in silico and experimentally. This involves quantum mechanical (QM calculations and chromatography experiments on locked nucleic acid (LNA phosphorothioate (PS oligonucleotides. iso-potential electrostatic surfaces are essential in this study and have been calculated from the wave functions derived from the QM calculations that provide binding information and other properties of these molecules. The QM calculations give details of the electronic structures in terms of e.g., energy and bonding, which make them distinguish or differentiate between the individual PS diastereoisomers determined by the position of sulfur atoms. Rules are derived from the electronic calculations of these molecules and include the effects of the phosphorothioate chirality and formation of electrostatic potential surfaces. Physical and electrochemical descriptors of the PS oligonucleotides are compared to the experiments in which chiral states on these molecules can be distinguished. The calculations demonstrate that electronic structure, electrostatic potential, and topology are highly sensitive to single PS configuration changes and can give a lead to understanding the activity of the molecules. Keywords: LNA phosphorothioate, DNA/LNA oligonucleotide, diastereoisomers, Hartree-Fock calculations, iso-potential surface, anion chromatograms

  8. Associating Oligonucleotides with Positively Charged Liposomes

    Czech Academy of Sciences Publication Activity Database

    Jurkiewicz, P.; Okruszek, A.; Hof, Martin; Langner, M.

    2003-01-01

    Roč. 8, č. 1 (2003), s. 77-84 ISSN 1425-8153 R&D Projects: GA MŠk LN00A032 Institutional research plan: CEZ:AV0Z4040901 Keywords : oligonucleotides * fluorescence correlation spectroscopy * DOTAP Subject RIV: CF - Physical ; Theoretical Chemistry Impact factor: 0.455, year: 2003

  9. Triplex-forming ability of modified oligonucleotides

    DEFF Research Database (Denmark)

    Højland, Torben; Babu, Bolle Ravindra; Bryld, Torsten

    2007-01-01

    We present our studies on the ability of several different nucleotide analogs as triplex-forming oligonucleotides. The modifications tested include 4'-C-hydroxymethyl, LNA, 2'-amino-LNA and N2'-functionalized 2'-amino-LNA. Triplexes containing monomers of N2'-glycyl-functionalized 2'-amino-LNA ar...

  10. Modulation of 5' splice site selection using tailed oligonucleotides carrying splicing signals

    Directory of Open Access Journals (Sweden)

    Elela Sherif

    2006-01-01

    Full Text Available Abstract Background We previously described the use of tailed oligonucleotides as a means of reprogramming alternative pre-mRNA splicing in vitro and in vivo. The tailed oligonucleotides that were used interfere with splicing because they contain a portion complementary to sequences immediately upstream of the target 5' splice site combined with a non-hybridizing 5' tail carrying binding sites for the hnRNP A1/A2 proteins. In the present study, we have tested the inhibitory activity of RNA oligonucleotides carrying different tail structures. Results We show that an oligonucleotide with a 5' tail containing the human β-globin branch site sequence inhibits the use of the 5' splice site of Bcl-xL, albeit less efficiently than a tail containing binding sites for the hnRNP A1/A2 proteins. A branch site-containing tail positioned at the 3' end of the oligonucleotide also elicited splicing inhibition but not as efficiently as a 5' tail. The interfering activity of a 3' tail was improved by adding a 5' splice site sequence next to the branch site sequence. A 3' tail carrying a Y-shaped branch structure promoted similar splicing interference. The inclusion of branch site or 5' splice site sequences in the Y-shaped 3' tail further improved splicing inhibition. Conclusion Our in vitro results indicate that a variety of tail architectures can be used to elicit splicing interference at low nanomolar concentrations, thereby broadening the scope and the potential impact of this antisense technology.

  11. Cellular Internalization of Therapeutic Oligonucleotides by Peptide Amphiphile Nanofibers and Nanospheres.

    Science.gov (United States)

    Mumcuoglu, Didem; Sardan Ekiz, Melis; Gunay, Gokhan; Tekinay, Turgay; Tekinay, Ayse B; Guler, Mustafa O

    2016-05-11

    Oligonucleotides are promising drug candidates due to the exceptionally high specificity they exhibit toward their target DNA and RNA sequences. However, their poor pharmacokinetic and pharmacodynamic properties, in conjunction with problems associated with their internalization by cells, necessitates their delivery through specialized carrier systems for efficient therapy. Here, we investigate the effects of carrier morphology on the cellular internalization mechanisms of oligonucleotides by using self-assembled fibrous or spherical peptide nanostructures. Size and geometry were both found to be important parameters for the oligonucleotide internalization process; direct penetration was determined to be the major mechanism for the internalization of nanosphere carriers, whereas nanofibers were internalized by clathrin- and dynamin-dependent endocytosis pathways. We further showed that glucose conjugation to carrier nanosystems improved cellular internalization in cancer cells due to the enhanced glucose metabolism associated with oncogenesis, and the internalization of the glucose-conjugated peptide/oligonucleotide complexes was found to be dependent on glucose transporters present on the surface of the cell membrane.

  12. Spatially Defined Oligonucleotide Arrays. Technical Report for Phase II; FINAL

    International Nuclear Information System (INIS)

    None

    2000-01-01

    ,4,1 2). The hybridization pattern of a fluorescently labeled nucleic acid target is used to gain primary structure information of the target. This format can be applied to a broad range of nucleic acid sequence analysis problems including pathogen identification, polymorphism detection, human identification, mRNA expression monitoring and de novo sequencing. In this review, we briefly describe the method of light-directed chemid synthesis to create high-density arrays of oligonucleotide probes, the method of fluorescently labeling target nucleic acids for hybridization to the probe arrays, the detection of hybridized targets by epi-fluorescence confocal scanning and the data analysis procedures used to interpret the hybridization signals. To illustrate the use of specific high-density oligonucleotide probe arrays, we describe their application to screening the reverse transcriptase (rt) and protease (pro) genes of HIV-I for polymorphisms and drug-resistance conferring mutations

  13. Correlating In Vitro Splice Switching Activity With Systemic In Vivo Delivery Using Novel ZEN-modified Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Suzan M Hammond

    2014-01-01

    Full Text Available Splice switching oligonucleotides (SSOs induce alternative splicing of pre-mRNA and typically employ chemical modifications to increase nuclease resistance and binding affinity to target pre-mRNA. Here we describe a new SSO non-base modifier (a naphthyl-azo group, “ZEN™” to direct exon exclusion in mutant dystrophin pre-mRNA to generate functional dystrophin protein. The ZEN modifier is placed near the ends of a 2′-O-methyl (2′OMe oligonucleotide, increasing melting temperature and potency over unmodified 2′OMe oligonucleotides. In cultured H2K cells, a ZEN-modified 2′OMe phosphorothioate (PS oligonucleotide delivered by lipid transfection greatly enhanced dystrophin exon skipping over the same 2′OMePS SSO lacking ZEN. However, when tested using free gymnotic uptake in vitro and following systemic delivery in vivo in dystrophin deficient mdx mice, the same ZEN-modified SSO failed to enhance potency. Importantly, we show for the first time that in vivo activity of anionic SSOs is modelled in vitro only when using gymnotic delivery. ZEN is thus a novel modifier that enhances activity of SSOs in vitro but will require improved delivery methods before its in vivo clinical potential can be realized.

  14. Hammerhead ribozyme activity and oligonucleotide duplex stability in mixed solutions of water and organic compounds

    Directory of Open Access Journals (Sweden)

    Shu-ichi Nakano

    2014-01-01

    Full Text Available Nucleic acids are useful for biomedical targeting and sensing applications in which the molecular environment is different from that of a dilute aqueous solution. In this study, the influence of various types of mixed solutions of water and water-soluble organic compounds on RNA was investigated by measuring the catalytic activity of the hammerhead ribozyme and the thermodynamic stability of an oligonucleotide duplex. The compounds with a net neutral charge, such as poly(ethylene glycol, small primary alcohols, amide compounds, and aprotic solvent molecules, added at high concentrations changed the ribozyme-catalyzed RNA cleavage rate, with the magnitude of the effect dependent on the NaCl concentration. These compounds also changed the thermodynamic stability of RNA base pairs of an oligonucleotide duplex and its dependence on the NaCl concentration. Specific interactions with RNA molecules and reduced water activity could account for the inhibiting effects on the ribozyme catalysis and destabilizing effects on the duplex stability. The salt concentration dependence data correlated with the dielectric constant, but not with water activity, viscosity, and the size of organic compounds. This observation suggests the significance of the dielectric constant effects on the RNA reactions under molecular crowding conditions created by organic compounds.

  15. A non-covalent peptide-based strategy for ex vivo and in vivo oligonucleotide delivery.

    Science.gov (United States)

    Crombez, Laurence; Morris, May C; Heitz, Frederic; Divita, Gilles

    2011-01-01

    The dramatic acceleration in identification of new nucleic acid-based therapeutic molecules such as short interfering RNA (siRNA) and peptide-nucleic acid (PNA) analogues has provided new perspectives for therapeutic targeting of specific genes responsible for pathological disorders. However, the poor cellular uptake of nucleic acids together with the low permeability of the cell membrane to negatively charged molecules remain major obstacles to their clinical development. Several non-viral strategies have been proposed to improve the delivery of synthetic short oligonucleotides both in cultured cells and in vivo. Cell-penetrating peptides constitute very promising tools for non-invasive cellular import of oligonucleotides and analogs. We recently described a non-covalent strategy based on short amphiphatic peptides (MPG8/PEP3) that have been successfully applied ex vivo and in vivo for the delivery of therapeutic siRNA and PNA molecules. PEP3 and MPG8 form stable nanoparticles with PNA analogues and siRNA, respectively, and promote their efficient cellular uptake, independently of the endosomal pathway, into a wide variety of cell lines, including primary and suspension lines, without any associated cytotoxicity. This chapter describes easy-to-handle protocols for the use of MPG-8 or PEP-3-nanoparticle technologies for PNA and siRNA delivery into adherent and suspension cell lines as well as in vivo into cancer mouse models.

  16. Fluorescent oligonucleotides containing a novel perylene 2′-amino-α-L-LNA monomer: Synthesis and analytical potential

    DEFF Research Database (Denmark)

    Astakhova, Irina; Kumar, Santhosh T.; Wengel, Jesper

    2011-01-01

    efficiency of the resulting perylene-2'-amino-alpha-L-LNA monomer (T*) into synthetic oligonucleotides was significantly improved by replacement of the typically used 1H-tetrazole activator with pyridine hydrochloride. Generally, oligonucleotides containing monomer T* showed high binding affinity towards...... incorporations of monomers T* was quenched (quantum yield Phi(F) = 0.21) relative to duplexes of this probe with complementary DNA and RNA (Phi(F) = 0.42 and 0.35, respectively). On the contrary, a strong fluorescence quenching upon target binding was demonstrated by two short oligonucleotides of analogues...... sequences containing monomers T* at 5'- and 3'-terminal positions. We explain the hybridization-induced light-up effect observed for double-labeled probe by a reduction of fluorescence quenching due to precise positioning of the fluorophores within the double-stranded complexes. Furthermore, we propose...

  17. Characterization of adjacent breast tumors using oligonucleotide microarrays

    International Nuclear Information System (INIS)

    Unger, Meredith A; Rishi, Mazhar; Clemmer, Virginia B; Hartman, Jennifer L; Keiper, Elizabeth A; Greshock, Joel D; Chodosh, Lewis A; Liebman, Michael N; Weber, Barbara L

    2001-01-01

    Current methodology often cannot distinguish second primary breast cancers from multifocal disease, a potentially important distinction for clinical management. In the present study we evaluated the use of oligonucleotide-based microarray analysis in determining the clonality of tumors by comparing gene expression profiles. Total RNA was extracted from two tumors with no apparent physical connection that were located in the right breast of an 87-year-old woman diagnosed with invasive ductal carcinoma (IDC). The RNA was hybridized to the Affymetrix Human Genome U95A Gene Chip ® (12,500 known human genes) and analyzed using the Gene Chip Analysis Suite ® 3.3 (Affymetrix, Inc, Santa Clara, CA, USA) and JMPIN ® 3.2.6 (SAS Institute, Inc, Cary, NC, USA). Gene expression profiles of tumors from five additional patients were compared in order to evaluate the heterogeneity in gene expression between tumors with similar clinical characteristics. The adjacent breast tumors had a pairwise correlation coefficient of 0.987, and were essentially indistinguishable by microarray analysis. Analysis of gene expression profiles from different individuals, however, generated a pairwise correlation coefficient of 0.710. Transcriptional profiling may be a useful diagnostic tool for determining tumor clonality and heterogeneity, and may ultimately impact on therapeutic decision making

  18. The use of oligonucleotide aptamers in cancer therapy

    Directory of Open Access Journals (Sweden)

    Adrian Odrzywolski

    2016-05-01

    Full Text Available Aptamers are a new class of molecules which originated in the 1990s. They are usually RNA or DNA oligonucleotides, the length of which ranges between 20 and 80 nt. They are produced using the SELEX method that allows one to obtain aptamers that bind to virtually any molecule of interest, providing a high specificity. Aptamers are an alternative to antibodies because on the one hand, their sensitivity is at a similar or sometimes even higher level, while on the other hand they do not show immunogenicity, and may be synthesized in vitro. To date, a broad range of different applications of aptamers has been described: as components of biosensors, or use in various laboratory techniques, such as microarrays or chromatography. One of the most important is the use of aptamers in medicine, especially in the fight against cancer. They can be used both for diagnosis and for the eradication of cancers – particularly through the delivery of drugs. Currently, most transport-related research is devoted to the delivery of chemotherapeutic drugs, such as doxorubicin. This was used in research on liver cancer cells, prostate, and acute lymphoblastic leukemia blast cells. Another possibility is to use aptamers to deliver siRNAs. In this way inhibition of the quality control process of the mRNA in tumor cells is possible. An aptamer complex with the drug allows for direct delivery of the active substance in a particular cell type, substantially eliminating the non-specific effect of the drug.

  19. Pharmacokinetics on a microscale: visualizing Cy5-labeled oligonucleotide release from poly(n-butylcyanoacrylate nanocapsules in cells

    Directory of Open Access Journals (Sweden)

    Tomcin S

    2014-11-01

    Full Text Available Stephanie Tomcin,1 Grit Baier,1 Katharina Landfester,1 Volker Mailänder1,21Max Planck Institute for Polymer Research, 2University Medical Center of the Johannes Gutenberg University, III Medical Clinic, Mainz, GermanyAbstract: For successful design of a nanoparticulate drug delivery system, the fate of the carrier and cargo need to be followed. In this work, we fluorescently labeled poly(n-butylcyanoacrylate (PBCA nanocapsules as a shell and separately an oligonucleotide (20 mer as a payload. The nanocapsules were formed by interfacial anionic polymerization on aqueous droplets generated by an inverse miniemulsion process. After uptake, the PBCA capsules were shown to be round-shaped, endosomal structures and the payload was successfully released. Cy5-labeled oligonucleotides accumulated at the mitochondrial membrane due to a combination of the high mitochondrial membrane potential and the specific molecular structure of Cy5. The specificity of this accumulation at the mitochondria was shown as the uncoupler dinitrophenol rapidly diminished the accumulation of the Cy5-labeled oligonucleotide. Importantly, a fluorescence resonance energy transfer investigation showed that the dye-labeled cargo (Cy3/Cy5-labeled oligonucleotides reached its target site without degradation during escape from an endosomal compartment to the cytoplasm. The time course of accumulation of fluorescent signals at the mitochondria was determined by evaluating the colocalization of Cy5-labeled oligonucleotides and mitochondrial markers for up to 48 hours. As oligonucleotides are an ideal model system for small interfering RNA PBCA nanocapsules demonstrate to be a versatile delivery platform for small interfering RNA to treat a variety of diseases.Keywords: drug delivery, mitochondria, miniemulsion, colocalization

  20. Template-Directed Ligation of Peptides to Oligonucleotides

    Science.gov (United States)

    Bruick, Richard K.; Dawson, Philip E.; Kent, Stephen BH; Usman, Nassim; Joyce, Gerald F.

    1996-01-01

    Synthetic oligonucleotides and peptides have enjoyed a wide range of applications in both biology and chemistry. As a consequence, oligonucleotide-peptide conjugates have received considerable attention, most notably in the development of antisense constructs with improved pharmacological properties. In addition, oligonucleotide-peptide conjugates have been used as molecular tags, in the assembly of supramolecular arrays and in the construction of encoded combinatorial libraries. To make these chimeric molecules more accessible for a broad range of investigations, we sought to develop a facile method for joining fully deprotected oligonucleotides and peptides through a stable amide bond linkage. Furthermore, we wished to make this ligation reaction addressable, enabling one to direct the ligation of specific oligonucleotide and peptide components.To confer specificity and accelerate the rate of the reaction, the ligation process was designed to be dependent on the presence of a complementary oligonucleotide template.

  1. Functionalized bioengineered spider silk spheres improve nuclease resistance and activity of oligonucleotide therapeutics providing a strategy for cancer treatment.

    Science.gov (United States)

    Kozlowska, Anna Karolina; Florczak, Anna; Smialek, Maciej; Dondajewska, Ewelina; Mackiewicz, Andrzej; Kortylewski, Marcin; Dams-Kozlowska, Hanna

    2017-09-01

    Cell-selective delivery and sensitivity to serum nucleases remain major hurdles to the clinical application of RNA-based oligonucleotide therapeutics, such as siRNA. Spider silk shows great potential as a biomaterial due to its biocompatibility and biodegradability. Self-assembling properties of silk proteins allow for processing into several different morphologies such as fibers, scaffolds, films, hydrogels, capsules and spheres. Moreover, bioengineering of spider silk protein sequences can functionalize silk by adding peptide moieties with specific features including binding or cell recognition domains. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel oligonucleotide delivery system that can be utilized to improve pharmacokinetics of RNA-based therapeutics, such as CpG-siRNA. The MS2 bioengineered silk was functionalized with poly-lysine domain (KN) to generate hybrid silk MS2KN. CpG-siRNA efficiently bound to MS2KN in contrary to control MS2. Both MS2KN complexes and spheres protected CpG-siRNA from degradation by serum nucleases. CpG-siRNA molecules encapsulated into MS2KN spheres were efficiently internalized and processed by TLR9-positive macrophages. Importantly, CpG-STAT3siRNA loaded in silk spheres showed delayed and extended target gene silencing compared to naked oligonucleotides. The prolonged Stat3 silencing resulted in the more pronounced downregulation of interleukin 6 (IL-6), a proinflammatory cytokine and upstream activator of STAT3, which limits the efficacy of TLR9 immunostimulation. Our results demonstrate the feasibility of using spider silk spheres as a carrier of therapeutic nucleic acids. Moreover, the modified kinetic and activity of the CpG-STAT3siRNA embedded into silk spheres is likely to improve immunotherapeutic effects in vivo. We demonstrated that modification of silk protein by adding the nucleic acid binding domain enabled the development of a novel

  2. Distribution of Sulfate-Reducing Bacteria, O2, and H2s in Photosynthetic Biofilms Determined by Oligonucleotide Probes and Microelectrodes Rid A-1977-2009

    DEFF Research Database (Denmark)

    RAMSING, NB; KUHL, M.; JØRGENSEN, BB

    1993-01-01

    The vertical distribution of sulfate-reducing bacteria (SRB) in photosynthetic biofilms from the trickling filter of a sewage treatment plant was investigated with oligonucleotide probes binding to 16S rRNA. To demonstrate the effect of daylight and photosynthesis and thereby of increased oxygen....... Fluorescent-dye-conjugated oligonucleotides were used as ''phylogenetic'' probes to identify single cells in the slices. Oligonucleotide sequences were selected which were complementary to short sequence elements (16 to 20 nucleotides) within the 16S rRNA of sulfate-reducing bacteria. The probes were labeled...... with fluorescein or rhodamine derivatives for subsequent visualization by epifluorescence microscopy. Five probes were synthesized for eukaryotes, eubacteria, SRB (including most species of the delta group of purple bacteria), Desulfobacter spp., and a nonhybridizing control. The SRB were unevenly distributed...

  3. Advancements of antisense oligonucleotides in treatment of breast cancer

    Institute of Scientific and Technical Information of China (English)

    YANGShuan-Ping; SONGSan-Tai; 等

    2003-01-01

    Breast cancer is one kind of multi-gene related malignancy.Overexpression of some oncogenes such as HER-2(c-erbB-2,Neu),bcl-2/bcl-xL,protein kinase A(PKA),and transferrin receptor gene(TfR gene),etc significantly affect the prognosis of breast cancer.It was shown that specific suppression of the overexpressed genes above resulted in the improvement of the therapy of breast cancer.Antisense interference.one of useful tools for inhibiting the overexpression of specific oncogenes,was involved in the therapy of breast cancer in recent years. Data indicated that antisense oligonucleotides(ON)could inhibit specially the expression of the target genes on mRNA or protein levels in most of cases;some ON candidates showed encouraging therapeutic effects in vitro and in vivo on breast cancer cell lines or xenografts.Furthermore,the combination use of the antisense ON and normal chemotherapeutic agents indicated synergistic antitumor effects,which was probably the best utilization of antisense ON in the treatment of breast cancer.

  4. Respirable antisense oligonucleotides: a new drug class for respiratory disease

    Directory of Open Access Journals (Sweden)

    Tanaka Makoto

    2000-12-01

    Full Text Available Abstract Respirable antisense oligonucleotides (RASONs, which attenuate specific disease-associated mRNAs, represent a new class of respiratory therapeutics with considerable potential. RASONs overcome previous obstacles that have impeded the development of antisense therapeutics targeting diseases in other organ systems. RASONs are delivered directly to the target tissue via inhalation; their uptake seems to be enhanced by cationic properties inherent in pulmonary surfactant, and, because of the markedly different target properties of mRNA and proteins, they can have very long durations of effect compared with traditional drugs targeting the protein of the same gene. RASONs contain chemical modifications that decrease their degradation by cellular nucleases. However, total insensitivity to nucleases is probably not an optimal design criterion for RASONs, because moderate nuclease sensitivity can prevent their systemic delivery, decreasing the potential for systemic toxicity. EPI-2010 is a 21-mer phosphorothioate RASON that attenuates bronchoconstriction, inflammation and surfactant depletion in preclinical models of human asthma, has a duration of effect of seven days, and seems to undergo minimal systemic delivery.

  5. Recent advances in antisense oligonucleotide therapy in genetic neuromuscular diseases

    Directory of Open Access Journals (Sweden)

    Ashok Verma

    2018-01-01

    Full Text Available Genetic neuromuscular diseases are caused by defective expression of nuclear or mitochondrial genes. Mutant genes may reduce expression of wild-type proteins, and strategies to activate expression of the wild-type proteins might provide therapeutic benefits. Also, a toxic mutant protein may cause cell death, and strategies that reduce mutant gene expression may provide therapeutic benefit. Synthetic antisense oligonucleotide (ASO can recognize cellular RNA and control gene expression. In recent years, advances in ASO chemistry, creation of designer ASO molecules to enhance their safety and target delivery, and scientific controlled clinical trials to ascertain their therapeutic safety and efficacy have led to an era of plausible application of ASO technology to treat currently incurable neuromuscular diseases. Over the past 1 year, for the first time, the United States Food and Drug Administration has approved two ASO therapies in genetic neuromuscular diseases. This overview summarizes the recent advances in ASO technology, evolution and use of synthetic ASOs as a therapeutic platform, and the mechanism of ASO action by exon-skipping in Duchenne muscular dystrophy and exon-inclusion in spinal muscular atrophy, with comments on their advantages and limitations.

  6. Structure Activity Relationships of α-L-LNA Modified Phosphorothioate Gapmer Antisense Oligonucleotides in Animals

    Directory of Open Access Journals (Sweden)

    Punit P Seth

    2012-01-01

    Full Text Available We report the structure activity relationships of short 14-mer phosphorothioate gapmer antisense oligonucleotides (ASOs modified with α-L-locked nucleic acid (LNA and related modifications targeting phosphatase and tensin homologue (PTEN messenger RNA in mice. α-L-LNA represents the α-anomer of enantio-LNA and modified oligonucleotides show LNA like binding affinity for complementary RNA. In contrast to sequence matched LNA gapmer ASOs which showed elevations in plasma alanine aminotransferase (ALT levels indicative of hepatotoxicity, gapmer ASOs modified with α-L-LNA and related analogs in the flanks showed potent downregulation of PTEN messenger RNA in liver tissue without producing elevations in plasma ALT levels. However, the α-L-LNA ASO showed a moderate dose-dependent increase in liver and spleen weights suggesting a higher propensity for immune stimulation. Interestingly, replacing α-L-LNA nucleotides in the 3′- and 5′-flanks with R-5′-Me-α-L-LNA but not R-6′-Me- or 3′-Me-α-L-LNA nucleotides, reversed the drug induced increase in organ weights. Examination of structural models of dinucleotide units suggested that the 5′-Me group increases steric bulk in close proximity to the phosphorothioate backbone or produces subtle changes in the backbone conformation which could interfere with recognition of the ASO by putative immune receptors. Our data suggests that introducing steric bulk at the 5′-position of the sugar-phosphate backbone could be a general strategy to mitigate the immunostimulatory profile of oligonucleotide drugs. In a clinical setting, proinflammatory effects manifest themselves as injection site reactions and flu-like symptoms. Thus, a mitigation of these effects could increase patient comfort and compliance when treated with ASOs.

  7. CTG repeat-targeting oligonucleotides for down-regulating Huntingtin expression

    DEFF Research Database (Denmark)

    Zaghloul, Eman M; Gissberg, Olof; Moreno, Pedro M D

    2017-01-01

    Huntington's disease (HD) is a fatal, neurodegenerative disorder in which patients suffer from mobility, psychological and cognitive impairments. Existing therapeutics are only symptomatic and do not significantly alter the disease progression or increase life expectancy. HD is caused by expansion....... Thus, reduction of both muHTT mRNA and protein levels would ideally be the most useful therapeutic option. We herein present a novel strategy for HD treatment using oligonucleotides (ONs) directly targeting the HTT trinucleotide repeat DNA. A partial, but significant and potentially long-term, HTT...

  8. Regulation of Gene Expression with Double-Stranded Phosphorothioate Oligonucleotides

    Science.gov (United States)

    Bielinska, Anna; Shivdasani, Ramesh A.; Zhang, Liquan; Nabel, Gary J.

    1990-11-01

    Alteration of gene transcription by inhibition of specific transcriptional regulatory proteins is necessary for determining how these factors participate in cellular differentiation. The functions of these proteins can be antagonized by several methods, each with specific limitations. Inhibition of sequence-specific DNA-binding proteins was achieved with double-stranded (ds) phosphorothioate oligonucleotides that contained octamer or kappaB consensus sequences. The phosphorothioate oligonucleotides specifically bound either octamer transcription factor or nuclear factor (NF)-kappaB. The modified oligonucleotides accumulated in cells more effectively than standard ds oligonucleotides and modulated gene expression in a specific manner. Octamer-dependent activation of a reporter plasmid or NF-kappaB-dependent activation of the human immunodeficiency virus (HIV) enhancer was inhibited when the appropriate phosphorothioate oligonucleotide was added to a transiently transfected B cell line. Addition of phosphorothioate oligonucleotides that contained the octamer consensus to Jurkat T leukemia cells inhibited interleukin-2 (IL-2) secretion to a degree similar to that observed with a mutated octamer site in the IL-2 enhancer. The ds phosphorothioate oligonucleotides probably compete for binding of specific transcription factors and may provide anti-viral, immunosuppressive, or other therapeutic effects.

  9. Modern methods for the synthesis of peptide-oligonucleotide conjugates

    International Nuclear Information System (INIS)

    Zubin, Evgenii M; Oretskaya, Tat'yana S; Romanova, Elena A

    2002-01-01

    The published data on the methods of chemical solution and solid-phase synthesis of peptide-oligonucleotide conjugates are reviewed. The known methods are systematised and their advantages and disadvantages are considered. The approaches to the solution synthesis of peptide-oligonucleotide conjugates are systematised according to the type of chemical bonds between the fragments, whereas those to the solid-phase synthesis are classified according to the procedure used for the preparation of conjugates, viz., stepwise elongation of oligonucleotide and peptide chains on the same polymeric support or solid-phase condensation of two presynthesised fragments. The bibliography includes 141 references.

  10. Optical Characterization of Oligonucleotide DNA Influenced by Magnetic Fields

    Directory of Open Access Journals (Sweden)

    Seyedeh Maryam Banihashemian

    2013-09-01

    Full Text Available UV-VIS spectroscopic analysis of oligonucleotide DNA exposed to different magnetic fields was performed in order to investigate the relationship between DNA extinction coefficients and optical parameters according to magnetic-field strength. The results with the oligonucleotides adenine-thymine 100 mer (AT-100 DNA and cytosine-guanine 100 mer (CG-100 DNA indicate that the magnetic field influences DNA molar extinction coefficients and refractive indexes. The imaginary parts of the refractive index and molar extinction coefficients of the AT-100 and CG-100 DNA decreased after exposure to a magnetic field of 750 mT due to cleavage of the DNA oligonucleotides into smaller segments.

  11. QDs Supported on Langmuir-Blodgett Films of Polymers and Gemini Surfactant

    Directory of Open Access Journals (Sweden)

    T. Alejo

    2013-01-01

    Full Text Available Different LB films of poly(octadecene-co-maleic anhydride, PMAO, poly(styrene-co-maleic anhydride partial 2 butoxy ethyl ester cumene terminated, PS-MA-BEE, and Gemini surfactant ethyl-bis(dimethyl octadecylammonium bromide, 18-2-18, have been used to study the effect of the substrate coating on the surface self-assembly of CdSe quantum dots (QDs. Results show that all the “coating molecules” avoid the 3D aggregation of QDs observed when these nanoparticles are directly deposited on mica. Different morphologies were observed depending on the molecules used as coatings, and this was related to the surface properties, such as wetting ability, and the morphology of the coating LB films.

  12. Enzymatic production of single-molecule FISH and RNA capture probes.

    Science.gov (United States)

    Gaspar, Imre; Wippich, Frank; Ephrussi, Anne

    2017-10-01

    Arrays of singly labeled short oligonucleotides that hybridize to a specific target revolutionized RNA biology, enabling quantitative, single-molecule microscopy analysis and high-efficiency RNA/RNP capture. Here, we describe a simple and efficient method that allows flexible functionalization of inexpensive DNA oligonucleotides by different fluorescent dyes or biotin using terminal deoxynucleotidyl transferase and custom-made functional group conjugated dideoxy-UTP. We show that (i) all steps of the oligonucleotide labeling-including conjugation, enzymatic synthesis, and product purification-can be performed in a standard biology laboratory, (ii) the process yields >90%, often >95% labeled product with minimal carryover of impurities, and (iii) the oligonucleotides can be labeled with different dyes or biotin, allowing single-molecule FISH, RNA affinity purification, and Northern blot analysis to be performed. © 2017 Gaspar et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  13. Structural Determinants of Photoreactivity of Triplex Forming Oligonucleotides Conjugated to Psoralens

    Science.gov (United States)

    Krishnan, Rajagopal; Oh, Dennis H.

    2010-01-01

    Triplex-forming oligonucleotides (TFOs) with both DNA and 2′-O-methyl RNA backbones can direct psoralen photoadducts to specific DNA sequences. However, the functional consequences of these differing structures on psoralen photoreactivity are unknown. We designed TFO sequences with DNA and 2′-O-methyl RNA backbones conjugated to psoralen by 2-carbon linkers and examined their ability to bind and target damage to model DNA duplexes corresponding to sequences within the human HPRT gene. While TFO binding affinity was not dramatically affected by the type of backbone, psoralen photoreactivity was completely abrogated by the 2′-O-methyl RNA backbone. Photoreactivity was restored when the psoralen was conjugated to the RNA TFO via a 6-carbon linker. In contrast to the B-form DNA of triplexes formed by DNA TFOs, the CD spectra of triplexes formed with 2′-O-methyl RNA TFOs exhibited features of A-form DNA. These results indicate that 2′-O-methyl RNA TFOs induce a partial B-to-A transition in their target DNA sequences which may impair the photoreactivity of a conjugated psoralen and suggest that optimal design of TFOs to target DNA damage may require a balance between binding ability and drug reactivity. PMID:20725628

  14. Intravitreal Injection of Splice-switching Oligonucleotides to Manipulate Splicing in Retinal Cells

    Directory of Open Access Journals (Sweden)

    Xavier Gérard

    2015-01-01

    Full Text Available Leber congenital amaurosis is a severe hereditary retinal dystrophy responsible for neonatal blindness. The most common disease-causing mutation (c.2991+1655A>G; 10–15% creates a strong splice donor site that leads to insertion of a cryptic exon encoding a premature stop codon. Recently, we reported that splice-switching oligonucleotides (SSO allow skipping of the mutant cryptic exon and the restoration of ciliation in fibroblasts of affected patients, supporting the feasibility of a SSO-mediated exon skipping strategy to correct the aberrant splicing. Here, we present data in the wild-type mouse, which demonstrate that intravitreal administration of 2’-OMePS-SSO allows selective alteration of Cep290 splicing in retinal cells, including photoreceptors as shown by successful alteration of Abca4 splicing using the same approach. We show that both SSOs and Cep290 skipped mRNA were detectable for at least 1 month and that intravitreal administration of oligonucleotides did not provoke any serious adverse event. These data suggest that intravitreal injections of SSO should be considered to bypass protein truncation resulting from the c.2991+1655A>G mutation as well as other truncating mutations in genes which like CEP290 or ABCA4 have a mRNA size that exceed cargo capacities of US Food and Drug Administration (FDA-approved adeno-associated virus (AAV-vectors, thus hampering gene augmentation therapy.

  15. Sequence-dependent theory of oligonucleotide hybridization kinetics

    International Nuclear Information System (INIS)

    Marimuthu, Karthikeyan; Chakrabarti, Raj

    2014-01-01

    A theoretical approach to the prediction of the sequence and temperature-dependent rate constants for oligonucleotide hybridization reactions has been developed based on the theory of relaxation kinetics. One-sided and two-sided melting reaction mechanisms for oligonucleotide hybridization reactions have been considered, analyzed, modified, and compared to select a physically consistent as well as robust model for prediction of the relaxation times of DNA hybridization reactions that agrees with the experimental evidence. The temperature- and sequence-dependent parameters of the proposed model have been estimated using available experimental data. The relaxation time model that we developed has been combined with the nearest neighbor model of hybridization thermodynamics to estimate the temperature- and sequence-dependent rate constants of an oligonucleotide hybridization reaction. The model-predicted rate constants are compared to experimentally determined rate constants for the same oligonucleotide hybridization reactions. Finally, we consider a few important applications of kinetically controlled DNA hybridization reactions

  16. Hole hopping rates in single strand oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Borrelli, Raffaele [Dipartimento di Scienze Agrarie, Forestali e Alimentari, Università di Torino, Largo Paolo Braccini 2, I-10095 Grugliasco, TO (Italy); Capobianco, Amedeo [Dipartimento di Chimica e Biologia, Università di Salerno, Via Giovanni Paolo II, I-84084 Fisciano, SA (Italy); Peluso, Andrea, E-mail: apeluso@unisa.it [Dipartimento di Chimica e Biologia, Università di Salerno, Via Giovanni Paolo II, I-84084 Fisciano, SA (Italy)

    2014-08-31

    Highlights: • DNA hole transfer rates have been computed. • Delocalized adenine domains significantly affect hole transfer rates in DNA. • Franck–Condon weighted density of state from DFT normal modes. • DNA application in molecular electronics. - Abstract: The rates of hole transfer between guanine and adenine in single strand DNA have been evaluated by using Fermi’s golden rule and Kubo’s generating function approach for the Franck–Condon weighted density of states. The whole sets of the normal modes and vibrational frequencies of the two nucleobases, obtained at DFT/B3LYP level of calculation, have been considered in computations. The results show that in single strand the pyramidalization/planarization mode of the amino groups of both nucleobases plays the major role. At room temperature, the Franck–Condon density of states extends over a wide range of hole site energy difference, 0–1 eV, giving some hints about the design of oligonucleotides of potential technological interest.

  17. A dystrophic Duchenne mouse model for testing human antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Marcel Veltrop

    Full Text Available Duchenne muscular dystrophy (DMD is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.

  18. Profiled support vector machines for antisense oligonucleotide efficacy prediction

    Directory of Open Access Journals (Sweden)

    Martín-Guerrero José D

    2004-09-01

    Full Text Available Abstract Background This paper presents the use of Support Vector Machines (SVMs for prediction and analysis of antisense oligonucleotide (AO efficacy. The collected database comprises 315 AO molecules including 68 features each, inducing a problem well-suited to SVMs. The task of feature selection is crucial given the presence of noisy or redundant features, and the well-known problem of the curse of dimensionality. We propose a two-stage strategy to develop an optimal model: (1 feature selection using correlation analysis, mutual information, and SVM-based recursive feature elimination (SVM-RFE, and (2 AO prediction using standard and profiled SVM formulations. A profiled SVM gives different weights to different parts of the training data to focus the training on the most important regions. Results In the first stage, the SVM-RFE technique was most efficient and robust in the presence of low number of samples and high input space dimension. This method yielded an optimal subset of 14 representative features, which were all related to energy and sequence motifs. The second stage evaluated the performance of the predictors (overall correlation coefficient between observed and predicted efficacy, r; mean error, ME; and root-mean-square-error, RMSE using 8-fold and minus-one-RNA cross-validation methods. The profiled SVM produced the best results (r = 0.44, ME = 0.022, and RMSE= 0.278 and predicted high (>75% inhibition of gene expression and low efficacy (http://aosvm.cgb.ki.se/. Conclusions The SVM approach is well suited to the AO prediction problem, and yields a prediction accuracy superior to previous methods. The profiled SVM was found to perform better than the standard SVM, suggesting that it could lead to improvements in other prediction problems as well.

  19. Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel.

    Science.gov (United States)

    Tang, Weizhong; Zhou, Huafu; Li, Wei

    2015-01-01

    To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. It was unexpected that oligo (dT) was hard to be silver-stained. Moreover, the silver staining of an oligonucleotide containing base T could be partially or completely inhibited by base T. The inhibition of silver staining by base T was a competitive inhibition which could be affected by the amounts of the argyrophil nucleobase and base T, the cis-distance between the argyrophil nucleobase and base T, and the gel concentration. The changes of the intensity of an oligonucleotide band caused by the changes of DNA base composition were diverse and interesting. The intensity of some oligonucleotide bands would significantly change when the changes of DNA base composition accumulated to a certain extent (usually ≥ 4 nt). The sensitivity of cyanine staining of ≤ 11-nt long oligonucleotides could be enhanced about 250-fold by fixing the gels with methanol fixing solution.

  20. Chemosensitization of Human Renal Cell Cancer Using Antisense Oligonucleotides Targeting the Antiapoptotic Gene Clusterin

    Directory of Open Access Journals (Sweden)

    Tobias Zellweger

    2001-01-01

    Full Text Available BACKGROUND: Renal cell cancer (RCC is a chemoresistant disease with no active chemotherapeutic agent achieving objective response rates higher than 15%. Clusterin is a cell survival gene that increases in human renal tubular epithelial cells after various states of injury and disease. Downregulation of clusterin, using antisense oligonucleotides (ASO, has recently been shown to increase chemosensitivity in several prostate cancer models. The objectives in this study were to evaluate clusterin expression levels in human RCC and normal kidney tissue, and to test whether clusterin ASO could also enhance chemosensitivity in human RCC Caki-2 cells both in vitro and in vivo. METHODS: Immunohistochemical staining was used to characterize clusterin expression in 67 RCC and normal kidney tissues obtained from radical nephrectomy specimens. Northern blot analysis was used to assess changes in clusterin mRNA expression after ASO and paclitaxel treatment. The effects of combined clusterin ASO and paclitaxel treatment on Caki-2 cell growth was examined using an MTT assay. Athymic mice bearing Caki-2 tumors were treated with clusterin ASO alone, clusterin ASO plus paclitaxel, and mismatch control oligonucleotides plus paclitaxel, over a period of 28 days with measurement of tumor volumes once weekly over 8 weeks. RESULTS: Immunohistochemistry of normal and malignant kidney tissue sections of 67 patients demonstrated positive clusterin staining for almost all RCC (98% and an overexpression, compared to normal tissue, in a majority of RCC (69%. Clusterin ASO, but not mismatch control oligonucleotides, decreased clusterin mRNA expression in Caki-2 cells in a dosedependent and sequence-specific manner. Pretreatment of Caki-2 cells with clusterin ASO significantly enhanced chemosensitivity to paclitaxel in vitro. Characteristic apoptotic DNA laddering was observed after combined treatment with ASO plus paclitaxel, but not with either agent alone. In vivo

  1. Development of specific oligonucleotide probes for the identification and in situ detection of hydrocarbon-degrading Alcanivorax strains.

    Science.gov (United States)

    Syutsubo, K; Kishira, H; Harayama, S

    2001-06-01

    The genus Alcanivorax comprises diverse hydrocarbon-degrading marine bacteria. Novel 16S rRNA-targeted oligonucleotide DNA probes (ALV735 and ALV735-b) were developed to quantify two subgroups of the Alcanivorax/Fundibacter group by fluorescence in situ hybridization (FISH), and the conditions for the single-mismatch discrimination of the probes were optimized. The specificity of the probes was improved further using a singly mismatched oligonucleotide as a competitor. The growth of Alcanivorax cells in crude oil-contaminated sea water under the biostimulation condition was investigated by FISH with the probe ALV735, which targeted the main cluster of the Alcanivorax/Fundibacter group. The size of the Alcanivorax population increased with increasing incubation time and accounted for 91% of the 4',6-diamidino-2-phenylindole (DAPI) count after incubation for 2 weeks. The probes developed in this study are useful for detecting Alcanivorax populations in petroleum hydrocarbon-degrading microbial consortia.

  2. Defibrotide: An Oligonucleotide for Sinusoidal Obstruction Syndrome.

    Science.gov (United States)

    Aziz, May T; Kakadiya, Payal P; Kush, Samantha M; Weigel, Kylie; Lowe, Denise K

    2018-02-01

    To review the efficacy and safety of defibrotide as well as its pharmacology, mechanism of action, pharmacokinetics (PK), drug-drug interactions, dosing, cost considerations, and place in therapy. A PubMed search was performed through August 2017 using the terms defibrotide, oligonucleotide, hepatic veno-occlusive disease (VOD), sinusoidal obstruction syndrome (SOS), and hematopoietic cell transplantation (HCT). Other data sources were from references of identified studies, review articles, and conference abstracts plus manufacturer product labeling and website, the Food and Drug Administration website, and clinicaltrials.gov. English-language trials that examined defibrotide's pharmacodynamics, mechanism, PK, efficacy, safety, dosing, and cost-effectiveness were included. Trials have confirmed the safety and efficacy of defibrotide for treatment of VOD/SOS in adult and pediatric HCT patients, with complete response rates and day +100 overall survival rates ranging from 25.5% to 76% and 35% to 64%, respectively. The British Committee for Standards in Haematology/British Society for Blood and Marrow Transplantation Guidelines recommend defibrotide prophylaxis in pediatric and adult HCT patients with risk factors for VOD/SOS; however, its prophylactic use in the United States is controversial. Although there are efficacy data to support this strategy, cost-effectiveness data have not shown it to be cost-effective. Defibrotide has manageable toxicities, with low rates of grade 3 to 4 adverse effects. Defibrotide is the first medication approved in the United States for the treatment of adults and children with hepatic VOD/SOS, with renal or pulmonary dysfunction following HCT. Data evaluating defibrotide for VOD/SOS prevention are conflicting and have not shown cost-effectiveness.

  3. Biominetic High Density Lipoproteins for the Delivery of Therapeutic Oligonucleotides

    Science.gov (United States)

    Tripathy, Sushant

    Advances in nanotechnology have brought about novel inorganic and hybrid nanoparticles with unique physico-chemical properties that make them suitable for a broad range of applications---from nano-circuitry to drug delivery. A significant part of those advancements have led to ground-breaking discoveries that have changed the approaches to formulation of therapeutics against diseases, such as cancer. Now-a-days the focus does not lie solely on finding a candidate small-molecule therapeutic with minimal adverse effects, but researchers are looking up to nanoparticles to improve biodistribution and biocompatibility profile of clinically proven therapeutics. The plethora of conjugation chemistries offered by currently extant inorganic nanoparticles have, in recent years, led to great leaps in the field of biomimicry---a modality that promises high biocompatibility. Further, in the pursuit of highly specific therapeutic molecules, researchers have turned to silencing oligonucleotides and some have already brought together the strengths of nanoparticles and silencing oligonucleotides in search of an efficacious therapy for cancer with minimal adverse effects. This dissertation work focuses on such a biomimetic platform---a gold nanoparticle based high density lipoprotein biomimetic (HDL NP), for the delivery of therapeutic oligonucleotides. The first chapter of this body of work introduces the molecular target of the silencing oligonucleotides---VEGFR2, and its role in the progression of solid tumor cancers. The background information also covers important aspects of natural high density lipoproteins (HDL), especially their innate capacity to bind and deliver exogenous and endogenous silencing oligonucleotides to tissues that express their high affinity receptor SRB1. We subsequently describe the synthesis of the biomimetic HDL NP and its oligonucleotide conjugates, and establish their biocompatibility. Further on, experimental data demonstrate the efficacy of silencing

  4. Initiation, elongation, and realignment during influenza virus mRNA synthesis

    NARCIS (Netherlands)

    Velthuis, te Aartjan J.W.; Oymans, Judith

    2018-01-01

    The RNA-dependent RNA polymerase (RdRp) of the influenza A virus replicates and transcribes the viral genome segments in the nucleus of the host cell. To transcribe these viral genome segments, the RdRp "snatches" capped RNA oligonucleotides from nascent host cell mRNAs and aligns these primers to

  5. Mismatch oligonucleotides in human and yeast: guidelines for probe design on tiling microarrays

    Directory of Open Access Journals (Sweden)

    Jee Justin

    2008-12-01

    Full Text Available Abstract Background Mismatched oligonucleotides are widely used on microarrays to differentiate specific from nonspecific hybridization. While many experiments rely on such oligos, the hybridization behavior of various degrees of mismatch (MM structure has not been extensively studied. Here, we present the results of two large-scale microarray experiments on S. cerevisiae and H. sapiens genomic DNA, to explore MM oligonucleotide behavior with real sample mixtures under tiling-array conditions. Results We examined all possible nucleotide substitutions at the central position of 36-nucleotide probes, and found that nonspecific binding by MM oligos depends upon the individual nucleotide substitutions they incorporate: C→A, C→G and T→A (yielding purine-purine mispairs are most disruptive, whereas A→X were least disruptive. We also quantify a marked GC skew effect: substitutions raising probe GC content exhibit higher intensity (and vice versa. This skew is small in highly-expressed regions (± 0.5% of total intensity range and large (± 2% or more elsewhere. Multiple mismatches per oligo are largely additive in effect: each MM added in a distributed fashion causes an additional 21% intensity drop relative to PM, three-fold more disruptive than adding adjacent mispairs (7% drop per MM. Conclusion We investigate several parameters for oligonucleotide design, including the effects of each central nucleotide substitution on array signal intensity and of multiple MM per oligo. To avoid GC skew, individual substitutions should not alter probe GC content. RNA sample mixture complexity may increase the amount of nonspecific hybridization, magnify GC skew and boost the intensity of MM oligos at all levels.

  6. Development and production of an oligonucleotide MuscleChip: use for validation of ambiguous ESTs

    Directory of Open Access Journals (Sweden)

    Lanfranchi Gerolamo

    2002-10-01

    Full Text Available Abstract Background We describe the development, validation, and use of a highly redundant 120,000 oligonucleotide microarray (MuscleChip containing 4,601 probe sets representing 1,150 known genes expressed in muscle and 2,075 EST clusters from a non-normalized subtracted muscle EST sequencing project (28,074 EST sequences. This set included 369 novel EST clusters showing no match to previously characterized proteins in any database. Each probe set was designed to contain 20–32 25 mer oligonucleotides (10–16 paired perfect match and mismatch probe pairs per gene, with each probe evaluated for hybridization kinetics (Tm and similarity to other sequences. The 120,000 oligonucleotides were synthesized by photolithography and light-activated chemistry on each microarray. Results Hybridization of human muscle cRNAs to this MuscleChip (33 samples showed a correlation of 0.6 between the number of ESTs sequenced in each cluster and hybridization intensity. Out of 369 novel EST clusters not showing any similarity to previously characterized proteins, we focused on 250 EST clusters that were represented by robust probe sets on the MuscleChip fulfilling all stringent rules. 102 (41% were found to be consistently "present" by analysis of hybridization to human muscle RNA, of which 40 ESTs (39% could be genome anchored to potential transcription units in the human genome sequence. 19 ESTs of the 40 ESTs were furthermore computer-predicted as exons by one or more than three gene identification algorithms. Conclusion Our analysis found 40 transcriptionally validated, genome-anchored novel EST clusters to be expressed in human muscle. As most of these ESTs were low copy clusters (duplex and triplex in the original 28,000 EST project, the identification of these as significantly expressed is a robust validation of the transcript units that permits subsequent focus on the novel proteins encoded by these genes.

  7. Simplified Paper Format for Detecting HIV Drug Resistance in Clinical Specimens by Oligonucleotide Ligation

    Science.gov (United States)

    Panpradist, Nuttada; Beck, Ingrid A.; Chung, Michael H.; Kiarie, James N.; Frenkel, Lisa M.; Lutz, Barry R.

    2016-01-01

    Human immunodeficiency virus (HIV) is a chronic infection that can be managed by antiretroviral treatment (ART). However, periods of suboptimal viral suppression during lifelong ART can select for HIV drug resistant (DR) variants. Transmission of drug resistant virus can lessen or abrogate ART efficacy. Therefore, testing of individuals for drug resistance prior to initiation of treatment is recommended to ensure effective ART. Sensitive and inexpensive HIV genotyping methods are needed in low-resource settings where most HIV infections occur. The oligonucleotide ligation assay (OLA) is a sensitive point mutation assay for detection of drug resistance mutations in HIV pol. The current OLA involves four main steps from sample to analysis: (1) lysis and/or nucleic acid extraction, (2) amplification of HIV RNA or DNA, (3) ligation of oligonucleotide probes designed to detect single nucleotide mutations that confer HIV drug resistance, and (4) analysis via oligonucleotide surface capture, denaturation, and detection (CDD). The relative complexity of these steps has limited its adoption in resource-limited laboratories. Here we describe a simplification of the 2.5-hour plate-format CDD to a 45-minute paper-format CDD that eliminates the need for a plate reader. Analysis of mutations at four HIV-1 DR codons (K103N, Y181C, M184V, and G190A) in 26 blood specimens showed a strong correlation of the ratios of mutant signal to total signal between the paper CDD and the plate CDD. The assay described makes the OLA easier to perform in low resource laboratories. PMID:26751207

  8. Antitumor effects of radioiodinated antisense oligonucleotide mediated by VIP receptor

    International Nuclear Information System (INIS)

    Ou Xiaohong; Tan Tianzhi; Li Yunchun; Kuang Anren

    2004-01-01

    Purpose: we had constructed a targeting delivery system based on intestinal peptide (VIP) for antisense oligonucleotide (ASON) transfer into VIP receptor-positive cells in previous study. The aims of present studies are to observe the antitumor effect of VIP-131I-ASON in HT29 human colon adenocarcinoma xenografts. Methods: A 15-met phosphorothioate ASON, which was complementary to the translation start region of the C-myc oncogene mRNA, was labeled with 131I and the labelled compound was linked to the VIP bound covalently 'to a polylysine chain so as to deliver oligonucleotide into tumor cells. Distribution experiments for evaluating the radiolabeled antisense complexe uptake in tumor tissue were performed in BALB/c nude mice bearing with HT29 tumor xenografts. Nude mice beating HT29 tumor xenografts were adminstered VIP-131I-ASON (3.7,7.4 MBq) or 131I-ASON (3.7 MBq), 131I labeled control sense and nosense DNA (3.7 MBq), or saline. Antitumor effects were assessed using endpoints of tumor growth delay. C-myc-encoded protein expression of tumor was measured by immunocytohistochemical staining. Results: Distribution experiment performed with athymic mice bearing human colon tumor xenografts revealed maximal accumulation of conjugated ASON in the tumor tissue 2 h after administration and significantly higher than that in nude mice injected unconjngated ASON [(5.89±1.03)%ID/g and(1.56±0.31)%ID/g, respectively; t=7.7954 P<0.001]. The radioratio of tumor to muscle was peaked 4h after administration. VIP-131I-ASON exhibited strong antitumor effects against HT29 xenografts, decreasing their growth rate 7-fold compare with that in saline-treated mice(tumor growth delay, 25.4±0.89 day). The antitumor effects of unconjugated 131I-ASON were much less profound than VIP-131I-ASON (tumor growth delay, 3.2±1.3 and 25.4±0.89 day, respectively; q=51.4126 P<0.01). Sense, nosense control ON with VIP carder caused no therapeutic effect. There was no progressive weight loss or

  9. Four Ways to Oligonucleotides Without Phosphoimidazolides

    Czech Academy of Sciences Publication Activity Database

    Šponer, Judit E.; Šponer, Jiří; Di Mauro, E.

    2016-01-01

    Roč. 82, č. 1 (2016), s. 5-10 ISSN 0022-2844 R&D Projects: GA ČR(CZ) GA14-12010S Institutional support: RVO:68081707 Keywords : RNA * Polymerization * Origin of life Subject RIV: BO - Biophysics Impact factor: 2.434, year: 2016

  10. Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides.

    Science.gov (United States)

    Yoga, Yano M K; Traore, Daouda A K; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R; Barker, Andrew; Leedman, Peter J; Wilce, Jacqueline A; Wilce, Matthew C J

    2012-06-01

    Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA and DNA, KH3 binds with intermediate affinity and KH2 only interacts detectibly with DNA. The crystal structure of KH1 bound to a 5'-CCCTCCCT-3' DNA sequence shows a 2:1 protein:DNA stoichiometry and demonstrates a molecular arrangement of KH domains bound to immediately adjacent oligonucleotide target sites. SPR experiments, with a series of poly-C-sequences reveals that cytosine is preferred at all four positions in the oligonucleotide binding cleft and that a C-tetrad binds KH1 with 10 times higher affinity than a C-triplet. The basis for this high affinity interaction is finally detailed with the structure determination of a KH1.W.C54S mutant bound to 5'-ACCCCA-3' DNA sequence. Together, these data establish the lead role of KH1 in oligonucleotide binding by αCP1 and reveal the molecular basis of its specificity for a C-rich tetrad.

  11. Technetium-99m labeled antisense oligonucleotide-noninvasive tumor imaging in mice

    International Nuclear Information System (INIS)

    Qin, G.M.; Zhang, Y.X.; An, R.; Gao, Z.R.; Cao, W.; Cao, G.X.; Hnatowich, D.J.

    2002-01-01

    Single-stranded RNA and DNA oligonucleotides may be useful as radiopharmaceuticals for antisense and other in vivo applications if convenient methods for stably attaching radionuclides such as 99m Tc can be developed. The c-myc oncogene works in cooperation with other oncogenes in a variety of malignant tumors. The concentration of c-myc messenger RNA increases rapidly 30 to 50 fold during DNA synthesis, thus making it a suitable target for following the progression of malignancy by noninvasive imaging with radiolabeled antisense oligonucleotide probes. Methods: 1 Oligonucleotide Conjugation: A solution of single stranded amine-derivatized DNA (100-1000μg) was prepared at a concentration of 2 mg/ml in 0.25M sodium bicarbonate, 1 M sodium chloride, 1mM EDTA, pH8.5. 2 Oligonucleotide Labeling: A fresh 50mg/ml solution of sodium tartrate was prepared in sterile 0.5 M ammonium The ability of the labeled DNA to hybridize to its complement was analyzed by Sep-Pak column chromatography before and after the addition of the complementary DNA. 3 Biodistribution and Tumor Imaging Studies: A colony of KM mice (15-20g) were inoculated with 1x10 6 Ehrlich carcinoma tumor cells in the right thigh, and the tumors were allowed to grow for 6-7 days to a size of 1.0-1.5 cm in diameter. Biodistribution studies were performed in 32 KM mice after 50 μCi per mouse of 99m Tc-labeled oncogene probes were injected intravenously. A total of 8 mice were injected intravenously in the tail vein with 1-2 mCi of 99m Tc-labeled sense or antisense probes, immobilized with ketamine hydrochloride and imaged periodically from 0.5hr to 24hr with a gamma camera. Results: Essentially complete conjugation was achieved by reverse-phase Sep-Pak C18 chromatography analysis. The labeled antisense DNA still remained the ability to hybridize with its complementary DNA. The highest accumulation of label was in the liver first, with the kidney and small bowel next. The injected activity localized in the lesion

  12. Detection of chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes

    International Nuclear Information System (INIS)

    Lloyd, R.V.; Jin, L.; Fields, K.

    1990-01-01

    We analyzed the distribution of chromogranins A and B in normal and neoplastic endocrine tissues with secretory granules using 35 S-labeled and biotin-labeled oligonucleotide probes by in situ hybridization (ISH). Both radioactive and nonradioactive probes detected messenger RNAs (mRNAs) in frozen and paraffin tissue sections. Endocrine tissues with variable immunoreactivities for chromogranin A protein, such as small-cell lung carcinomas, neuroblastomas, insulinomas, and parathyroid adenomas, expressed the mRNA for chromogranins A and B in most cells. Some technical problems with the biotinylated probes included nonspecific nuclear staining and endogenous alkaline phosphatase, which was not completely abolished by levamisole pretreatment. A differential distribution of chromogranins A and B was seen in pituitary prolactinomas, which expressed abundant chromogranin B but not chromogranin A mRNAs, and in parathyroid adenomas, which expressed abundant chromogranin A but only small amounts of chromogranin B mRNAs. These results indicate that ISH can be used to detect chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes and that the mRNAs for chromogranin A and B are demonstrable in some tumors even when the chromogranin proteins cannot be detected by immunohistochemistry

  13. Detection of chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes

    Energy Technology Data Exchange (ETDEWEB)

    Lloyd, R.V.; Jin, L.; Fields, K. (Univ. of Michigan, Ann Arbor (USA))

    1990-01-01

    We analyzed the distribution of chromogranins A and B in normal and neoplastic endocrine tissues with secretory granules using {sup 35}S-labeled and biotin-labeled oligonucleotide probes by in situ hybridization (ISH). Both radioactive and nonradioactive probes detected messenger RNAs (mRNAs) in frozen and paraffin tissue sections. Endocrine tissues with variable immunoreactivities for chromogranin A protein, such as small-cell lung carcinomas, neuroblastomas, insulinomas, and parathyroid adenomas, expressed the mRNA for chromogranins A and B in most cells. Some technical problems with the biotinylated probes included nonspecific nuclear staining and endogenous alkaline phosphatase, which was not completely abolished by levamisole pretreatment. A differential distribution of chromogranins A and B was seen in pituitary prolactinomas, which expressed abundant chromogranin B but not chromogranin A mRNAs, and in parathyroid adenomas, which expressed abundant chromogranin A but only small amounts of chromogranin B mRNAs. These results indicate that ISH can be used to detect chromogranins A and B in endocrine tissues with radioactive and biotinylated oligonucleotide probes and that the mRNAs for chromogranin A and B are demonstrable in some tumors even when the chromogranin proteins cannot be detected by immunohistochemistry.

  14. Challenges to oligonucleotides-based therapeutics for Duchenne muscular dystrophy

    Directory of Open Access Journals (Sweden)

    Goyenvalle Aurélie

    2011-02-01

    Full Text Available Abstract Antisense oligonucleotides are short nucleic acids designed to bind to specific messenger RNAs in order to modulate splicing patterns or inhibit protein translation. As such, they represent promising therapeutic tools for many disorders and have been actively developed for more than 20 years as a form of molecular medicine. Although significant progress has been made in developing these agents as drugs, they are yet not recognized as effective therapeutics and several hurdles remain to be overcome. Within the last few years, however, the prospect of successful oligonucleotides-based therapies has moved a step closer, in particular for Duchenne muscular dystrophy. Clinical trials have recently been conducted for this myopathy, where exon skipping is being used to achieve therapeutic outcomes. In this review, the recent developments and clinical trials using antisense oligonucleotides for Duchenne muscular dystrophy are discussed, with emphasis on the challenges ahead for this type of therapy, especially with regards to delivery and regulatory issues.

  15. Rapid and accurate synthesis of TALE genes from synthetic oligonucleotides.

    Science.gov (United States)

    Wang, Fenghua; Zhang, Hefei; Gao, Jingxia; Chen, Fengjiao; Chen, Sijie; Zhang, Cuizhen; Peng, Gang

    2016-01-01

    Custom synthesis of transcription activator-like effector (TALE) genes has relied upon plasmid libraries of pre-fabricated TALE-repeat monomers or oligomers. Here we describe a novel synthesis method that directly incorporates annealed synthetic oligonucleotides into the TALE-repeat units. Our approach utilizes iterative sets of oligonucleotides and a translational frame check strategy to ensure the high efficiency and accuracy of TALE-gene synthesis. TALE arrays of more than 20 repeats can be constructed, and the majority of the synthesized constructs have perfect sequences. In addition, this novel oligonucleotide-based method can readily accommodate design changes to the TALE repeats. We demonstrated an increased gene targeting efficiency against a genomic site containing a potentially methylated cytosine by incorporating non-conventional repeat variable di-residue (RVD) sequences.

  16. Simultaneous visualization of the subfemtomolar expression of microRNA and microRNA target gene using HILO microscopy† †Electronic supplementary information (ESI) available: The LED device for the sample photobleaching, a schematic presentation of HILO microscopy, fluorescence spectra and hybridization curves of the molecular beacons, the linear correlation between the miRNA fluorescence intensity and the miRNA copy number, a validation of the miRNA adsorption and miRNA target gene expression via RT-qPCR, a validation of RT-qPCR using capillary electrophoresis, the reproducibility of RT-qPCR and Poisson distribution of the miRNA pipetting as well as a complete list of the oligonucleotides used in this study. See DOI: 10.1039/c7sc02701j Click here for additional data file.

    Science.gov (United States)

    Lin, Yi-Zhen; Ou, Da-Liang; Chang, Hsin-Yuan; Lin, Wei-Yu; Hsu, Chiun

    2017-01-01

    The family of microRNAs (miRNAs) not only plays an important role in gene regulation but is also useful for the diagnosis of diseases. A reliable method with high sensitivity may allow researchers to detect slight fluctuations in ultra-trace amounts of miRNA. In this study, we propose a sensitive imaging method for the direct probing of miR-10b (miR-10b-3p, also called miR-10b*) and its target (HOXD10 mRNA) in fixed cells based on the specific recognition of molecular beacons combined with highly inclined and laminated optical sheet (HILO) fluorescence microscopy. The designed dye-quencher-labelled molecular beacons offer excellent efficiencies of fluorescence resonance energy transfer that allow us to detect miRNA and the target mRNA simultaneously in hepatocellular carcinoma cells using HILO fluorescence microscopy. Not only can the basal trace amount of miRNA be observed in each individual cell, but the obtained images also indicate that this method is useful for monitoring the fluctuations in ultra-trace amounts of miRNA when the cells are transfected with a miRNA precursor or a miRNA inhibitor (anti-miR). Furthermore, a reasonable causal relation between the miR-10b and HOXD10 expression levels was observed in miR-10b* precursor-transfected cells and miR-10b* inhibitor-transfected cells. The trends of the miRNA alterations obtained using HILO microscopy completely matched the RT-qPCR data and showed remarkable reproducibility (the coefficient of variation [CV] = 0.86%) and sensitivity (<1.0 fM). This proposed imaging method appears to be useful for the simultaneous visualisation of ultra-trace amounts of miRNA and target mRNA and excludes the procedures for RNA extraction and amplification. Therefore, the visualisation of miRNA and the target mRNA should facilitate the exploration of the functions of ultra-trace amounts of miRNA in fixed cells in biological studies and may serve as a powerful tool for diagnoses based on circulating cancer cells. PMID:28989695

  17. Novel complex MAD phasing and RNase H structural insights using selenium oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Abdur, Rob; Gerlits, Oksana O.; Gan, Jianhua; Jiang, Jiansheng; Salon, Jozef; Kovalevsky, Andrey Y.; Chumanevich, Alexander A.; Weber, Irene T.; Huang, Zhen, E-mail: huang@gsu.edu [Georgia State University, Atlanta, GA 30303 (United States)

    2014-02-01

    Selenium-derivatized oligonucleotides may facilitate phase determination and high-resolution structure determination for protein–nucleic acid crystallography. The Se atom-specific mutagenesis (SAM) strategy may also enhance the study of nuclease catalysis. The crystal structures of protein–nucleic acid complexes are commonly determined using selenium-derivatized proteins via MAD or SAD phasing. Here, the first protein–nucleic acid complex structure determined using selenium-derivatized nucleic acids is reported. The RNase H–RNA/DNA complex is used as an example to demonstrate the proof of principle. The high-resolution crystal structure indicates that this selenium replacement results in a local subtle unwinding of the RNA/DNA substrate duplex, thereby shifting the RNA scissile phosphate closer to the transition state of the enzyme-catalyzed reaction. It was also observed that the scissile phosphate forms a hydrogen bond to the water nucleophile and helps to position the water molecule in the structure. Consistently, it was discovered that the substitution of a single O atom by a Se atom in a guide DNA sequence can largely accelerate RNase H catalysis. These structural and catalytic studies shed new light on the guide-dependent RNA cleavage.

  18. Argonaute pull-down and RISC analysis using 2'-O-methylated oligonucleotides affinity matrices.

    Science.gov (United States)

    Jannot, Guillaume; Vasquez-Rifo, Alejandro; Simard, Martin J

    2011-01-01

    During the last decade, several novel small non-coding RNA pathways have been unveiled, which reach out to many biological processes. Common to all these pathways is the binding of a small RNA molecule to a protein member of the Argonaute family, which forms a minimal core complex called the RNA-induced silencing complex or RISC. The RISC targets mRNAs in a sequence-specific manner, either to induce mRNA cleavage through the intrinsic activity of the Argonaute protein or to abrogate protein synthesis by a mechanism that is still under investigation. We describe here, in details, a method for the affinity chromatography of the let-7 RISC starting from extracts of the nematode Caenorhabditis elegans. Our method exploits the sequence specificity of the RISC and makes use of biotinylated and 2'-O-methylated oligonucleotides to trap and pull-down small RNAs and their associated proteins. Importantly, this technique may easily be adapted to target other small RNAs expressed in different cell types or model organisms. This method provides a useful strategy to identify the proteins associated with the RISC, and hence gain insight in the functions of small RNAs.

  19. Versatile functionalization of nanoelectrodes by oligonucleotides via pyrrole electrochemistry.

    Science.gov (United States)

    Descamps, Emeline; Nguyen, Khoa; Bouchain-Gautier, Christelle; Filoramo, Arianna; Goux-Capes, Laurence; Goffman, Marcello; Bourgoin, Jean-Philippe; Mailley, Pascal; Livache, Thierry

    2010-11-15

    Surface modification at the nanometer scale is a challenge for the future of molecular electronics. In particular, the precise anchoring and electrical addressing of biological scaffolds such as complex DNA nanonetworks is of importance for generating bio-directed assemblies of nano-objects for nanocircuit purposes. Herein, we consider the individual modification of nanoelectrodes with different oligonucleotide sequences by an electrochemically driven co-polymerization process of pyrrole and modified oligonucleotide sequences bearing pyrrole monomers. We demonstrate that this one-step technique presents the advantages of simplicity, localization of surface modification, mechanical, biological and chemical stability of the coatings, and high lateral resolution.

  20. Summarization on the synthesis and radionuclide-labeling of peptide nucleic acid for an oligonucleotide analogue

    International Nuclear Information System (INIS)

    Song, Hongtao; Zhang, Huaming; Gao, Hui

    2009-04-01

    Peptide nucleic acid (PNA), which is one kind of antisense nucleic acid compounds and an oligonucleotide analogue that binds strongly to DNA and RNA in a sequence specific manner, has its unique advantages in the field of molecular diagnostics and treatment of diseases. Now, people gradually attach more importance to PNA. To optimize the application of PNA in genetic re- search and therapy, a great number of backbone modifications on the newly- type structures of PNA were synthesized to improve its physicochemical proper- ties, such as hybridization speciality, solubility in biofluid, or cell permeability. The modified PNA labeled with radionuclides, which can obtain the aim at specific target and minimal non-target trauma, has important role in research and application of tumorous genitherapy. Here a review on the basic synthesis idea and several primary synthetic methods of PNA analogs was given, and also correlative studies and expectation on the compounds belonging to PNA series labeled with radionuclides were included. (authors)

  1. Detection of autoimmune antibodies in localized scleroderma by synthetic oligonucleotide antigens

    DEFF Research Database (Denmark)

    Samuelsen, Simone; Jørgensen, Christian Damsgaard; Mellins, Elizabeth D

    2018-01-01

    In this study, we developed a series of synthetic oligonucleotides that allowed us to investigate the details on the antigen recognition by autoimmune antibodies in localized scleroderma subjects. Besides dramatically improved analytical specificity of the assay, our data suggests a potential...... linking for antibodies to DNA to the biological status of disease state in localized scleroderma. Moreover, introducing chemical modifications into short synthetic deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) molecules completely changed the binding titers of corresponding antibodies...... and their clinical relevance. The strongest observed effect was registered for the localized scleroderma skin damage index (LoSDI) on the IgG antibodies to TC dinucleotide-rich double-stranded antigen (p

  2. Whole-cell hybridization of Methanosarcina cells with two new oligonucleotide probes

    DEFF Research Database (Denmark)

    Sørensen, A.H.; Torsvik, V.L.; Torsvik, T.

    1997-01-01

    Two new oligonucleotide probes targeting the 16S rRNA of the methanogenic genus Methanosarcina were developed. The probes have the following sequences (Escherichia coli numbering): probe SARCI551, 5'-GAC CCAATAATCACGATCAC-3', and probe SARCI645, 5'-TCCCGGTTCCAAGTCTGGC-3'. In situ hybridization...... with the fluorescently labelled probes required several modifications of standard procedures. Cells of Methanosarcina mazeii S-6 were found to lyse during the hybridization step if fixed in 3% formaldehyde and stored in 50% ethanol. Lysis was, however, not observed with cells fixed and stored in 1.6% formaldehyde-0.......85% NaCl. Extensive autofluorescence of the cells was found upon hybridization in the presence of 5 mM EDTA, but successful hybridization could be obtained without addition of this compound. The mounting agent Citifluor AF1, often used in conjugation with the fluorochrome fluorescein, was found to wash...

  3. Development of dansyl-modified oligonucleotide probes responding to structural changes in a duplex.

    Science.gov (United States)

    Suzuki, Yoshio; Kowata, Keiko; Komatsu, Yasuo

    2013-11-15

    We have synthesized a nonnucleoside amidite block of dansyl fluorophore to prepare dansyl-modified oligonucleotides (ONTs). The fluorescence intensities of dansyl-ONT specifically increased by the presence of adjacent guanosine residues but, significantly reduced in a dansyl-flipping duplex. These changes were caused by solvatochromism effect due to the number of guanine which is hydrophobic functional group and the external environment of dansyl group. The fluorescence intensities could be plotted as a function of the ONTs concentrations and the increase in the fluorescence was observed to equimolar concentrations of target DNA. This duplex exhibited higher melting temperature relative to the corresponding duplexes containing other base pairs. Similar changes in fluorescence could be detected upon hybridization with complementary RNAs. Thus, the dansyl-modified ONTs provide sequence specific fluorescent probe of DNA and RNA. Copyright © 2013 Elsevier Ltd. All rights reserved.

  4. A pilot study of transcription unit analysis in rice using oligonucleotide tiling-path microarray

    DEFF Research Database (Denmark)

    Stolc, Viktor; Li, Lei; Wang, Xiangfeng

    2005-01-01

    As the international efforts to sequence the rice genome are completed, an immediate challenge and opportunity is to comprehensively and accurately define all transcription units in the rice genome. Here we describe a strategy of using high-density oligonucleotide tiling-path microarrays to map...... transcription of the japonica rice genome. In a pilot experiment to test this approach, one array representing the reverse strand of the last 11.2 Mb sequence of chromosome 10 was analyzed in detail based on a mathematical model developed in this study. Analysis of the array data detected 77% of the reference...... gene models in a mixture of four RNA populations. Moreover, significant transcriptional activities were found in many of the previously annotated intergenic regions. These preliminary results demonstrate the utility of genome tiling microarrays in evaluating annotated rice gene models...

  5. Factor XI Antisense Oligonucleotide for Prevention of Venous Thrombosis

    NARCIS (Netherlands)

    Büller, Harry R.; Bethune, Claudette; Bhanot, Sanjay; Gailani, David; Monia, Brett P.; Raskob, Gary E.; Segers, Annelise; Verhamme, Peter; Weitz, Jeffrey I.; Weitz, Jeffrey; Prins, Martin; Beenen, Ludo; Otten, Hans-Martin; Roos, Yvo; Slagboom, Ton; Vandenbriele, Christophe; Vanassche, Thomas; Dani, Vidhi; Schulz, Dan; Shapiro, Cara; Kwoh, Katherine; Jung, Bill; Gawinek-Samelczak, Agata; Kaemmer, Christina; Angelov, S.; Stavrev, V.; Kinov, P.; Dessouki, E.; Abuzgaya, F.; Baurovskis, A.; Peredistijs, A.; Petronis, S.; Danilyak, V.; Driagin, V.; Kuropatkin, G.; Parfeev, S.; Safronov, A.; Ankin, M.; Korzh, M.; Olinichenko, G.; Polivoda, A.; Shevchenko, V.; Sulyma, V.

    2015-01-01

    Background Experimental data indicate that reducing factor XI levels attenuates thrombosis without causing bleeding, but the role of factor XI in the prevention of postoperative venous thrombosis in humans is unknown. FXI-ASO (ISIS 416858) is a second-generation antisense oligonucleotide that

  6. Lipid-modified G4-decoy oligonucleotide anchored to nanoparticles

    DEFF Research Database (Denmark)

    Cogoi, S; Jakobsen, U; Pedersen, E B

    2016-01-01

    KRAS is mutated in >90% of pancreatic ductal adenocarcinomas. As its inactivation leads to tumour regression, mutant KRAS is considered an attractive target for anticancer drugs. In this study we report a new delivery strategy for a G4-decoy oligonucleotide that sequesters MAZ, a transcription fa...

  7. Effects of Atelocollagen Formulation Containing Oligonucleotide on Endothelial Permeability

    Directory of Open Access Journals (Sweden)

    Koji Hanai

    2012-01-01

    Full Text Available Atelocollagen is a major animal protein that is used as a highly biocompatible biomaterial. To date, atelocollagen has been used as an effective drug delivery technology to sustain the release of antitumor proteins and to enhance the antitumor activity of oligonucleotides in in vivo models. However, the biological effects of this technology are not fully understood. In the present study, we investigated the effects of atelocollagen on endothelial paracellular barrier function. An atelocollagen formulation containing oligonucleotides specifically increased the permeability of two types of endothelial cells, and the change was dependent on the molecular size, structure of the oligonucleotides used and the concentrations of the oligonucleotide and atelocollagen in the formulation. An immunohistochemical examination revealed that the formulation had effects on the cellular skeleton and intercellular structure although it did not affect the expression of adherens junction or tight junction proteins. These changes were induced through p38 MAP kinase signaling. It is important to elucidate the biological functions of atelocollagen in order to be able to exploit its drug delivery properties.

  8. mathFISH, a Web Tool That Uses Thermodynamics-Based Mathematical Models for In Silico Evaluation of Oligonucleotide Probes for Fluorescence In Situ Hybridization▿ †

    OpenAIRE

    Yilmaz, L. Safak; Parnerkar, Shreyas; Noguera, Daniel R.

    2010-01-01

    Mathematical models of RNA-targeted fluorescence in situ hybridization (FISH) for perfectly matched and mismatched probe/target pairs are organized and automated in web-based mathFISH (http://mathfish.cee.wisc.edu). Offering the users up-to-date knowledge of hybridization thermodynamics within a theoretical framework, mathFISH is expected to maximize the probability of success during oligonucleotide probe design.

  9. mathFISH, a web tool that uses thermodynamics-based mathematical models for in silico evaluation of oligonucleotide probes for fluorescence in situ hybridization.

    Science.gov (United States)

    Yilmaz, L Safak; Parnerkar, Shreyas; Noguera, Daniel R

    2011-02-01

    Mathematical models of RNA-targeted fluorescence in situ hybridization (FISH) for perfectly matched and mismatched probe/target pairs are organized and automated in web-based mathFISH (http://mathfish.cee.wisc.edu). Offering the users up-to-date knowledge of hybridization thermodynamics within a theoretical framework, mathFISH is expected to maximize the probability of success during oligonucleotide probe design.

  10. Fluorinated Nucleotide Modifications Modulate Allele Selectivity of SNP-Targeting Antisense Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Michael E. Østergaard

    2017-06-01

    Full Text Available Antisense oligonucleotides (ASOs have the potential to discriminate between subtle RNA mismatches such as SNPs. Certain mismatches, however, allow ASOs to bind at physiological conditions and result in RNA cleavage mediated by RNase H. We showed that replacing DNA nucleotides in the gap region of an ASO with other chemical modification can improve allele selectivity. Herein, we systematically substitute every position in the gap region of an ASO targeting huntingtin gene (HTT with fluorinated nucleotides. Potency is determined in cell culture against mutant HTT (mtHTT and wild-type HTT (wtHTT mRNA and RNase H cleavage intensities, and patterns are investigated. This study profiled five different fluorinated nucleotides and showed them to have predictable, site-specific effects on RNase H cleavage, and the cleavage patterns were rationalized from a published X-ray structure of human RNase H1. The results herein can be used as a guide for future projects where ASO discrimination of SNPs is important.

  11. Antisense Oligonucleotides Internally Labeled with Peptides Show Improved Target Recognition and Stability to Enzymatic Degradation

    DEFF Research Database (Denmark)

    Taskova, Maria; Madsen, Charlotte S.; Jensen, Knud J.

    2017-01-01

    Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, m...... and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.......Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide......, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides...

  12. Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

    Science.gov (United States)

    Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

    2016-03-01

    Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (Prooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer.

  13. Dramatically improved RNA in situ hybridization signals using LNA-modified probes

    DEFF Research Database (Denmark)

    Thomsen, Rune; Nielsen, Peter Stein; Jensen, Torben Heick

    2005-01-01

    . This increases the thermal stability of hybrids formed with RNA. The LNA-based probes detect specific RNAs in fixed yeast cells with an efficiency far better than conventional DNA oligonucleotide probes of the same sequence. Using this probe design, we were also able to detect poly(A)+ RNA accumulation within......In situ detection of RNA by hybridization with complementary probes is a powerful technique. Probe design is a critical parameter in successful target detection. We have evaluated the efficiency of fluorescent DNA oligonucleotides modified to contain locked nucleic acid (LNA) residues...

  14. Identification of a Peptide for Systemic Brain Delivery of a Morpholino Oligonucleotide in Mouse Models of Spinal Muscular Atrophy

    Science.gov (United States)

    Shabanpoor, Fazel; Hammond, Suzan M; Abendroth, Frank; Hazell, Gareth; Wood, Matthew J.A.

    2017-01-01

    Splice-switching antisense oligonucleotides are emerging treatments for neuromuscular diseases, with several splice-switching oligonucleotides (SSOs) currently undergoing clinical trials such as for Duchenne muscular dystrophy (DMD) and spinal muscular atrophy (SMA). However, the development of systemically delivered antisense therapeutics has been hampered by poor tissue penetration and cellular uptake, including crossing of the blood–brain barrier (BBB) to reach targets in the central nervous system (CNS). For SMA application, we have investigated the ability of various BBB-crossing peptides for CNS delivery of a splice-switching phosphorodiamidate morpholino oligonucleotide (PMO) targeting survival motor neuron 2 (SMN2) exon 7 inclusion. We identified a branched derivative of the well-known ApoE (141–150) peptide, which as a PMO conjugate was capable of exon inclusion in the CNS following systemic administration, leading to an increase in the level of full-length SMN2 transcript. Treatment of newborn SMA mice with this peptide-PMO (P-PMO) conjugate resulted in a significant increase in the average lifespan and gains in weight, muscle strength, and righting reflexes. Systemic treatment of adult SMA mice with this newly identified P-PMO also resulted in small but significant increases in the levels of SMN2 pre-messenger RNA (mRNA) exon inclusion in the CNS and peripheral tissues. This work provides proof of principle for the ability to select new peptide paradigms to enhance CNS delivery and activity of a PMO SSO through use of a peptide-based delivery platform for the treatment of SMA potentially extending to other neuromuscular and neurodegenerative diseases. PMID:28118087

  15. LNA-antisense rivals siRNA for gene silencing

    DEFF Research Database (Denmark)

    Jepsen, Jan Stenvang; Wengel, Jesper; Stenvang, Jan

    2004-01-01

    Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing unprecedented binding affinity toward complementary DNA and RNA while obeying the Watson-Crick base-pairing rules. For efficient gene silencing in vitro and in vivo, fully modified or chimeric LNA oligonucleotides have been a...

  16. Contribution of the first K-homology domain of poly(C)-binding protein 1 to its affinity and specificity for C-rich oligonucleotides

    OpenAIRE

    Yoga, Yano M. K.; Traore, Daouda A. K.; Sidiqi, Mahjooba; Szeto, Chris; Pendini, Nicole R.; Barker, Andrew; Leedman, Peter J.; Wilce, Jacqueline A.; Wilce, Matthew C. J.

    2012-01-01

    Poly-C-binding proteins are triple KH (hnRNP K homology) domain proteins with specificity for single stranded C-rich RNA and DNA. They play diverse roles in the regulation of protein expression at both transcriptional and translational levels. Here, we analyse the contributions of individual αCP1 KH domains to binding C-rich oligonucleotides using biophysical and structural methods. Using surface plasmon resonance (SPR), we demonstrate that KH1 makes the most stable interactions with both RNA...

  17. Oligonucleotide aptamers against tyrosine kinase receptors: Prospect for anticancer applications.

    Science.gov (United States)

    Camorani, Simona; Crescenzi, Elvira; Fedele, Monica; Cerchia, Laura

    2018-04-01

    Transmembrane receptor tyrosine kinases (RTKs) play crucial roles in cancer cell proliferation, survival, migration and differentiation. Area of intense research is searching for effective anticancer therapies targeting these receptors and, to date, several monoclonal antibodies and small-molecule tyrosine kinase inhibitors have entered the clinic. However, some of these drugs show limited efficacy and give rise to acquired resistance. Emerging highly selective compounds for anticancer therapy are oligonucleotide aptamers that interact with their targets by recognizing a specific three-dimensional structure. Because of their nucleic acid nature, the rational design of advanced strategies to manipulate aptamers for both diagnostic and therapeutic applications is greatly simplified over antibodies. In this manuscript, we will provide a comprehensive overview of oligonucleotide aptamers as next generation strategies to efficiently target RTKs in human cancers. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Thermoplastic polymers surfaces for Dip-Pen Nanolithography of oligonucleotides

    International Nuclear Information System (INIS)

    Suriano, Raffaella; Biella, Serena; Cesura, Federico; Levi, Marinella; Turri, Stefano

    2013-01-01

    Different thermoplastic polymers were spin-coated to prepare smooth surfaces for the direct deposition of end-group modified oligonucleotides by Dip-Pen Nanolithography. A study of the diffusion process was done in order to investigate the dependence of calibration coefficient and quality of deposited features on environmental parameters (temperature, relative humidity) and ink's molecular weight and functionality. The optimization of the process parameters led to the realization of high quality and density nanoarrays on plastics.

  19. Thermoplastic polymers surfaces for Dip-Pen Nanolithography of oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Suriano, Raffaella [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano (Italy); Biella, Serena, E-mail: serena.biella@polimi.it [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano (Italy); Cesura, Federico; Levi, Marinella; Turri, Stefano [Dipartimento di Chimica, Materiali e Ingegneria Chimica “Giulio Natta”, Politecnico di Milano, Piazza Leonardo da Vinci 32, 20133 Milano (Italy)

    2013-05-15

    Different thermoplastic polymers were spin-coated to prepare smooth surfaces for the direct deposition of end-group modified oligonucleotides by Dip-Pen Nanolithography. A study of the diffusion process was done in order to investigate the dependence of calibration coefficient and quality of deposited features on environmental parameters (temperature, relative humidity) and ink's molecular weight and functionality. The optimization of the process parameters led to the realization of high quality and density nanoarrays on plastics.

  20. Direct, rapid RNA sequence analysis

    International Nuclear Information System (INIS)

    Peattie, D.A.

    1987-01-01

    The original methods of RNA sequence analysis were based on enzymatic production and chromatographic separation of overlapping oligonucleotide fragments from within an RNA molecule followed by identification of the mononucleotides comprising the oligomer. Over the past decade the field of nucleic acid sequencing has changed dramatically, however, and RNA molecules now can be sequenced in a variety of more streamlined fashions. Most of the more recent advances in RNA sequencing have involved one-dimensional electrophoretic separation of 32 P-end-labeled oligoribonucleotides on polyacrylamide gels. In this chapter the author discusses two of these methods for determining the nucleotide sequences of RNA molecules rapidly: the chemical method and the enzymatic method. Both methods are direct and degradative, i.e., they rely on fragmatic and chemical approaches should be utilized. The single-strand-specific ribonucleases (A, T 1 , T 2 , and S 1 ) provide an efficient means to locate double-helical regions rapidly, and the chemical reactions provide a means to determine the RNA sequence within these regions. In addition, the chemical reactions allow one to assign interactions to specific atoms and to distinguish secondary interactions from tertiary ones. If the RNA molecule is small enough to be sequenced directly by the enzymatic or chemical method, the probing reactions can be done easily at the same time as sequencing reactions

  1. THERAPEUTIC ANTISENSE OLIGONUCLEOTIDES AGAINST CANCER: HURDLING TO THE CLINIC

    Directory of Open Access Journals (Sweden)

    Pedro Miguel Duarte Moreno

    2014-10-01

    Full Text Available Under clinical development since the early 90’s and with two successfully approved drugs (Fomivirsen and Mipomersen, oligonucleotide-based therapeutics have not yet delivered a clinical drug to the market in the cancer field. Whilst many pre-clinical data has been generated, a lack of understanding still exists on how to efficiently tackle all the different challenges presented for cancer targeting in a clinical setting. Namely, effective drug vectorization, careful choice of target gene or synergistic multi-gene targeting are surely decisive, while caution must be exerted to avoid potential toxic, often misleading off-target-effects. Here a brief overview will be given on the nucleic acid chemistry advances that established oligonucleotide technologies as a promising therapeutic alternative and ongoing cancer related clinical trials. Special attention will be given towards a perspective on the hurdles encountered specifically in the cancer field by this class of therapeutic oligonucleotides and a view on possible avenues for success is presented, with particular focus on the contribution from nanotechnology to the field.

  2. Peroxide-mediated desulfurization of phosphorothioate oligonucleotides and its prevention.

    Science.gov (United States)

    Krotz, Achim H; Mehta, Rahul C; Hardee, Gregory E

    2005-02-01

    Desulfurization at the internucleotide phosphorothioate linkage of antisense oligonucleotides (ASOs) in dermatological formulations has been investigated using strong ion exchange chromatography and mass spectroscopy. The formation of phosphate diester linkages appeared to arise from a reaction between the phosphorothioate oligonucleotide and a potent oxidizing agent. Screening of excipients used in the formulation indicated that the cause of desulfurization was related to the presence of polyethylene glycol-derived nonionic surfactants MYRJ 52 or BRIJ 58. Autoxidation of the polyethylene glycol chain is suggested as the probable origin for the observed incompatibility. The ability of various antioxidants to prevent oxidative degradation of ASO-1 in simple test systems and in oil-in-water emulsions is described. It is found that in test systems both lipophilic and hydrophilic antioxidants are effective. However, in cream formulation (oil-in-water emulsions) of ASO-1 the addition of hydrophilic antioxidants L-cysteine or DL-alpha-lipoic acid has been shown to be superior in protecting the oligonucleotide from desulfurization upon storage. Copyright 2004 Wiley-Liss, Inc.

  3. G-Quadruplex Forming Oligonucleotides as Anti-HIV Agents.

    Science.gov (United States)

    Musumeci, Domenica; Riccardi, Claudia; Montesarchio, Daniela

    2015-09-22

    Though a variety of different non-canonical nucleic acids conformations have been recognized, G-quadruplex structures are probably the structural motifs most commonly found within known oligonucleotide-based aptamers. This could be ascribed to several factors, as their large conformational diversity, marked responsiveness of their folding/unfolding processes to external stimuli, high structural compactness and chemo-enzymatic and thermodynamic stability. A number of G-quadruplex-forming oligonucleotides having relevant in vitro anti-HIV activity have been discovered in the last two decades through either SELEX or rational design approaches. Improved aptamers have been obtained by chemical modifications of natural oligonucleotides, as terminal conjugations with large hydrophobic groups, replacement of phosphodiester linkages with phosphorothioate bonds or other surrogates, insertion of base-modified monomers, etc. In turn, detailed structural studies have elucidated the peculiar architectures adopted by many G-quadruplex-based aptamers and provided insight into their mechanism of action. An overview of the state-of-the-art knowledge of the relevance of putative G-quadruplex forming sequences within the viral genome and of the most studied G-quadruplex-forming aptamers, selectively targeting HIV proteins, is here presented.

  4. A new oligonucleotide microarray for detection of pathogenic and non-pathogenic Legionella spp.

    Directory of Open Access Journals (Sweden)

    Boyang Cao

    Full Text Available Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp.

  5. A New Oligonucleotide Microarray for Detection of Pathogenic and Non-Pathogenic Legionella spp.

    Science.gov (United States)

    Cao, Boyang; Liu, Xiangqian; Yu, Xiang; Chen, Min; Feng, Lu; Wang, Lei

    2014-01-01

    Legionella pneumophila has been recognized as the major cause of legionellosis since the discovery of the deadly disease. Legionella spp. other than L. pneumophila were later found to be responsible to many non-pneumophila infections. The non-L. pneumophila infections are likely under-detected because of a lack of effective diagnosis. In this report, we have sequenced the 16S-23S rRNA gene internal transcribed spacer (ITS) of 10 Legionella species and subspecies, including L. anisa, L. bozemanii, L. dumoffii, L. fairfieldensis, L. gormanii, L. jordanis, L. maceachernii, L. micdadei, L. pneumophila subspp. fraseri and L. pneumophila subspp. pasculleii, and developed a rapid oligonucleotide microarray detection technique accordingly to identify 12 most common Legionella spp., which consist of 11 pathogenic species of L. anisa, L. bozemanii, L. dumoffii, L. gormanii, L. jordanis, L. longbeachae, L. maceachernii, L. micdadei, and L. pneumophila (including subspp. pneumophila, subspp. fraseri, and subspp. pasculleii) and one non-pathogenic species, L. fairfieldensis. Twenty-nine probes that reproducibly detected multiple Legionella species with high specificity were included in the array. A total of 52 strains, including 30 target pathogens and 22 non-target bacteria, were used to verify the oligonucleotide microarray assay. The sensitivity of the detection was at 1.0 ng with genomic DNA or 13 CFU/100 mL with Legionella cultures. The microarray detected seven samples of air conditioner-condensed water with 100% accuracy, validating the technique as a promising method for applications in basic microbiology, clinical diagnosis, food safety, and epidemiological surveillance. The phylogenetic study based on the ITS has also revealed that the non-pathogenic L. fairfieldensis is the closest to L. pneumophila than the nine other pathogenic Legionella spp. PMID:25469776

  6. An evaluation of oligonucleotide-based therapeutic strategies for polyQ diseases

    Directory of Open Access Journals (Sweden)

    Fiszer Agnieszka

    2012-03-01

    Full Text Available Abstract Background RNA interference (RNAi and antisense strategies provide experimental therapeutic agents for numerous diseases, including polyglutamine (polyQ disorders caused by CAG repeat expansion. We compared the potential of different oligonucleotide-based strategies for silencing the genes responsible for several polyQ diseases, including Huntington's disease and two spinocerebellar ataxias, type 1 and type 3. The strategies included nonallele-selective gene silencing, gene replacement, allele-selective SNP targeting and CAG repeat targeting. Results Using the patient-derived cell culture models of polyQ diseases, we tested various siRNAs, and antisense reagents and assessed their silencing efficiency and allele selectivity. We showed considerable allele discrimination by several SNP targeting siRNAs based on a weak G-G or G-U pairing with normal allele and strong G-C pairing with mutant allele at the site of RISC-induced cleavage. Among the CAG repeat targeting reagents the strongest allele discrimination is achieved by miRNA-like functioning reagents that bind to their targets and inhibit their translation without substantial target cleavage. Also, morpholino analog performs well in mutant and normal allele discrimination but its efficient delivery to cells at low effective concentration still remains a challenge. Conclusions Using three cellular models of polyQ diseases and the same experimental setup we directly compared the performance of different oligonucleotide-based treatment strategies that are currently under development. Based on the results obtained by us and others we discussed the advantages and drawbacks of these strategies considering them from several different perspectives. The strategy aimed at nonallele-selective inhibiting of causative gene expression by targeting specific sequence of the implicated gene is the easiest to implement but relevant benefits are still uncertain. The gene replacement strategy that

  7. Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

    International Nuclear Information System (INIS)

    Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

    2016-01-01

    Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer. - Highlights: • Core/shell type Iron oxide nanoparticles were used as a novel and efficient method. • This method increases exogenous DNA uptake by rooster spermatozoa. • Static magnetic field decreased DNA uptake by rooster spermatozoa.

  8. Static magnetic field reduced exogenous oligonucleotide uptake by spermatozoa using magnetic nanoparticle gene delivery system

    Energy Technology Data Exchange (ETDEWEB)

    Katebi, Samira; Esmaeili, Abolghasem, E-mail: aesmaeili@sci.ui.ac.ir; Ghaedi, Kamran

    2016-03-15

    Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (P<0.001) when spermatozoa were incubated in exogenous oligonucleotide solution and MNPs. However, by applying static magnetic field during magnetofection method, a significant decrease in exogenous oligonucleotide uptake was observed (P<0.05). Findings of this study showed that MNPs were effective to increase exogenous oligonucleotide uptake by rooster spermatozoa; however unlike others studies, static magnetic field, was not only ineffective to enhance exogenous oligonucleotide uptake by rooster spermatozoa but also led to reduction in efficiency of magnetic nanoparticles in gene transfer. - Highlights: • Core/shell type Iron oxide nanoparticles were used as a novel and efficient method. • This method increases exogenous DNA uptake by rooster spermatozoa. • Static magnetic field decreased DNA uptake by rooster spermatozoa.

  9. Oligoadenylate is present in the mitochondrial RNA of Saccharomyces cerevisiae

    International Nuclear Information System (INIS)

    Yuckenberg, P.D.; Phillips, S.L.

    1982-01-01

    The authors examined Saccharomyces cerevisiae mitochondrial RNA for polyadenylate. Using hybridization to [/sup 3/H]polyuridylate as the assay for adenylate sequences, they found adenylate-rich oligonucleotides approximately 8 residues long. Longer polyadenylate was not detected. Most of the adenylate-rich sequence is associated with the large mitochondrial rRNA. The remainder is associated with the 10-12S group of transcripts

  10. Survival of the fittest before the beginning of life: selection of the first oligonucleotide-like polymers by UV light

    Directory of Open Access Journals (Sweden)

    Cherepanov Dmitry A

    2003-05-01

    Full Text Available Abstract Background A key event in the origin of life on this planet has been formation of self-replicating RNA-type molecules, which were complex enough to undergo a Darwinian-type evolution (origin of the "RNA world". However, so far there has been no explanation of how the first RNA-like biopolymers could originate and survive on the primordial Earth. Results As condensation of sugar phosphates and nitrogenous bases is thermodynamically unfavorable, these compounds, if ever formed, should have undergone rapid hydrolysis. Thus, formation of oligonucleotide-like structures could have happened only if and when these structures had some selective advantage over simpler compounds. It is well known that nitrogenous bases are powerful quenchers of UV quanta and effectively protect the pentose-phosphate backbones of RNA and DNA from UV cleavage. To check if such a protection could play a role in abiogenic evolution on the primordial Earth (in the absence of the UV-protecting ozone layer, we simulated, by using Monte Carlo approach, the formation of the first oligonucleotides under continuous UV illumination. The simulations confirmed that UV irradiation could have worked as a selective factor leading to a relative enrichment of the system in longer sugar-phosphate polymers carrying nitrogenous bases as UV-protectors. Partial funneling of the UV energy into the condensation reactions could provide a further boost for the oligomerization. Conclusion These results suggest that accumulation of the first polynucleotides could be explained by their abiogenic selection as the most UV-resistant biopolymers.

  11. Antisense oligonucleotides targeting translation inhibitory elements in 5' UTRs can selectively increase protein levels.

    Science.gov (United States)

    Liang, Xue-Hai; Sun, Hong; Shen, Wen; Wang, Shiyu; Yao, Joyee; Migawa, Michael T; Bui, Huynh-Hoa; Damle, Sagar S; Riney, Stan; Graham, Mark J; Crooke, Rosanne M; Crooke, Stanley T

    2017-09-19

    A variety of diseases are caused by deficiencies in amounts or activity of key proteins. An approach that increases the amount of a specific protein might be of therapeutic benefit. We reasoned that translation could be specifically enhanced using trans-acting agents that counter the function of negative regulatory elements present in the 5' UTRs of some mRNAs. We recently showed that translation can be enhanced by antisense oligonucleotides (ASOs) that target upstream open reading frames. Here we report the amount of a protein can also be selectively increased using ASOs designed to hybridize to other translation inhibitory elements in 5' UTRs. Levels of human RNASEH1, LDLR, and ACP1 and of mouse ACP1 and ARF1 were increased up to 2.7-fold in different cell types and species upon treatment with chemically modified ASOs targeting 5' UTR inhibitory regions in the mRNAs encoding these proteins. The activities of ASOs in enhancing translation were sequence and position dependent and required helicase activity. The ASOs appear to improve the recruitment of translation initiation factors to the target mRNA. Importantly, ASOs targeting ACP1 mRNA significantly increased the level of ACP1 protein in mice, suggesting that this approach has therapeutic and research potentials. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Specific Increase of Protein Levels by Enhancing Translation Using Antisense Oligonucleotides Targeting Upstream Open Frames.

    Science.gov (United States)

    Liang, Xue-Hai; Shen, Wen; Crooke, Stanley T

    2017-01-01

    A number of diseases are caused by low levels of key proteins; therefore, increasing the amount of specific proteins in human bodies is of therapeutic interest. Protein expression is downregulated by some structural or sequence elements present in the 5' UTR of mRNAs, such as upstream open reading frames (uORF). Translation initiation from uORF(s) reduces translation from the downstream primary ORF encoding the main protein product in the same mRNA, leading to a less efficient protein expression. Therefore, it is possible to use antisense oligonucleotides (ASOs) to specifically inhibit translation of the uORF by base-pairing with the uAUG region of the mRNA, redirecting translation machinery to initiate from the primary AUG site. Here we review the recent findings that translation of specific mRNAs can be enhanced using ASOs targeting uORF regions. Appropriately designed and optimized ASOs are highly specific, and they act in a sequence- and position-dependent manner, with very minor off-target effects. Protein levels can be increased using this approach in different types of human and mouse cells, and, importantly, also in mice. Since uORFs are present in around half of human mRNAs, the uORF-targeting ASOs may thus have valuable potential as research tools and as therapeutics to increase the levels of proteins for a variety of genes.

  13. Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice

    Directory of Open Access Journals (Sweden)

    Dominic Jauvin

    2017-06-01

    Full Text Available Myotonic dystrophy type 1 (DM1, a dominant hereditary muscular dystrophy, is caused by an abnormal expansion of a (CTGn trinucleotide repeat in the 3′ UTR of the human dystrophia myotonica protein kinase (DMPK gene. As a consequence, mutant transcripts containing expanded CUG repeats are retained in nuclear foci and alter the function of splicing regulatory factors members of the MBNL and CELF families, resulting in alternative splicing misregulation of specific transcripts in affected DM1 tissues. In the present study, we treated DMSXL mice systemically with a 2′-4′-constrained, ethyl-modified (ISIS 486178 antisense oligonucleotide (ASO targeted to the 3′ UTR of the DMPK gene, which led to a 70% reduction in CUGexp RNA abundance and foci in different skeletal muscles and a 30% reduction in the heart. Furthermore, treatment with ISIS 486178 ASO improved body weight, muscle strength, and muscle histology, whereas no overt toxicity was detected. This is evidence that the reduction of CUGexp RNA improves muscle strength in DM1, suggesting that muscle weakness in DM1 patients may be improved following elimination of toxic RNAs.

  14. Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice.

    Science.gov (United States)

    Jauvin, Dominic; Chrétien, Jessina; Pandey, Sanjay K; Martineau, Laurie; Revillod, Lucille; Bassez, Guillaume; Lachon, Aline; MacLeod, A Robert; Gourdon, Geneviève; Wheeler, Thurman M; Thornton, Charles A; Bennett, C Frank; Puymirat, Jack

    2017-06-16

    Myotonic dystrophy type 1 (DM1), a dominant hereditary muscular dystrophy, is caused by an abnormal expansion of a (CTG) n trinucleotide repeat in the 3' UTR of the human dystrophia myotonica protein kinase (DMPK) gene. As a consequence, mutant transcripts containing expanded CUG repeats are retained in nuclear foci and alter the function of splicing regulatory factors members of the MBNL and CELF families, resulting in alternative splicing misregulation of specific transcripts in affected DM1 tissues. In the present study, we treated DMSXL mice systemically with a 2'-4'-constrained, ethyl-modified (ISIS 486178) antisense oligonucleotide (ASO) targeted to the 3' UTR of the DMPK gene, which led to a 70% reduction in CUG exp RNA abundance and foci in different skeletal muscles and a 30% reduction in the heart. Furthermore, treatment with ISIS 486178 ASO improved body weight, muscle strength, and muscle histology, whereas no overt toxicity was detected. This is evidence that the reduction of CUG exp RNA improves muscle strength in DM1, suggesting that muscle weakness in DM1 patients may be improved following elimination of toxic RNAs. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  15. Adenylylation of small RNA sequencing adapters using the TS2126 RNA ligase I.

    Science.gov (United States)

    Lama, Lodoe; Ryan, Kevin

    2016-01-01

    Many high-throughput small RNA next-generation sequencing protocols use 5' preadenylylated DNA oligonucleotide adapters during cDNA library preparation. Preadenylylation of the DNA adapter's 5' end frees from ATP-dependence the ligation of the adapter to RNA collections, thereby avoiding ATP-dependent side reactions. However, preadenylylation of the DNA adapters can be costly and difficult. The currently available method for chemical adenylylation of DNA adapters is inefficient and uses techniques not typically practiced in laboratories profiling cellular RNA expression. An alternative enzymatic method using a commercial RNA ligase was recently introduced, but this enzyme works best as a stoichiometric adenylylating reagent rather than a catalyst and can therefore prove costly when several variant adapters are needed or during scale-up or high-throughput adenylylation procedures. Here, we describe a simple, scalable, and highly efficient method for the 5' adenylylation of DNA oligonucleotides using the thermostable RNA ligase 1 from bacteriophage TS2126. Adapters with 3' blocking groups are adenylylated at >95% yield at catalytic enzyme-to-adapter ratios and need not be gel purified before ligation to RNA acceptors. Experimental conditions are also reported that enable DNA adapters with free 3' ends to be 5' adenylylated at >90% efficiency. © 2015 Lama and Ryan; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  16. Efficient assessment of modified nucleoside stability under conditions of automated oligonucleotide synthesis: characterization of the oxidation and oxidative desulfurization of 2-thiouridine.

    Science.gov (United States)

    Sochacka, E

    2001-01-01

    In order to efficiently assess the chemical stability of modified nucleosides to the reagents and conditions of automated oligonucleotide synthesis, we designed, developed and tested a scheme in which the modified nucleoside, directly attached to a solid support, is exposed to the cyclic chemistry of the instrument. Stability of 2-thiouridine against different oxidizers was investigated. Tertbutyl hydroperoxide (1 M) in anhydrous acetonitrile was a more effective oxidizer for the incorporation of 2-thiouridine into oligonucleotide chains than the same oxidizer in methylene chloride. Carbon tetrachloride/water in the presence of a basic catalyst was superior in maintaining the thiocarbonyl function, but its utility for RNA synthesis has yet to be fully tested, whereas 2-phenylsulfonyloxaziridine was a very efficient reagent for oxidative desulfurization of 2-thiouridine.

  17. Quantification of syntrophic fatty acid-{beta}-oxidizing bacteria in a mesophilic biogas reactor by oligonucleotide probe hybridization

    Energy Technology Data Exchange (ETDEWEB)

    Hansen, K.H.; Ahring, B.K.; Raskin, L.

    1999-11-01

    Small-subunit rRNA sequences were obtained for two saturated fatty acid-{beta}-oxidizing syntrophic bacteria, Syntrophomonas sapovorans and Syntrophomonas wolfei LYB, and sequence analysis confirmed their classification as members of the family Syntrophomonadaceae. S.wolfei LYB was closely related to S.wolfei subsp. solfei, but S. sapovorans did not cluster with the other members of the genus Syntrophomonas. Five oligonucleotide probes targeting the small-subunit rRNA of different groups within the family Syntrophomonadaceae, which contains all currently known saturated fatty acid-{beta}-oxidizing syntrophic bacteria, were developed and characterized. The probes were designed to be specific at the family, genus, and species levels and were characterized by temperature-of-dissociation and specificity studies. To demonstrate the usefulness of the probes for the detection and quantification of saturated fatty acid-{beta}-oxidizing syntrophic bacteria in methanogenic environments, the microbial community structure of a sample from a full-scale biogas plant was determined. Hybridization results with probes for syntrophic bacteria and methanogens were compared to specific methanogenic activities and microbial numbers determined with most-probable-number estimates. Most of the methanogenic rRNA was comprised of Methanomicrobiales rRNA, suggesting that members of this order served as the main hydrogen-utilizing microorganisms. Between 0.2 and 1% of the rRNA was attributed to the Syntrophomonadaceae, or which the majority was accounted for by the genus Syntrophomonas.

  18. Antisense Oligonucleotide (AON-based Therapy for Leber Congenital Amaurosis Caused by a Frequent Mutation in CEP290

    Directory of Open Access Journals (Sweden)

    Rob WJ Collin

    2012-01-01

    Full Text Available Leber congenital amaurosis (LCA is the most severe form of inherited retinal degeneration, with an onset in the first year of life. The most frequent mutation that causes LCA, present in at least 10% of individuals with LCA from North-American and Northern-European descent, is an intronic mutation in CEP290 that results in the inclusion of an aberrant exon in the CEP290 mRNA. Here, we describe a genetic therapy approach that is based on antisense oligonucleotides (AONs, small RNA molecules that are able to redirect normal splicing of aberrantly processed pre-mRNA. Immortalized lymphoblastoid cells of individuals with LCA homozygously carrying the intronic CEP290 mutation were transfected with several AONs that target the aberrant exon that is incorporated in the mutant CEP290 mRNA. Subsequent RNA isolation and reverse transcription-PCR analysis revealed that a number of AONs were capable of almost fully redirecting normal CEP290 splicing, in a dose-dependent manner. Other AONs however, displayed no effect on CEP290 splicing at all, indicating that the rescue of aberrant CEP290 splicing shows a high degree of sequence specificity. Together, our data show that AON-based therapy is a promising therapeutic approach for CEP290-associated LCA that warrants future research in animal models to develop a cure for this blinding disease.

  19. Pharmacokinetic Profiling of Conjugated Therapeutic Oligonucleotides: A High-Throughput Method Based Upon Serial Blood Microsampling Coupled to Peptide Nucleic Acid Hybridization Assay.

    Science.gov (United States)

    Godinho, Bruno M D C; Gilbert, James W; Haraszti, Reka A; Coles, Andrew H; Biscans, Annabelle; Roux, Loic; Nikan, Mehran; Echeverria, Dimas; Hassler, Matthew; Khvorova, Anastasia

    2017-12-01

    Therapeutic oligonucleotides, such as small interfering RNAs (siRNAs), hold great promise for the treatment of incurable genetically defined disorders by targeting cognate toxic gene products for degradation. To achieve meaningful tissue distribution and efficacy in vivo, siRNAs must be conjugated or formulated. Clear understanding of the pharmacokinetic (PK)/pharmacodynamic behavior of these compounds is necessary to optimize and characterize the performance of therapeutic oligonucleotides in vivo. In this study, we describe a simple and reproducible methodology for the evaluation of in vivo blood/plasma PK profiles and tissue distribution of oligonucleotides. The method is based on serial blood microsampling from the saphenous vein, coupled to peptide nucleic acid hybridization assay for quantification of guide strands. Performed with minimal number of animals, this method allowed unequivocal detection and sensitive quantification without the need for amplification, or further modification of the oligonucleotides. Using this methodology, we compared plasma clearances and tissue distribution profiles of two different hydrophobically modified siRNAs (hsiRNAs). Notably, cholesterol-hsiRNA presented slow plasma clearances and mainly accumulated in the liver, whereas, phosphocholine-docosahexaenoic acid-hsiRNA was rapidly cleared from the plasma and preferably accumulated in the kidney. These data suggest that the PK/biodistribution profiles of modified hsiRNAs are determined by the chemical nature of the conjugate. Importantly, the method described in this study constitutes a simple platform to conduct pilot assessments of the basic clearance and tissue distribution profiles, which can be broadly applied for evaluation of new chemical variants of siRNAs and micro-RNAs.

  20. Oligonucleotide assisted light-emitting Alq3 microrods: energy transfer effect with fluorescent dyes.

    Science.gov (United States)

    Cui, Chunzhi; Park, Dong Hyuk; Kim, Jeongyong; Joo, Jinsoo; Ahn, Dong June

    2013-06-14

    Oligonucleotide assisted tri(8-hydroxyquinoline) aluminium (Alq3) microrods were prepared for the first time. When hybridized with oligonucleotide labeled by Cy3 fluorescent dye, a significant photoluminescence variation of the Alq3 microrods was observed due to Förster resonance energy transfer, unlike when Cy5-oligonucleotide was used. Versatile nucleotide manipulation would open up wider applications of Alq3-based materials, based on this fundamental observation.

  1. The hormone response element mimic sequence of GAS5 lncRNA is sufficient to induce apoptosis in breast cancer cells

    Science.gov (United States)

    Pickard, Mark R.; Williams, Gwyn T.

    2016-01-01

    Growth arrest-specific 5 (GAS5) lncRNA promotes apoptosis, and its expression is down-regulated in breast cancer. GAS5 lncRNA is a decoy of glucocorticoid/related receptors; a stem-loop sequence constitutes the GAS5 hormone response element mimic (HREM), which is essential for the regulation of breast cancer cell apoptosis. This preclinical study aimed to determine if the GAS5 HREM sequence alone promotes the apoptosis of breast cancer cells. Nucleofection of hormone-sensitive and –insensitive breast cancer cell lines with a GAS5 HREM DNA oligonucleotide increased both basal and ultraviolet-C-induced apoptosis, and decreased culture viability and clonogenic growth, similar to GAS5 lncRNA. The HREM oligonucleotide demonstrated similar sequence specificity to the native HREM for its functional activity and had no effect on endogenous GAS5 lncRNA levels. Certain chemically modified HREM oligonucleotides, notably DNA and RNA phosphorothioates, retained pro-apoptotic. activity. Crucially the HREM oligonucleotide could overcome apoptosis resistance secondary to deficient endogenous GAS5 lncRNA levels. Thus, the GAS5 lncRNA HREM sequence alone is sufficient to induce apoptosis in breast cancer cells, including triple-negative breast cancer cells. These findings further suggest that emerging knowledge of structure/function relationships in the field of lncRNA biology can be exploited for the development of entirely novel, oligonucleotide mimic-based, cancer therapies. PMID:26862727

  2. Folding Topology of a Short Coiled-Coil Peptide Structure Templated by an Oligonucleotide Triplex

    DEFF Research Database (Denmark)

    Lou, Chenguang; Christensen, Niels Johan; Martos Maldonado, Manuel Cristo

    2017-01-01

    by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex......, small-angle X-ray scattering (SAXS), and molecular modeling. Stabilizing cooperativity was observed between the trimeric peptide and the oligonucleotide triplex domains, and the overall molecular size (ca. 12nm) in solution was revealed to be independent of concentration. The topological folding...

  3. Characterization of peptide-oligonucleotide heteroconjugates by mass spectrometry.

    OpenAIRE

    Jensen, O N; Kulkarni, S; Aldrich, J V; Barofsky, D F

    1996-01-01

    Two peptide-oligothymidylic acids, prepared by joining an 11 residue synthetic peptide containing one internal carboxyl group (Asp side chain) to amino-linker-5'pdT6 and amino-linker-5'pdT10 oligonucleotides, were analyzed by matrix-assisted laser desorption/ionization (MALDI) on a linear time-of-flight mass spectrometer and by electrospray ionization (ESI) on a triple-quadrupole system. These synthetic compounds model peptide-nucleic acid heteroconjugates encountered in antisense research an...

  4. PCR amplification on microarrays of gel immobilized oligonucleotides

    Science.gov (United States)

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  5. Effect of mutations in the A site of 16 S rRNA on aminoglycoside antibiotic-ribosome interaction

    DEFF Research Database (Denmark)

    Recht, M I; Douthwaite, S; Dahlquist, K D

    1999-01-01

    antibiotics, which also interact with this region of rRNA. Mutations of certain nucleotides in rRNA reduce aminoglycoside binding affinity, as previously demonstrated using a model RNA oligonucleotide system. Here, predictions from the oligonucleotide system were tested in the ribosome by mutation...... for the aminoglycoside paromomycin, whereas no discernible reduction in affinity was observed with 1406 mutant ribosomes. These data are consistent with prior NMR structural determination of aminoglycoside interaction with the decoding region, and further our understanding of how aminoglycoside resistance can...

  6. RNA interference for performance enhancement and detection in doping control.

    Science.gov (United States)

    Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

    2011-10-01

    RNA interference represents a comparably new route of regulating and manipulating specific gene expression. Promising results were obtained in experimental therapies aim at the treatment of different kinds of diseases including cancer, diabetes mellitus or Dychenne muscular dystrophy. While studies on down-regulation efficiency are often performed by analyzing the regulated protein, the direct detection of small, interfering RNA molecules and antisense oligonucleotides is of great interest for the investigation of the metabolism and degradation and also for the detection of a putative misuse of these molecules in sports. Myostatin down-regulation was shown to result in increased performance and muscle growth and the regulation of several other proteins could be relevant for performance enhancement. This mini-review summarizes current approaches for the mass spectrometric analysis of siRNA and antisense oligonucleotides from biological matrices and the available data on biodistribution, metabolism, and half-life of relevant substances are discussed. Copyright © 2011 John Wiley & Sons, Ltd.

  7. A simple and robust vector-based shRNA expression system used for RNA interference.

    Science.gov (United States)

    Wang, Xue-jun; Li, Ying; Huang, Hai; Zhang, Xiu-juan; Xie, Pei-wen; Hu, Wei; Li, Dan-dan; Wang, Sheng-qi

    2013-01-01

    RNA interference (RNAi) mediated by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs) has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV) knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

  8. A simple and robust vector-based shRNA expression system used for RNA interference.

    Directory of Open Access Journals (Sweden)

    Xue-jun Wang

    Full Text Available BACKGROUND: RNA interference (RNAi mediated by small interfering RNAs (siRNAs or short hairpin RNAs (shRNAs has become a powerful genetic tool for conducting functional studies. Previously, vector-based shRNA-expression strategies capable of inducing RNAi in viable cells have been developed, however, these vector systems have some disadvantages, either because they were error-prone or cost prohibitive. RESULTS: In this report we described the development of a simple, robust shRNA expression system utilizing 1 long oligonucleotide or 2 short oligonucleotides for half the cost of conventional shRNA construction methods and with a >95% cloning success rate. The shRNA loop sequence and stem structure were also compared and carefully selected for better RNAi efficiency. Furthermore, an easier strategy was developed based on isocaudomers which permit rapid combination of the most efficient promoter-shRNA cassettes. Finally, using this method, the conservative target sites for hepatitis B virus (HBV knockdown were systemically screened and HBV antigen expression shown to be successfully suppressed in the presence of connected multiple shRNAs both in vitro and in vivo. CONCLUSION: This novel design describes an inexpensive and effective way to clone and express single or multiple shRNAs from the same vector with the capacity for potent and effective silencing of target genes.

  9. PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice

    Science.gov (United States)

    Zinker, Bradley A.; Rondinone, Cristina M.; Trevillyan, James M.; Gum, Rebecca J.; Clampit, Jill E.; Waring, Jeffrey F.; Xie, Nancy; Wilcox, Denise; Jacobson, Peer; Frost, Leigh; Kroeger, Paul E.; Reilly, Regina M.; Koterski, Sandra; Opgenorth, Terry J.; Ulrich, Roger G.; Crosby, Seth; Butler, Madeline; Murray, Susan F.; McKay, Robert A.; Bhanot, Sanjay; Monia, Brett P.; Jirousek, Michael R.

    2002-01-01

    The role of protein-tyrosine phosphatase 1B (PTP1B) in diabetes was investigated using an antisense oligonucleotide in ob/ob and db/db mice. PTP1B antisense oligonucleotide treatment normalized plasma glucose levels, postprandial glucose excursion, and HbA1C. Hyperinsulinemia was also reduced with improved insulin sensitivity. PTP1B protein and mRNA were reduced in liver and fat with no effect in skeletal muscle. Insulin signaling proteins, insulin receptor substrate 2 and phosphatidylinositol 3 (PI3)-kinase regulatory subunit p50α, were increased and PI3-kinase p85α expression was decreased in liver and fat. These changes in protein expression correlated with increased insulin-stimulated protein kinase B phosphorylation. The expression of liver gluconeogenic enzymes, phosphoenolpyruvate carboxykinase, and fructose-1,6-bisphosphatase was also down-regulated. These findings suggest that PTP1B modulates insulin signaling in liver and fat, and that therapeutic modalities targeting PTP1B inhibition may have clinical benefit in type 2 diabetes. PMID:12169659

  10. In vivo biodistribution and pharmacokinetics of18F-labelled Spiegelmers: a new class of oligonucleotidic radiopharmaceuticals

    International Nuclear Information System (INIS)

    Boisgard, Raphael; Younes, Cheraz; Tavitian, Bertrand; Kuhnast, Bertrand; Hinnen, Francoise; Dolle, Frederic; Vonhoff, Stefan; Wlotzka, Britta; Klussmann, Sven; Verbavatz, Jean-Marc; Rousseau, Bernard; Fuerste, Jens Peter

    2005-01-01

    Single-stranded mirror-image oligonucleotides (Spiegelmers) are highly resistant to nuclease degradation and are capable of tightly and specifically binding to protein targets. Here we explored the potential of Spiegelmers as in vivo imaging probes for positron emission tomography (PET). We investigated the biodistribution and pharmacokinetics of [ 18 F]-l-DNA and [ 18 F]-l-RNA Spiegelmers by dynamic quantitative whole-body PET imaging after intravenous administration in non-human primates. Their metabolic profile was explored in primates and rats, and ex vivo autoradiography of [ 125 I]-l-RNA was performed in rat kidneys, the major organ for Spiegelmer uptake. Both [ 18 F]-l-DNA and [ 18 F]-l-RNA Spiegelmers were metabolically stable in plasma during 2 h after injection. No evidence of non-specific binding was found with either type of Spiegelmer in any tissue. The biodistribution and metabolic profiles of [ 18 F]-l-DNA and [ 18 F]-l-RNA Spiegelmers highlight their potential as radiotracers for in vivo imaging applications. (orig.)

  11. Facilitating RNA structure prediction with microarrays.

    Science.gov (United States)

    Kierzek, Elzbieta; Kierzek, Ryszard; Turner, Douglas H; Catrina, Irina E

    2006-01-17

    Determining RNA secondary structure is important for understanding structure-function relationships and identifying potential drug targets. This paper reports the use of microarrays with heptamer 2'-O-methyl oligoribonucleotides to probe the secondary structure of an RNA and thereby improve the prediction of that secondary structure. When experimental constraints from hybridization results are added to a free-energy minimization algorithm, the prediction of the secondary structure of Escherichia coli 5S rRNA improves from 27 to 92% of the known canonical base pairs. Optimization of buffer conditions for hybridization and application of 2'-O-methyl-2-thiouridine to enhance binding and improve discrimination between AU and GU pairs are also described. The results suggest that probing RNA with oligonucleotide microarrays can facilitate determination of secondary structure.

  12. The use of oligonucleotide probes for meningococcal serotype characterization

    Directory of Open Access Journals (Sweden)

    SACCHI Claudio Tavares

    1998-01-01

    Full Text Available In the present study we examine the potential use of oligonucleotide probes to characterize Neisseria meningitidis serotypes without the use of monoclonal antibodies (MAbs. Antigenic diversity on PorB protein forms the bases of serotyping method. However, the current panel of MAbs underestimated, by at least 50% the PorB variability, presumably because reagents for several PorB variable regions (VRs are lacking, or because a number of VR variants are not recognized by serotype-defining MAbs12. We analyzed the use of oligonucleotide probes to characterize serotype 10 and serotype 19 of N. meningitidis. The porB gene sequence for the prototype strain of serotype 10 was determined, aligned with 7 other porB sequences from different serotypes, and analysis of individual VRs were performed. The results of DNA probes 21U (VR1-A and 615U (VR3-B used against 72 N. meningitidis strains confirm that VR1 type A and VR3 type B encode epitopes for serotype-defined MAbs 19 and 10, respectively. The use of probes for characterizing serotypes possible can type 100% of the PorB VR diversity. It is a simple and rapid method specially useful for analysis of large number of samples.

  13. Differentiation of the seven major lyssavirus species by oligonucleotide microarray.

    Science.gov (United States)

    Xi, Jin; Guo, Huancheng; Feng, Ye; Xu, Yunbin; Shao, Mingfu; Su, Nan; Wan, Jiayu; Li, Jiping; Tu, Changchun

    2012-03-01

    An oligonucleotide microarray, LyssaChip, has been developed and verified as a highly specific diagnostic tool for differentiation of the 7 major lyssavirus species. As with conventional typing microarray methods, the LyssaChip relies on sequence differences in the 371-nucleotide region coding for the nucleoprotein. This region was amplified using nested reverse transcription-PCR primers that bind to the 7 major lyssaviruses. The LyssaChip includes 57 pairs of species typing and corresponding control oligonucleotide probes (oligoprobes) immobilized on glass slides, and it can analyze 12 samples on a single slide within 8 h. Analysis of 111 clinical brain specimens (65 from animals with suspected rabies submitted to the laboratory and 46 of butchered dog brain tissues collected from restaurants) showed that the chip method was 100% sensitive and highly consistent with the "gold standard," a fluorescent antibody test (FAT). The chip method could detect rabies virus in highly decayed brain tissues, whereas the FAT did not, and therefore the chip test may be more applicable to highly decayed brain tissues than the FAT. LyssaChip may provide a convenient and inexpensive alternative for diagnosis and differentiation of rabies and rabies-related diseases.

  14. Synthesis of 3'-, or 5'-, or internal methacrylamido-modified oligonucleotides

    Science.gov (United States)

    Golova, Julia B.; Chernov, Boris K.

    2010-04-27

    New modifiers were synthesized for incorporation of a methacrylic function in 3'-, 5'- and internal positions of oligonucleotides during solid phase synthesis. A modifier was used for synthesis of 5'-methacrylated oligonucleotides for preparation of microarrays by a co-polymerization method.

  15. [Inheritable phenotypic normalization of rodent cells transformed by simian adenovirus SA7 E1 oncogenes by singled-stranded oligonucleotides complementary to a long region of integrated oncogenes].

    Science.gov (United States)

    Grineva, N I; Borovkova, T V; Sats, N V; Kurabekova, R M; Rozhitskaia, O S; Solov'ev, G Ia; Pantin, V I

    1995-08-01

    G11 mouse cells and SH2 rat cells transformed with simian adenovirus SA7 DNA showed inheritable oncogen-specific phenotypic normalization when treated with sense and antisense oligonucleotides complementary to long RNA sequences, plus or minus strands of the integrated adenovirus oncogenes E1A and E1B. Transitory treatment of the cells with the oligonucleotides in the absence of serum was shown to cause the appearance of normalized cell lines with fibroblastlike morphology, slower cell proliferation, and lack of ability to form colonies in soft agar. Proliferative activity and adhesion of the normalized cells that established cell lines were found to depend on the concentration of growth factors in the cultural medium. In some of the cell lines, an inhibition of transcription of the E1 oncogenes was observed. The normalization also produced cells that divided 2 - 5 times and died and cells that reverted to a transformed phenotype in 2 - 10 days. The latter appeared predominantly upon the action of the antisense oligonucleotides.

  16. Nano and Microtechnologies for the Delivery of Oligonucleotides with Gene Silencing Properties

    Directory of Open Access Journals (Sweden)

    Giuseppe De Rosa

    2009-07-01

    Full Text Available Oligonucleotides (ONs are synthetic fragments of nucleic acid designed to modulate the expression of target proteins. DNA-based ONs (antisense, antigene, aptamer or decoy and more recently a new class of RNA-based ONs, the small interfering RNAs (siRNAs, have gained great attention for the treatment of different disease states, such as viral infections, inflammation, diabetes, and cancer. However, the development of therapeutic strategies based on ONs is hampered by their low bioavailability, poor intracellular uptake and rapid degradation in biological fluids. The use of a non-viral carrier can be a powerful tool to overcome these drawbacks. Lipid or polymer-based nanotechnologies can improve biological stability and cellular uptake of ONs, with possibility of tissue and/or cellular targeting. The use of polymeric devices can also produce a prolonged release of the ON, thus reducing the need of frequent administrations. This review summarizes advantages and issues related to the main non-viral vectors used for ON delivery.

  17. Inhaled ENaC antisense oligonucleotide ameliorates cystic fibrosis-like lung disease in mice.

    Science.gov (United States)

    Crosby, Jeff R; Zhao, Chenguang; Jiang, Chong; Bai, Dong; Katz, Melanie; Greenlee, Sarah; Kawabe, Hiroshi; McCaleb, Michael; Rotin, Daniela; Guo, Shuling; Monia, Brett P

    2017-11-01

    Epithelial sodium channel (ENaC, Scnn1) hyperactivity in the lung leads to airway surface dehydration and mucus accumulation in cystic fibrosis (CF) patients and in mice with CF-like lung disease. We identified several potent ENaC specific antisense oligonucleotides (ASOs) and tested them by inhalation in mouse models of CF-like lung disease. The inhaled ASOs distributed into lung airway epithelial cells and decreased ENaC expression by inducing RNase H1-dependent degradation of the targeted Scnn1a mRNA. Aerosol delivered ENaC ASO down-regulated mucus marker expression and ameliorated goblet cell metaplasia, inflammation, and airway hyper-responsiveness. Lack of systemic activity of ASOs delivered via the aerosol route ensures the safety of this approach. Our results demonstrate that antisense inhibition of ENaC in airway epithelial cells could be an effective and safe approach for the prevention and reversal of lung symptoms in CF and potentially other inflammatory diseases of the lung. Copyright © 2017 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.

  18. Antisense Oligonucleotide-mediated Exon Skipping as a Systemic Therapeutic Approach for Recessive Dystrophic Epidermolysis Bullosa.

    Science.gov (United States)

    Bremer, Jeroen; Bornert, Olivier; Nyström, Alexander; Gostynski, Antoni; Jonkman, Marcel F; Aartsma-Rus, Annemieke; van den Akker, Peter C; Pasmooij, Anna Mg

    2016-10-18

    The "generalized severe" form of recessive dystrophic epidermolysis bullosa (RDEB-gen sev) is caused by bi-allelic null mutations in COL7A1, encoding type VII collagen. The absence of type VII collagen leads to blistering of the skin and mucous membranes upon the slightest trauma. Because most patients carry exonic point mutations or small insertions/deletions, most exons of COL7A1 are in-frame, and low levels of type VII collagen already drastically improve the disease phenotype, this gene seems a perfect candidate for antisense oligonucleotide (AON)-mediated exon skipping. In this study, we examined the feasibility of AON-mediated exon skipping in vitro in primary cultured keratinocytes and fibroblasts, and systemically in vivo using a human skin-graft mouse model. We show that treatment with AONs designed against exon 105 leads to in-frame exon 105 skipping at the RNA level and restores type VII collagen protein production in vitro. Moreover, we demonstrate that systemic delivery in vivo induces de novo expression of type VII collagen in skin grafts generated from patient cells. Our data demonstrate strong proof-of-concept for AON-mediated exon skipping as a systemic therapeutic strategy for RDEB.

  19. Nucleobase-modified antisense oligonucleotides containing 5-(phenyltriazol)-2′-deoxyuridine nucleotides induce exon-skipping

    DEFF Research Database (Denmark)

    Le, Bao T.; Hornum, Mick; Sharma, Pawan K.

    2017-01-01

    Chemically-modified antisense oligonucleotide-mediated exon-skipping has been validated as a therapeutic strategy for tackling several disease pathologies, particularly duchenne muscular dystrophy. To date, only sugar-modified and internucleotide linkage-modified oligonucleotide chemistries have...

  20. Fluoroscence in situ hybridization of chicken intestinal samples with bacterial rRNA targeted oligonucleotide probes

    DEFF Research Database (Denmark)

    Olsen, Katja Nyholm; Francesch, M.; Christensen, Henrik

    2006-01-01

    were hybridized for 24-72h, centrifuged, washed with pre-heated hybridization buffer, centrifuged and resuspended in Millipore quality water before filtration onto a 0.22 µm black polycarbonate filter. The probes used in this study were, LGC354A, LGC354B, LGC354C, Strc493, Bacto1080, Sal3, Chis150, EUB...

  1. Inhibition of microRNA function by antimiR oligonucleotides

    DEFF Research Database (Denmark)

    Stenvang, Jan; Petri, Andreas; Lindow, Morten

    2012-01-01

    MicroRNAs (miRNAs) have emerged as important post-transcriptional regulators of gene expression in many developmental and cellular processes. Moreover, there is now ample evidence that perturbations in the levels of individual or entire families of miRNAs are strongly associated...

  2. [Oligonucleotide derivatives in the nucleic acid hybridization analysis. I. Covalent immobilization of oligonucleotide probes onto the nylon].

    Science.gov (United States)

    Dmitrienko, E V; Pyshnaia, I A; Pyshnyĭ, D V

    2010-01-01

    The features of UV-induced immobilization of oligonucleotides on a nylon membranes and the effectiveness of enzymatic labeling of immobilized probes at heterophase detection of nucleic acids are studied. Short terminal oligothymidilate (up to 10 nt) sequences are suggested to attach to the probe via a flexible ethylene glycol based linker. The presence of such fragment enhances the intensity of immobilization and reduces UV-dependent degradation of the targeted (sequence-specific) part of the probe by reducing the dose needed for the immobilization of DNA. The optimum dose of UV-irradiation is determined to be ~0.4 J/cm(2) at the wavelength 254 nm. This dose provides high level of hybridization signal for immobilized probes with various nucleotide composition of the sequence specific moiety. The amide groups of the polyamide are shown to play the key role in the photoinduced immobilization of nucleic acids, whereas the primary amino groups in the structure of PA is not the center responsible for the covalent binding of DNA by UV-irradiation, as previously believed. Various additives in the soaking solution during the membrane of UV-dependent immobilization of probes are shown to influence its effectiveness. The use of alternative to UV-irradiation system of radical generation are shown to provide the immobilization of oligonucleotides onto the nylon membrane.

  3. Design of oligonucleotides for microarrays and perspectives for design of multi-transcriptome arrays

    DEFF Research Database (Denmark)

    Nielsen, Henrik Bjørn; Wernersson, Rasmus; Knudsen, Steen

    2003-01-01

    with an overview of these parameters. We present here a flexible tool named OligoWiz for designing oligonucleotides for multiple purposes. OligoWiz presents a set of parameter scores in a graphical interface to facilitate an overview for the user. Additional custom parameter scores can easily be added......Optimal design of oligonucleotides for microarrays involves tedious and laborious work evaluating potential oligonucleotides relative to a series of parameters. The currently available tools for this purpose are limited in their flexibility and do not present the oligonucleotide designer...... to the program to extend the default parameters: homology, DeltaTm, low-complexity, position and GATC-only. Furthermore we present an analysis of the limitations in designing oligonucleotide sets that can detect transcripts from multiple organisms. OligoWiz is available at www.cbs.dtu.dk/services/OligoWiz/....

  4. Personalized RNA Medicine for Pancreatic Cancer.

    Science.gov (United States)

    Gilles, Maud-Emmanuelle; Hao, Liangliang; Huang, Ling; Rupaimoole, Rajesha; Lopez-Casas, Pedro P; Pulver, Emilia; Jeong, Jong Cheol; Muthuswamy, Senthil K; Hidalgo, Manuel; Bhatia, Sangeeta N; Slack, Frank J

    2018-04-01

    Purpose: Since drug responses vary between patients, it is crucial to develop pre-clinical or co-clinical strategies that forecast patient response. In this study, we tested whether RNA-based therapeutics were suitable for personalized medicine by using patient-derived-organoid (PDO) and patient-derived-xenograft (PDX) models. Experimental Design: We performed microRNA (miRNA) profiling of PDX samples to determine the status of miRNA deregulation in individual pancreatic ductal adenocarcinoma (PDAC) patients. To deliver personalized RNA-based-therapy targeting oncogenic miRNAs that form part of this common PDAC miRNA over-expression signature, we packaged antimiR oligonucleotides against one of these miRNAs in tumor-penetrating nanocomplexes (TPN) targeting cell surface proteins on PDAC tumors. Results: As a validation for our pre-clinical strategy, the therapeutic potential of one of our nano-drugs, TPN-21, was first shown to decrease tumor cell growth and survival in PDO avatars for individual patients, then in their PDX avatars. Conclusions: This general approach appears suitable for co-clinical validation of personalized RNA medicine and paves the way to prospectively identify patients with eligible miRNA profiles for personalized RNA-based therapy. Clin Cancer Res; 24(7); 1734-47. ©2018 AACR . ©2018 American Association for Cancer Research.

  5. RNA Crystallization

    Science.gov (United States)

    Golden, Barbara L.; Kundrot, Craig E.

    2003-01-01

    RNA molecules may be crystallized using variations of the methods developed for protein crystallography. As the technology has become available to syntheisize and purify RNA molecules in the quantities and with the quality that is required for crystallography, the field of RNA structure has exploded. The first consideration when crystallizing an RNA is the sequence, which may be varied in a rational way to enhance crystallizability or prevent formation of alternate structures. Once a sequence has been designed, the RNA may be synthesized chemically by solid-state synthesis, or it may be produced enzymatically using RNA polymerase and an appropriate DNA template. Purification of milligram quantities of RNA can be accomplished by HPLC or gel electrophoresis. As with proteins, crystallization of RNA is usually accomplished by vapor diffusion techniques. There are several considerations that are either unique to RNA crystallization or more important for RNA crystallization. Techniques for design, synthesis, purification, and crystallization of RNAs will be reviewed here.

  6. Modulation of splicing of the preceding intron by antisense oligonucleotide complementary to intra-exon sequence deleted in dystrophin Kobe

    Energy Technology Data Exchange (ETDEWEB)

    Takeshima, Y.; Matuso, M.; Sakamoto, H.; Nishio, H. [Kobe Univ. School of Medicine and Science (Japan)

    1994-09-01

    Molecular analysis of dystrophin Kobe showed that exon 19 of the dystrophin gene bearing a 52 bp deletion was skipped during splicing, although the known consensus sequences at the 5{prime} and 3{prime} splice site of exon 19 were maintained. These data suggest that the deleted sequence of exon 19 may function as a cis-acting factor for exact splicing for the upstream intron. To investigate this potential role, an in vitro splicing system using dystrophin precursors was established. A two-exon precursor containing exon 18, truncated intron 18, and exon 19 was accurately spliced. However, splicing of intron 18 was dramatically inhibited when wild exon 19 was replaced with mutated exon 19. Even though the length of exon 19 was restored to normal by replacing the deleted sequence with other sequence, splicing of intron 18 was not fully reactivated. Characteristically, splicing of intron 18 was inactivated more markedly when the replaced sequence contained less polypurine stretches. These data suggested that modification of the exon sequence would result in a splicing abnormality. Antisense 31 mer 2`-O-methyl ribonucleotide was targeted against 5{prime} end of deleted region of exon 19 to modulate splicing of the mRNA precursor. Splicing of intron 18 was inhibited in a dose- and time-dependent manner. This is the first in vitro evidence to show splicing of dystrophin pre-mRNA can be managed by antisense oligonucleotides. These experiments represent an approach in which antisense oligonucleotides are used to restore the function of a defective dystrophin gene in Duchenne muscular dystrophy by inducing skipping of certain exons during splicing.

  7. Oral tongue cancer gene expression profiling: Identification of novel potential prognosticators by oligonucleotide microarray analysis

    International Nuclear Information System (INIS)

    Estilo, Cherry L; Boyle, Jay O; Kraus, Dennis H; Patel, Snehal; Shaha, Ashok R; Wong, Richard J; Huryn, Joseph M; Shah, Jatin P; Singh, Bhuvanesh; O-charoenrat, Pornchai; Talbot, Simon; Socci, Nicholas D; Carlson, Diane L; Ghossein, Ronald; Williams, Tijaana; Yonekawa, Yoshihiro; Ramanathan, Yegnanarayana

    2009-01-01

    The present study is aimed at identifying potential candidate genes as prognostic markers in human oral tongue squamous cell carcinoma (SCC) by large scale gene expression profiling. The gene expression profile of patients (n=37) with oral tongue SCC were analyzed using Affymetrix HG-U95Av2 high-density oligonucleotide arrays. Patients (n=20) from which there were available tumor and matched normal mucosa were grouped into stage (early vs. late) and nodal disease (node positive vs. node negative) subgroups and genes differentially expressed in tumor vs. normal and between the subgroups were identified. Three genes, GLUT3, HSAL2, and PACE4, were selected for their potential biological significance in a larger cohort of 49 patients via quantitative real-time RT-PCR. Hierarchical clustering analyses failed to show significant segregation of patients. In patients (n=20) with available tumor and matched normal mucosa, 77 genes were found to be differentially expressed (P< 0.05) in the tongue tumor samples compared to their matched normal controls. Among the 45 over-expressed genes, MMP-1 encoding interstitial collagenase showed the highest level of increase (average: 34.18 folds). Using the criterion of two-fold or greater as overexpression, 30.6%, 24.5% and 26.5% of patients showed high levels of GLUT3, HSAL2 and PACE4, respectively. Univariate analyses demonstrated that GLUT3 over-expression correlated with depth of invasion (P<0.0001), tumor size (P=0.024), pathological stage (P=0.009) and recurrence (P=0.038). HSAL2 was positively associated with depth of invasion (P=0.015) and advanced T stage (P=0.047). In survival studies, only GLUT3 showed a prognostic value with disease-free (P=0.049), relapse-free (P=0.002) and overall survival (P=0.003). PACE4 mRNA expression failed to show correlation with any of the relevant parameters. The characterization of genes identified to be significant predictors of prognosis by oligonucleotide microarray and further validation by

  8. Electrical manipulation of oligonucleotides grafted to charged surfaces.

    Science.gov (United States)

    Rant, Ulrich; Arinaga, Kenji; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard; Tornow, Marc

    2006-09-21

    The electrical manipulation of short DNA molecules on surfaces offers novel functionalities with fascinating possibilities in the field of bio-interfaces. Here we present systematic investigations of the electrical interactions which govern the structure of oligonucleotides on charged gold surfaces. Successively, we address influences of the applied field strength, the role of DC electrode potentials, in particular for polycrystalline surfaces, as well as screening effects of the surrounding electrolyte solution. Data obtained for single and double stranded DNA exhibit differences which can be attributed to the dissimilar flexibility of the different molecular conformations. A comparison of the experimental results with a basic model shows how the alignment of the molecules adjusts according to a balance between electrically induced ordering and stochastic thermal motions. The presented conclusions are expected to be of general relevance for the behaviour of polyelectrolytes exposed to localized electric fields at interfaces.

  9. Direct microcontact printing of oligonucleotides for biochip applications

    Directory of Open Access Journals (Sweden)

    Trévisiol E

    2005-07-01

    Full Text Available Abstract Background A critical step in the fabrication of biochips is the controlled placement of probes molecules on solid surfaces. This is currently performed by sequential deposition of probes on a target surface with split or solid pins. In this article, we present a cost-effective procedure namely microcontact printing using stamps, for a parallel deposition of probes applicable for manufacturing biochips. Results Contrary to a previous work, we showed that the stamps tailored with an elastomeric poly(dimethylsiloxane material did not require any surface modification to be able to adsorb oligonucleotides or PCR products. The adsorbed DNA molecules are subsequently printed efficiently on a target surface with high sub-micron resolution. Secondly, we showed that successive stamping is characterized by an exponential decay of the amount of transferred DNA molecules to the surface up the 4th print, then followed by a second regime of transfer that was dependent on the contact time and which resulted in reduced quality of the features. Thus, while consecutive stamping was possible, this procedure turned out to be less reproducible and more time consuming than simply re-inking the stamps between each print. Thirdly, we showed that the hybridization signals on arrays made by microcontact printing were 5 to 10-times higher than those made by conventional spotting methods. Finally, we demonstrated the validity of this microcontact printing method in manufacturing oligonucleotides arrays for mutations recognition in a yeast gene. Conclusion The microcontact printing can be considered as a new potential technology platform to pattern DNA microarrays that may have significant advantages over the conventional spotting technologies as it is easy to implement, it uses low cost material to make the stamp, and the arrays made by this technology are 10-times more sensitive in term of hybridization signals than those manufactured by conventional spotting

  10. Dissecting the hybridization of oligonucleotides to structured complementary sequences.

    Science.gov (United States)

    Peracchi, Alessio

    2016-06-01

    When oligonucleotides hybridize to long target molecules, the process is slowed by the secondary structure in the targets. The phenomenon has been analyzed in several previous studies, but many details remain poorly understood. I used a spectrofluorometric strategy, focusing on the formation/breaking of individual base pairs, to study the kinetics of association between a DNA hairpin and >20 complementary oligonucleotides ('antisenses'). Hybridization rates differed by over three orders of magnitude. Association was toehold-mediated, both for antisenses binding to the target's ends and for those designed to interact with the loop. Binding of these latter, besides being consistently slower, was affected to variable, non-uniform extents by the asymmetric loop structure. Divalent metal ions accelerated hybridization, more pronouncedly when nucleation occurred at the loop. Incorporation of locked nucleic acid (LNA) residues in the antisenses substantially improved the kinetics only when LNAs participated to the earliest hybridization steps. The effects of individual LNAs placed along the antisense indicated that the reaction transition state occurred after invading at least the first base pair of the stem. The experimental approach helps dissect hybridization reactions involving structured nucleic acids. Toehold-dependent, nucleation-invasion models appear fully appropriate for describing such reactions. Estimating the stability of nucleation complexes formed at internal toeholds is the major hurdle for the quantitative prediction of hybridization rates. While analyzing the mechanisms of a fundamental biochemical process (hybridization), this work also provides suggestions for the improvement of technologies that rely on such process. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Combining gene expression data from different generations of oligonucleotide arrays

    Directory of Open Access Journals (Sweden)

    Kong Sek

    2004-10-01

    Full Text Available Abstract Background One of the important challenges in microarray analysis is to take full advantage of previously accumulated data, both from one's own laboratory and from public repositories. Through a comparative analysis on a variety of datasets, a more comprehensive view of the underlying mechanism or structure can be obtained. However, as we discover in this work, continual changes in genomic sequence annotations and probe design criteria make it difficult to compare gene expression data even from different generations of the same microarray platform. Results We first describe the extent of discordance between the results derived from two generations of Affymetrix oligonucleotide arrays, as revealed in cluster analysis and in identification of differentially expressed genes. We then propose a method for increasing comparability. The dataset we use consists of a set of 14 human muscle biopsy samples from patients with inflammatory myopathies that were hybridized on both HG-U95Av2 and HG-U133A human arrays. We find that the use of the probe set matching table for comparative analysis provided by Affymetrix produces better results than matching by UniGene or LocusLink identifiers but still remains inadequate. Rescaling of expression values for each gene across samples and data filtering by expression values enhance comparability but only for few specific analyses. As a generic method for improving comparability, we select a subset of probes with overlapping sequence segments in the two array types and recalculate expression values based only on the selected probes. We show that this filtering of probes significantly improves the comparability while retaining a sufficient number of probe sets for further analysis. Conclusions Compatibility between high-density oligonucleotide arrays is significantly affected by probe-level sequence information. With a careful filtering of the probes based on their sequence overlaps, data from different

  12. RNA Origami

    DEFF Research Database (Denmark)

    Sparvath, Steffen Lynge

    introducerede vores gruppe den enkeltstrengede RNA-origami metode, der giver mulighed for cotranscriptional foldning af veldefinerede nanostrukturer, og er en central del af arbejdet præsenteret heri. Denne ph.d.-afhandling udforsker potentielle anvendelser af RNA-origami nanostrukturer, som nanomedicin eller...... biosensorer. Afhandlingen består af en introduktion til RNA-nanoteknologi feltet, en introduktion af enkeltstrenget RNA-origami design, og fire studier, der beskriver design, produktion og karakterisering af både strukturelle og funktionelle RNA-origamier. Flere RNA-origami designs er blevet undersøgt, og...... projekterne, der indgår i denne afhandling, inkluderer de nyeste fremskridt indenfor strukturel RNA-nanoteknologi og udvikling af funktionelle RNA-baserede enheder. Det første studie beskriver konstruktion og karakterisering af en enkeltstrenget 6-helix RNA-origami stuktur, som er den første demonstration af...

  13. Thermal Stability of siRNA Modulates Aptamer- conjugated siRNA Inhibition

    Directory of Open Access Journals (Sweden)

    Alexey Berezhnoy

    2012-01-01

    Full Text Available Oligonucleotide aptamer-mediated in vivo cell targeting of small interfering RNAs (siRNAs is emerging as a useful approach to enhance the efficacy and reduce the adverse effects resulting from siRNA-mediated genetic interference. A current main impediment in aptamer-mediated siRNA targeting is that the activity of the siRNA is often compromised when conjugated to an aptamer, often requiring labor intensive and time consuming design and testing of multiple configurations to identify a conjugate in which the siRNA activity has not been significantly reduced. Here, we show that the thermal stability of the siRNA is an important parameter of siRNA activity in its conjugated form, and that siRNAs with lower melting temperature (Tm are not or are minimally affected when conjugated to the 3′ end of 2′F-pyrimidine-modified aptamers. In addition, the configuration of the aptamer-siRNA conjugate retains activity comparable with the free siRNA duplex when the passenger strand is co-transcribed with the aptamer and 3′ overhangs on the passenger strand are removed. The approach described in this paper significantly reduces the time and effort necessary to screening siRNA sequences that retain biological activity upon aptamer conjugation, facilitating the process of identifying candidate aptamer-siRNA conjugates suitable for in vivo testing.

  14. Evaluation of 2’-Deoxy-2’-fluoro Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Silvana M G Jirka

    2015-01-01

    Full Text Available Duchenne muscular dystrophy (DMD is a severe muscle wasting disorder typically caused by frame-shifting mutations in the DMD gene. Restoration of the reading frame would allow the production of a shorter but partly functional dystrophin protein as seen in Becker muscular dystrophy patients. This can be achieved with antisense oligonucleotides (AONs that induce skipping of specific exons during pre-mRNA splicing. Different chemical modifications have been developed to improve AON properties. The 2’-deoxy-2’-fluoro (2F RNA modification is attractive for exon skipping due to its ability to recruit ILF2/3 proteins to the 2F/pre-mRNA duplex, which resulted in enhanced exon skipping in spinal muscular atrophy models. In this study, we examined the effect of two different 2’-substituted AONs (2’-F phosphorothioate (2FPS and 2’-O-Me phosphorothioate (2OMePS on exon skipping in DMD cell and animal models. In human cell cultures, 2FPS AONs showed higher exon skipping levels than their isosequential 2OMePS counterparts. Interestingly, in the mdx mouse model, 2FPS was less efficient than 2OMePS and suggested safety issues as evidenced by increased spleen size and weight loss. Our results do not support a clinical application for 2FPS AON.

  15. Disruption of Specific RNA-RNA Interactions in a Double-Stranded RNA Virus Inhibits Genome Packaging and Virus Infectivity.

    Science.gov (United States)

    Fajardo, Teodoro; Sung, Po-Yu; Roy, Polly

    2015-12-01

    Bluetongue virus (BTV) causes hemorrhagic disease in economically important livestock. The BTV genome is organized into ten discrete double-stranded RNA molecules (S1-S10) which have been suggested to follow a sequential packaging pathway from smallest to largest segment during virus capsid assembly. To substantiate and extend these studies, we have investigated the RNA sorting and packaging mechanisms with a new experimental approach using inhibitory oligonucleotides. Putative packaging signals present in the 3'untranslated regions of BTV segments were targeted by a number of nuclease resistant oligoribonucleotides (ORNs) and their effects on virus replication in cell culture were assessed. ORNs complementary to the 3' UTR of BTV RNAs significantly inhibited virus replication without affecting protein synthesis. Same ORNs were found to inhibit complex formation when added to a novel RNA-RNA interaction assay which measured the formation of supramolecular complexes between and among different RNA segments. ORNs targeting the 3'UTR of BTV segment 10, the smallest RNA segment, were shown to be the most potent and deletions or substitution mutations of the targeted sequences diminished the RNA complexes and abolished the recovery of viable viruses using reverse genetics. Cell-free capsid assembly/RNA packaging assay also confirmed that the inhibitory ORNs could interfere with RNA packaging and further substitution mutations within the putative RNA packaging sequence have identified the recognition sequence concerned. Exchange of 3'UTR between segments have further demonstrated that RNA recognition was segment specific, most likely acting as part of the secondary structure of the entire genomic segment. Our data confirm that genome packaging in this segmented dsRNA virus occurs via the formation of supramolecular complexes formed by the interaction of specific sequences located in the 3' UTRs. Additionally, the inhibition of packaging in-trans with inhibitory ORNs

  16. Synthetic Method for Oligonucleotide Block by Using Alkyl-Chain-Soluble Support.

    Science.gov (United States)

    Matsuno, Yuki; Shoji, Takao; Kim, Shokaku; Chiba, Kazuhiro

    2016-02-19

    A straightforward method for the synthesis of oligonucleotide blocks using a Cbz-type alkyl-chain-soluble support (Z-ACSS) attached to the 3'-OH group of 3'-terminal nucleosides was developed. The Z-ACSS allowed for the preparation of fully protected deoxyribo- and ribo-oligonucleotides without chromatographic purification and released dimer- to tetramer-size oligonucleotide blocks via hydrogenation using a Pd/C catalyst without significant loss or migration of protective groups such as 5'-end 4,4'-dimethoxtrityl, 2-cyanoethyl on internucleotide bonds, or 2'-TBS.

  17. Solid-phase synthesis of 2{sup '}-O-methoxyethyl oligonucleotides using dimeric phosphoramidate blocks

    Energy Technology Data Exchange (ETDEWEB)

    Yu, Gi Weon; Kang, Yong Han [Dept. of Applied Chemistry, Hanyang University, Ansan (Korea, Republic of)

    2016-11-15

    This research focused on the method of using dimeric phosphoramidite blocks to synthesize oligonucleotides for development as oligonucleotide drugs. A 16-mer oligonucleotide with the randomly selected sequence of C*C*T*C*G*C *T*C*T*C*G*C*C* C*G*C was synthesized using CC, GC, and TC dimers, a combination of monomers and dimers, or only monomers as building blocks. Using dimer blocks in this synthetic method provided a significant decrease in critical impurities that had similar properties to the main product, which was confirmed by LC-MS and HPLC analysis.

  18. Dielectric properties of DNA oligonucleotides on the surface of silicon nanostructures

    Energy Technology Data Exchange (ETDEWEB)

    Bagraev, N. T., E-mail: bagraev@mail.ioffe.ru [St. Petersburg Polytechnic University (Russian Federation); Chernev, A. L. [Russian Academy of Sciences, St. Petersburg Academic University—Nanotechnology Research and Education Center (Russian Federation); Klyachkin, L. E. [St. Petersburg Polytechnic University (Russian Federation); Malyarenko, A. M. [Russian Academy of Sciences, Ioffe Physical–Technical Institute (Russian Federation); Emel’yanov, A. K.; Dubina, M. V. [Russian Academy of Sciences, St. Petersburg Academic University—Nanotechnology Research and Education Center (Russian Federation)

    2016-10-15

    Planar silicon nanostructures that are formed as a very narrow silicon quantum well confined by δ barriers heavily doped with boron are used to study the dielectric properties of DNA oligonucleotides deposited onto the surface of the nanostructures. The capacitance characteristics of the silicon nanostructures with oligonucleotides deposited onto their surface are determined by recording the local tunneling current–voltage characteristics by means of scanning tunneling microscopy. The results show the possibility of identifying the local dielectric properties of DNA oligonucleotide segments consisting of repeating G–C pairs. These properties apparently give grounds to correlate the segments with polymer molecules exhibiting the properties of multiferroics.

  19. Differential distribution of calcineurin Aα isoenzyme mRNA's in rat brain

    NARCIS (Netherlands)

    Buttini, M.; Limonta, S.; Luyten, M.; Boddeke, H.

    1993-01-01

    Specific antisense oligonucleotide probes for the α isoforms of the catalytic subunit (A-subunit) of calcineurin were prepared and the distribution of Aα1 and Aα2 mRNA's has been studied in rat brain using in situ hybridization histochemistry. Clear regional differences have been observed for the

  20. Synthesis and methylation of ribosomal RNA in HeLa cells infected with the herpes virus pseudorabies virus

    International Nuclear Information System (INIS)

    Furlong, J.C.; Kyriakidis, S.; Stevely, W.S.

    1982-01-01

    The effects of infection with the herpes virus pseudorabies virus on the metabolism of HeLa cell ribosomal RNA were examined. There is a decline both in the synthesis of nucleolar 45S ribosomal precursor RNA and in its processing to mature cytoplasmic RNA. The methylated oligonucleotides in the ribosomal RNA species were studied. The methylation of cytoplasmic ribosomal RNA was essentially unchanged. However there was some undermethylation of the nucleolar precursor. If undermethylated RNA does not mature then this may partly explain the reduced processing in the infected cells. (Author)

  1. Covalent Strategies for Targeting Messenger and Non-Coding RNAs: An Updated Review on siRNA, miRNA and antimiR Conjugates

    Directory of Open Access Journals (Sweden)

    Santiago Grijalvo

    2018-02-01

    Full Text Available Oligonucleotide-based therapy has become an alternative to classical approaches in the search of novel therapeutics involving gene-related diseases. Several mechanisms have been described in which demonstrate the pivotal role of oligonucleotide for modulating gene expression. Antisense oligonucleotides (ASOs and more recently siRNAs and miRNAs have made important contributions either in reducing aberrant protein levels by sequence-specific targeting messenger RNAs (mRNAs or restoring the anomalous levels of non-coding RNAs (ncRNAs that are involved in a good number of diseases including cancer. In addition to formulation approaches which have contributed to accelerate the presence of ASOs, siRNAs and miRNAs in clinical trials; the covalent linkage between non-viral vectors and nucleic acids has also added value and opened new perspectives to the development of promising nucleic acid-based therapeutics. This review article is mainly focused on the strategies carried out for covalently modifying siRNA and miRNA molecules. Examples involving cell-penetrating peptides (CPPs, carbohydrates, polymers, lipids and aptamers are discussed for the synthesis of siRNA conjugates whereas in the case of miRNA-based drugs, this review article makes special emphasis in using antagomiRs, locked nucleic acids (LNAs, peptide nucleic acids (PNAs as well as nanoparticles. The biomedical applications of siRNA and miRNA conjugates are also discussed.

  2. Sequence relationships between the genome and the intracellular RNA species 1,3,6 and 7 of mouse hepatitis virus strain A59

    NARCIS (Netherlands)

    Horzinek, M.C.; Spaan, W.J.M.; Rottier, P.J.M.; Zeijst, B.A.M. van der

    1982-01-01

    We have shown by T1 oligonucleotide fingerprinting that the genome of mouse hepatitis virus strain A59 and its intracellular RNA 1 have identical fingerprints and that RNA 1 and the subgenomic RNAs 3, 6, and 7 contain common sequences. To localize the homologous region between the RNAs, we compared

  3. Localization of insulin receptor mRNA in rat brain by in situ hybridization

    International Nuclear Information System (INIS)

    Marks, J.L.; Porte, D. Jr.; Stahl, W.L.; Baskin, D.G.

    1990-01-01

    Insulin receptor mRNA was demonstrated in rat brain slices by in situ hybridization with three 35 S-oligonucleotide probes and contact film autoradiography. Specificity was confirmed by showing that (a) excess unlabeled probe abolished the signal, (b) an oligonucleotide probe for rat neuropeptide Y mRNA showed a different distribution of hybridization signal, and (c) the distribution of insulin receptor binding was consistent with the distribution of insulin receptor mRNA. Insulin receptor mRNA was most abundant in the granule cell layers of the olfactory bulb, cerebellum and dentate gyrus, in the pyramidal cell body layers of the pyriform cortex and hippocampus, in the choroid plexus and in the arcuate nucleus of the hypothalamus

  4. Hydrophobically Modified siRNAs Silence Huntingtin mRNA in Primary Neurons and Mouse Brain

    Directory of Open Access Journals (Sweden)

    Julia F Alterman

    2015-01-01

    Full Text Available Applications of RNA interference for neuroscience research have been limited by a lack of simple and efficient methods to deliver oligonucleotides to primary neurons in culture and to the brain. Here, we show that primary neurons rapidly internalize hydrophobically modified siRNAs (hsiRNAs added directly to the culture medium without lipid formulation. We identify functional hsiRNAs targeting the mRNA of huntingtin, the mutation of which is responsible for Huntington's disease, and show that direct uptake in neurons induces potent and specific silencing in vitro. Moreover, a single injection of unformulated hsiRNA into mouse brain silences Htt mRNA with minimal neuronal toxicity. Thus, hsiRNAs embody a class of therapeutic oligonucleotides that enable simple and straightforward functional studies of genes involved in neuronal biology and neurodegenerative disorders in a native biological context.

  5. Evaluation of fluorine-18-labeled alkylating agents as potential synthons for the labeling of oligonucleotides

    NARCIS (Netherlands)

    de Vries, EFJ; Vroegh, J; Elsinga, PH; Vaalburg, W

    Six fluorine-18-labeled alkylating agents were selected as potentially suitable synthons for the labeling of antisense oligonucleotides. The selected synthons were evaluated in a model reaction with the monomer adenosine 5'-O-thiomonophosphate. Of these synthons,

  6. Novel approaches to study low-energy electron-induced damage to DNA oligonucleotides

    International Nuclear Information System (INIS)

    Rackwitz, Jenny; Bald, Ilko; Ranković, Miloš Lj; Milosavljević, Aleksandar R

    2015-01-01

    The novel approach of DNA origami structures as templates for precise quantification of various well- defined oligonucleotides provides the opportunity to determine the sensitivity of complex DNA sequences towards low-energy electrons. (paper)

  7. Functionalization of PVC membrane with ss oligonucleotides for a potentiometric biosensor.

    Science.gov (United States)

    Shishkanova, T V; Volf, R; Krondak, M; Král, V

    2007-05-15

    A novel application of a single stranded (ss) oligonucleotide as an active component of polymeric membrane in an ion-selective electrode (ISE) is described. The original oligonucleotides, oligo(dA)(15), modified by cholesterol, triphenylmethyl and hexadecyl derivatives, were immobilized into poly(vinyl chloride) (PVC) membrane using extraction protocol. In parallel, the adsorption protocol was used to immobilize unmodified oligo(dA)(15) on the PVC membrane based on tridodecylmethyammonium chloride (TDDMA(+)Cl(-)). Immobilization of ss oligonucleotide probe through spacer was more effective for the potentiometric detection of the hybridization between complementary oligonucleotides. It was found that cholesterol-oligo(dA)(15) modified membranes were sensitive toward complementary oligo(dT)(15) in the concentration range 2-80 nM at pH 7. An explanation for the detection mechanism is proposed.

  8. Oligonucleotide-based biosensors for in vitro diagnostics and environmental hazard detection.

    Science.gov (United States)

    Jung, Il Young; Lee, Eun Hee; Suh, Ah Young; Lee, Seung Jin; Lee, Hyukjin

    2016-04-01

    Oligonucleotide-based biosensors have drawn much attention because of their broad applications in in vitro diagnostics and environmental hazard detection. They are particularly of interest to many researchers because of their high specificity as well as excellent sensitivity. Recently, oligonucleotide-based biosensors have been used to achieve not only genetic detection of targets but also the detection of small molecules, peptides, and proteins. This has further broadened the applications of these sensors in the medical and health care industry. In this review, we highlight various examples of oligonucleotide-based biosensors for the detection of diseases, drugs, and environmentally hazardous chemicals. Each example is provided with detailed schematics of the detection mechanism in addition to the supporting experimental results. Furthermore, future perspectives and new challenges in oligonucleotide-based biosensors are discussed.

  9. Thermal Stability of Modified i-Motif Oligonucleotides with Naphthalimide Intercalating Nucleic Acids

    DEFF Research Database (Denmark)

    El-Sayed, Ahmed Ali; Pedersen, Erik B.; Khaireldin, Nahid Y.

    2016-01-01

    In continuation of our investigation of characteristics and thermodynamic properties of the i-motif 5′-d[(CCCTAA)3CCCT)] upon insertion of intercalating nucleotides into the cytosine-rich oligonucleotide, this article evaluates the stabilities of i-motif oligonucleotides upon insertion of naphtha......In continuation of our investigation of characteristics and thermodynamic properties of the i-motif 5′-d[(CCCTAA)3CCCT)] upon insertion of intercalating nucleotides into the cytosine-rich oligonucleotide, this article evaluates the stabilities of i-motif oligonucleotides upon insertion...... of naphthalimide (1H-benzo[de]isoquinoline-1,3(2H)-dione) as the intercalating nucleic acid. The stabilities of i-motif structures with inserted naphthalimide intercalating nucleotides were studied using UV melting temperatures (Tm) and circular dichroism spectra at different pH values and conditions (crowding...

  10. Rapid Induction of Protective Immunity Against Biothreat Agents Using CPG-Based Oligonucleotides

    National Research Council Canada - National Science Library

    Klinman, Dennis

    2003-01-01

    This research project examines the ability of synthetic oligonucleotides (ODN) containing immunostimulatory "CpG motifs' to trigger the innate immune system, thereby improving the host's ability to survive infection by biowarfare agents...

  11. [Oligonucleotide derivatives in the nucleic acid hybridization analysis. III. Synthesis and investigation of properties of oligonucleotides, bearing bifunctional non-nucleotide insert].

    Science.gov (United States)

    Kupriushkin, M S; Pyshnyĭ, D V

    2012-01-01

    Non-nucleotide phosporamidites were synthetized, having branched backbone with different position of functional groups. Obtained phosphoramidite monomers contain intercalator moiety--6-chloro-2-methoxyacridine, and additional hydroxyl residue protected with dimethoxytrityl group or with tert-butyldimethylsilyl group for post-synthetic modification. Synthesized oligothymidilates contain one or more modified units in different positions of sequence. Melting temperature and thermodynamic parameters of formation of complementary duplexes formed by modified oligonucleotides was defined (change in enthalpy and entropy). The introduction of intercalating residue causes a significant stabilization of DNA duplexes. It is shown that the efficiency of the fluorescence of acridine residue in the oligonucleotide conjugate significantly changes upon hybridization with DNA.

  12. Oligonucleotide Length-Dependent Formation of Virus-Like Particles.

    Science.gov (United States)

    Maassen, Stan J; de Ruiter, Mark V; Lindhoud, Saskia; Cornelissen, Jeroen J L M

    2018-05-23

    Understanding the assembly pathway of viruses can contribute to creating monodisperse virus-based materials. In this study, the cowpea chlorotic mottle virus (CCMV) is used to determine the interactions between the capsid proteins of viruses and their cargo. The assembly of the capsid proteins in the presence of different lengths of short, single-stranded (ss) DNA is studied at neutral pH, at which the protein-protein interactions are weak. Chromatography, electrophoresis, microscopy, and light scattering data show that the assembly efficiency and speed of the particles increase with increasing length of oligonucleotides. The minimal length required for assembly under the conditions used herein is 14 nucleotides. Assembly of particles containing such short strands of ssDNA can take almost a month. This slow assembly process enabled the study of intermediate states, which confirmed a low cooperative assembly for CCMV and allowed for further expansion of current assembly theories. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Selective Covalent Conjugation of Phosphorothioate DNA Oligonucleotides with Streptavidin

    Directory of Open Access Journals (Sweden)

    Christof M. Niemeyer

    2011-08-01

    Full Text Available Protein-DNA conjugates have found numerous applications in the field of diagnostics and nanobiotechnology, however, their intrinsic susceptibility to DNA degradation by nucleases represents a major obstacle for many applications. We here report the selective covalent conjugation of the protein streptavidin (STV with phosphorothioate oligonucleotides (psDNA containing a terminal alkylthiolgroup as the chemically addressable linking unit, using a heterobifunctional NHS-/maleimide crosslinker. The psDNA-STV conjugates were synthesized in about 10% isolated yields. We demonstrate that the terminal alkylthiol group selectively reacts with the maleimide while the backbone sulfur atoms are not engaged in chemical conjugation. The novel psDNA-STV conjugates retain their binding capabilities for both biotinylated macromolecules and the complementary nucleic acid. Moreover, the psDNA-STV conjugate retained its binding capacity for complementary oligomers even after a nuclease digestion step, which effectively degrades deoxyribonucleotide oligomers and thus the binding capability of regular DNA-STV conjugates. The psDNA-STV therefore hold particular promise for applications e.g. in proteome research and novel biosensing devices, where interfering endogenous nucleic acids need to be removed from analytes by nuclease digestion.

  14. Antineoplastic Effect of Decoy Oligonucleotide Derived from MGMT Enhancer

    Science.gov (United States)

    Refael, Miri; Zrihan, Daniel; Siegal, Tali; Lavon, Iris

    2014-01-01

    Silencing of O(6)-methylguanine-DNA-methyltransferase (MGMT) in tumors, mainly through promoter methylation, correlates with a better therapeutic response and with increased survival. Therefore, it is conceivable to consider MGMT as a potential therapeutic target for the treatment of cancers. Our previous results demonstrated the pivotal role of NF-kappaB in MGMT expression, mediated mainly through p65/NF-kappaB homodimers. Here we show that the non-canonical NF-KappaB motif (MGMT-kappaB1) within MGMT enhancer is probably the major inducer of MGMT expression following NF-kappaB activation. Thus, in an attempt to attenuate the transcription activity of MGMT in tumors we designed locked nucleic acids (LNA) modified decoy oligonucleotides corresponding to the specific sequence of MGMT-kappaB1 (MGMT-kB1-LODN). Following confirmation of the ability of MGMT-kB1-LODN to interfere with the binding of p65/NF-kappaB to the NF-KappaB motif within MGMT enhancer, the efficacy of the decoy was studied in-vitro and in-vivo. The results of these experiments show that the decoy MGMT-kB1-LODN have a substantial antineoplastic effect when used either in combination with temozolomide or as monotherapy. Our results suggest that MGMT-kB1-LODN may provide a novel strategy for cancer therapy. PMID:25460932

  15. Antineoplastic effect of decoy oligonucleotide derived from MGMT enhancer.

    Directory of Open Access Journals (Sweden)

    Tamar Canello

    Full Text Available Silencing of O(6-methylguanine-DNA-methyltransferase (MGMT in tumors, mainly through promoter methylation, correlates with a better therapeutic response and with increased survival. Therefore, it is conceivable to consider MGMT as a potential therapeutic target for the treatment of cancers. Our previous results demonstrated the pivotal role of NF-kappaB in MGMT expression, mediated mainly through p65/NF-kappaB homodimers. Here we show that the non-canonical NF-KappaB motif (MGMT-kappaB1 within MGMT enhancer is probably the major inducer of MGMT expression following NF-kappaB activation. Thus, in an attempt to attenuate the transcription activity of MGMT in tumors we designed locked nucleic acids (LNA modified decoy oligonucleotides corresponding to the specific sequence of MGMT-kappaB1 (MGMT-kB1-LODN. Following confirmation of the ability of MGMT-kB1-LODN to interfere with the binding of p65/NF-kappaB to the NF-KappaB motif within MGMT enhancer, the efficacy of the decoy was studied in-vitro and in-vivo. The results of these experiments show that the decoy MGMT-kB1-LODN have a substantial antineoplastic effect when used either in combination with temozolomide or as monotherapy. Our results suggest that MGMT-kB1-LODN may provide a novel strategy for cancer therapy.

  16. Correction of Spatial Bias in Oligonucleotide Array Data

    Directory of Open Access Journals (Sweden)

    Philippe Serhal

    2013-01-01

    Full Text Available Background. Oligonucleotide microarrays allow for high-throughput gene expression profiling assays. The technology relies on the fundamental assumption that observed hybridization signal intensities (HSIs for each intended target, on average, correlate with their target’s true concentration in the sample. However, systematic, nonbiological variation from several sources undermines this hypothesis. Background hybridization signal has been previously identified as one such important source, one manifestation of which appears in the form of spatial autocorrelation. Results. We propose an algorithm, pyn, for the elimination of spatial autocorrelation in HSIs, exploiting the duality of desirable mutual information shared by probes in a common probe set and undesirable mutual information shared by spatially proximate probes. We show that this correction procedure reduces spatial autocorrelation in HSIs; increases HSI reproducibility across replicate arrays; increases differentially expressed gene detection power; and performs better than previously published methods. Conclusions. The proposed algorithm increases both precision and accuracy, while requiring virtually no changes to users’ current analysis pipelines: the correction consists merely of a transformation of raw HSIs (e.g., CEL files for Affymetrix arrays. A free, open-source implementation is provided as an R package, compatible with standard Bioconductor tools. The approach may also be tailored to other platform types and other sources of bias.

  17. Correction of Spatial Bias in Oligonucleotide Array Data

    Science.gov (United States)

    Lemieux, Sébastien

    2013-01-01

    Background. Oligonucleotide microarrays allow for high-throughput gene expression profiling assays. The technology relies on the fundamental assumption that observed hybridization signal intensities (HSIs) for each intended target, on average, correlate with their target's true concentration in the sample. However, systematic, nonbiological variation from several sources undermines this hypothesis. Background hybridization signal has been previously identified as one such important source, one manifestation of which appears in the form of spatial autocorrelation. Results. We propose an algorithm, pyn, for the elimination of spatial autocorrelation in HSIs, exploiting the duality of desirable mutual information shared by probes in a common probe set and undesirable mutual information shared by spatially proximate probes. We show that this correction procedure reduces spatial autocorrelation in HSIs; increases HSI reproducibility across replicate arrays; increases differentially expressed gene detection power; and performs better than previously published methods. Conclusions. The proposed algorithm increases both precision and accuracy, while requiring virtually no changes to users' current analysis pipelines: the correction consists merely of a transformation of raw HSIs (e.g., CEL files for Affymetrix arrays). A free, open-source implementation is provided as an R package, compatible with standard Bioconductor tools. The approach may also be tailored to other platform types and other sources of bias. PMID:23573083

  18. Effect of oligonucleotide primers in determining viral variability within hosts

    Directory of Open Access Journals (Sweden)

    Moya Andrés

    2004-12-01

    Full Text Available Abstract Background Genetic variability in viral populations is usually estimated by means of polymerase chain reaction (PCR based methods in which the relative abundance of each amplicon is assumed to be proportional to the frequency of the corresponding template in the initial sample. Although bias in template-to-product ratios has been described before, its relevance in describing viral genetic variability at the intrapatient level has not been fully assessed yet. Results To investigate the role of oligonucleotide design in estimating viral variability within hosts, genetic diversity in hepatitis C virus (HCV populations from eight infected patients was characterised by two parallel PCR amplifications performed with two slightly different sets of primers, followed by cloning and sequencing (mean = 89 cloned sequences per patient. Population genetics analyses of viral populations recovered by pairs of amplifications revealed that in seven patients statistically significant differences were detected between populations sampled with different set of primers. Conclusions Genetic variability analyses demonstrates that PCR selection due to the choice of primers, differing in their degeneracy degree at some nucleotide positions, can eclipse totally or partially viral variants, hence yielding significant different estimates of viral variability within a single patient and therefore eventually producing quite different qualitative and quantitative descriptions of viral populations within each host.

  19. Effect of oligonucleotide primers in determining viral variability within hosts.

    Science.gov (United States)

    Bracho, Maria Alma; García-Robles, Inmaculada; Jiménez, Nuria; Torres-Puente, Manuela; Moya, Andrés; González-Candelas, Fernando

    2004-12-09

    Genetic variability in viral populations is usually estimated by means of polymerase chain reaction (PCR) based methods in which the relative abundance of each amplicon is assumed to be proportional to the frequency of the corresponding template in the initial sample. Although bias in template-to-product ratios has been described before, its relevance in describing viral genetic variability at the intrapatient level has not been fully assessed yet. To investigate the role of oligonucleotide design in estimating viral variability within hosts, genetic diversity in hepatitis C virus (HCV) populations from eight infected patients was characterised by two parallel PCR amplifications performed with two slightly different sets of primers, followed by cloning and sequencing (mean = 89 cloned sequences per patient). Population genetics analyses of viral populations recovered by pairs of amplifications revealed that in seven patients statistically significant differences were detected between populations sampled with different set of primers. Genetic variability analyses demonstrates that PCR selection due to the choice of primers, differing in their degeneracy degree at some nucleotide positions, can eclipse totally or partially viral variants, hence yielding significant different estimates of viral variability within a single patient and therefore eventually producing quite different qualitative and quantitative descriptions of viral populations within each host.

  20. Methods for the preparation of protein-oligonucleotide-lipid constructs.

    Science.gov (United States)

    Takasaki, Jennifer; Raney, Sameersingh G; Chikh, Ghania; Sekirov, Laura; Brodsky, Irina; Tam, Ying; Ansell, Steven M

    2006-01-01

    A mixture of ionizable cationic lipids, steric barrier lipids, and colipids is used to encapsulate oligonucleotide DNA in lipidic particles called SALP. This material is under development as an adjuvant for vaccines. Previously we have shown that coupling the antigen directly to the surface of SALP can lead to enhanced immunological responses in vivo. Two different methods for preparing ovalbumin-SALP were assessed in this work. Originally the conjugates were prepared by treating SALP containing a maleimide-derivatized lipid with thiolated ovalbumin, a method we refer to as active coupling. This reaction was found to be difficult to control and generally resulted in low coupling efficiencies. The issues relating to this approach were characterized. We have recently developed alternative techniques based on first coupling ovalbumin to a micelle and then incubating the resultant product with SALP, methods we refer to as passive coupling. We have shown that this method allows accurate control of the levels of protein associated SALP and does not suffer from surface saturation effects seen with the active coupling method that places maximum limits on the amount of protein that can be coupled to the SALP surface. The products from the passive coupling protocol are shown to have activity comparable to those derived from the active coupling protocol in investigations of in vivo immune responses.

  1. Comparative analysis of human conjunctival and corneal epithelial gene expression with oligonucleotide microarrays.

    Science.gov (United States)

    Turner, Helen C; Budak, Murat T; Akinci, M A Murat; Wolosin, J Mario

    2007-05-01

    To determine global mRNA expression levels in corneal and conjunctival epithelia and identify transcripts that exhibit preferential tissue expression. cDNA samples derived from human conjunctival and corneal epithelia were hybridized in three independent experiments to a commercial oligonucleotide array representing more than 22,000 transcripts. The resultant signal intensities and microarray software transcript present/absent calls were used in conjunction with the local pooled error (LPE) statistical method to identify transcripts that are preferentially or exclusively expressed in one of the two tissues at significant levels (expression >1% of the beta-actin level). EASE (Expression Analysis Systematic Explorer software) was used to identify biological systems comparatively overrepresented in either epithelium. Immuno-, and cytohistochemistry was performed to validate or expand on selected results of interest. The analysis identified 332 preferential and 93 exclusive significant corneal epithelial transcripts. The corresponding numbers of conjunctival epithelium transcripts were 592 and 211, respectively. The overrepresented biological processes in the cornea were related to cell adhesion and oxiredox equilibria and cytoprotection activities. In the conjunctiva, the biological processes that were most prominent were related to innate immunity and melanogenesis. Immunohistochemistry for antigen-presenting cells and melanocytes was consistent with these gene signatures. The transcript comparison identified a substantial number of genes that have either not been identified previously or are not known to be highly expressed in these two epithelia, including testican-1, ECM1, formin, CRTAC1, and NQO1 in the cornea and, in the conjunctiva, sPLA(2)-IIA, lipocalin 2, IGFBP3, multiple MCH class II proteins, and the Na-Pi cotransporter type IIb. Comparative gene expression profiling leads to the identification of many biological processes and previously unknown genes that

  2. Syntheses of prodrug-type phosphotriester oligonucleotides responsive to intracellular reducing environment for improvement of cell membrane permeability and nuclease resistance.

    Science.gov (United States)

    Hayashi, Junsuke; Samezawa, Yusuke; Ochi, Yosuke; Wada, Shun-Ichi; Urata, Hidehito

    2017-07-15

    We synthesized prodrug-type phosphotriester (PTE) oligonucleotides containing the six-membered cyclic disulfide moiety by using phosphoramidite chemistry. Prodrug-type oligonucleotides named "Reducing-Environment-Dependent Uncatalyzed Chemical Transforming (REDUCT) PTE oligonucleotides" were converted into natural oligonucleotides under cytosol-mimetic reductive condition. Furthermore, the REDUCT PTE oligonucleotides were robust to nuclease digestion and exhibited good cell membrane permeability. Copyright © 2017 Elsevier Ltd. All rights reserved.

  3. In vitro transcription in the presence of DNA oligonucleotides can generate strong anomalous initiation sites.

    Science.gov (United States)

    Chow, C W; Clark, M P; Rinaldo, J E; Chalkley, R

    1996-03-01

    In the present study, we have explored an unexpected observation in transcription initiation that is mediated by single-stranded oligonucleotides. Initially, our goal was to understand the function of different upstream regulatory elements/initiation sites in the rat xanthine dehydrogenase/oxidase (XDH/XO) promoter. We performed in vitro transcription with HeLa nuclear extracts in the presence of different double-stranded oligonucleotides against upstream elements as competitors. A new and unusual transcription initiation site was detected by primer extension. This new initiation site maps to the downstream region of the corresponding competitor. Subsequent analyses have indicated that the induction of a new transcription initiation site is anomalous which is due to the presence of a small amount of single-stranded oligonucleotide in the competitor. We found that this anomalous initiation site is insensitive to the orientation of the promoter and requires only a small amount of single-stranded oligonucleotide (< 2-fold molar excess relative to template). We surmise that a complementary interaction between the single-stranded oligonucleotide and transiently denatured promoter template may be responsible for this sequence-specific transcription initiation artifact. To study the regulation of transcription initiation by in vitro transcription approaches, we propose that one should probe the effect of removing transacting factors by adding an excess of a cognate oligonucleotide which does not bear exact sequence identity to the template.

  4. Systematic validation and atomic force microscopy of non-covalent short oligonucleotide barcode microarrays.

    Directory of Open Access Journals (Sweden)

    Michael A Cook

    Full Text Available BACKGROUND: Molecular barcode arrays provide a powerful means to analyze cellular phenotypes in parallel through detection of short (20-60 base unique sequence tags, or "barcodes", associated with each strain or clone in a collection. However, costs of current methods for microarray construction, whether by in situ oligonucleotide synthesis or ex situ coupling of modified oligonucleotides to the slide surface are often prohibitive to large-scale analyses. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrate that unmodified 20mer oligonucleotide probes printed on conventional surfaces show comparable hybridization signals to covalently linked 5'-amino-modified probes. As a test case, we undertook systematic cell size analysis of the budding yeast Saccharomyces cerevisiae genome-wide deletion collection by size separation of the deletion pool followed by determination of strain abundance in size fractions by barcode arrays. We demonstrate that the properties of a 13K unique feature spotted 20 mer oligonucleotide barcode microarray compare favorably with an analogous covalently-linked oligonucleotide array. Further, cell size profiles obtained with the size selection/barcode array approach recapitulate previous cell size measurements of individual deletion strains. Finally, through atomic force microscopy (AFM, we characterize the mechanism of hybridization to unmodified barcode probes on the slide surface. CONCLUSIONS/SIGNIFICANCE: These studies push the lower limit of probe size in genome-scale unmodified oligonucleotide microarray construction and demonstrate a versatile, cost-effective and reliable method for molecular barcode analysis.

  5. U1 Adaptor Oligonucleotides Targeting BCL2 and GRM1 Suppress Growth of Human Melanoma Xenografts In Vivo

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    Rafal Goraczniak

    2013-01-01

    Full Text Available U1 Adaptor is a recently discovered oligonucleotide-based gene-silencing technology with a unique mechanism of action that targets nuclear pre-mRNA processing. U1 Adaptors have two distinct functional domains, both of which must be present on the same oligonucleotide to exert their gene-silencing function. Here, we present the first in vivo use of U1 Adaptors by targeting two different human genes implicated in melanomagenesis, B-cell lymphoma 2 (BCL2 and metabotropic glutamate receptor 1 (GRM1, in a human melanoma cell xenograft mouse model system. Using a newly developed dendrimer delivery system, anti-BCL2 U1 Adaptors were very potent and suppressed tumor growth at doses as low as 34 µg/kg with twice weekly intravenous (iv administration. Anti-GRM1 U1 Adaptors suppressed tumor xenograft growth with similar potency. Mechanism of action was demonstrated by showing target gene suppression in tumors and by observing that negative control U1 Adaptors with just one functional domain show no tumor suppression activity. The anti-BCL2 and anti-GRM1 treatments were equally effective against cell lines harboring either wild-type or a mutant V600E B-RAF allele, the most common mutation in melanoma. Treatment of normal immune-competent mice (C57BL6 indicated no organ toxicity or immune stimulation. These proof-of-concept studies represent an in-depth (over 800 mice in ~108 treatment groups validation that U1 Adaptors are a highly potent gene-silencing therapeutic and open the way for their further development to treat other human diseases.

  6. Short (16-mer locked nucleic acid splice-switching oligonucleotides restore dystrophin production in Duchenne Muscular Dystrophy myotubes.

    Directory of Open Access Journals (Sweden)

    Vanessa Borges Pires

    Full Text Available Splice-switching antisense oligonucleotides (SSOs offer great potential for RNA-targeting therapies, and two SSO drugs have been recently approved for treating Duchenne Muscular Dystrophy (DMD and Spinal Muscular Atrophy (SMA. Despite promising results, new developments are still needed for more efficient chemistries and delivery systems. Locked nucleic acid (LNA is a chemically modified nucleic acid that presents several attractive properties, such as high melting temperature when bound to RNA, potent biological activity, high stability and low toxicity in vivo. Here, we designed a series of LNA-based SSOs complementary to two sequences of the human dystrophin exon 51 that are most evolutionary conserved and evaluated their ability to induce exon skipping upon transfection into myoblasts derived from a DMD patient. We show that 16-mers with 60% of LNA modification efficiently induce exon skipping and restore synthesis of a truncated dystrophin isoform that localizes to the plasma membrane of patient-derived myotubes differentiated in culture. In sum, this study underscores the value of short LNA-modified SSOs for therapeutic applications.

  7. Orotidine-Containing RNA: Implications for the Hierarchical Selection (Systems Chemistry Emergence) of RNA.

    Science.gov (United States)

    Kim, Eun-Kyong; Martin, Vincent; Krishnamurthy, Ramanarayanan

    2017-09-12

    The prebiotic synthesis of canonical nucleobases from HCN is a cornerstone for the RNA world hypothesis. However, their role in the primordial pathways to RNA is still debated. The very same process starting from HCN also gives rise to orotic acid, which (via orotidine) plays a crucial role in extant biology in the de novo synthesis of uridine and cytidine, the informational base-pairs in RNA. However, orotidine itself is absent in RNA. Given the prebiotic and biological relevance of orotic acid vis-à-vis uracil, we investigated orotidine-containing RNA oligonucleotides and show that they have severely compromised base-pairing properties. While not unexpected, these results suggest that the emergence of extant RNA cannot just be a consequence of the plausible prebiotic formation of its chemical constituents/building blocks. In combination with other investigations on alternative prebiotic nucleobases, sugars, and linkers, these findings imply that the selection of the components of extant RNA occurred at a higher hierarchical level of an oligomer/polymer based on its functional properties-pointing to a systems chemistry emergence of RNA from a library of precursors. © 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  8. Application of heteronuclear couplings to conformational analysis of oligonucleotides

    International Nuclear Information System (INIS)

    Zhu, G.; Live, D.; Bax, A.

    1994-01-01

    The value of vicinal coupling constants extracted from NMR spectra in deducing torsion angles for conformational analysis is well recognized. Due to the abundance of protons, their couplings have been mostly widely used. In many instances, couplings between protons and other nuclei may be a valuable complement to proton-proton couplings or, in some instances, may be the only coupling available to characterize the torsion angle about a bond. Recently, heteronuclear couplings have been used to great benefit in studies of isotopically enriched proteins, and this general approach has been extended to peptides at natural abundance. The possibility of using this approach to study oligonucleotides is also attractive but has not as yet been widely exploited. With the development of strategies for labeling such molecules, particularly RNAs, this may become an important component in conformational analysis. For DNA, labeling is less accessible, but sufficient quantities of unlabeled material are readily available for measuring these couplings at natural abundance. We chose several DNA systems to explore the usefulness of heteronuclear couplings in addressing the sugar conformation and the glycosidic torsion angle. Intensities of cross peaks in long-range HMQC experiments can be related to the couplings. Crosspeaks involving H1' and C1' atoms have been emphasized because of the superior shift dispersion at these positions between sugar protons and carbon atoms. Results will be shown for the self-complementary Dickerson duplex dodecamer sequence d(CGCGAATTCGCG) and for d(GGTCGG), which dimerizes to form a G-tetrad structure incorporating both syn and anti base orientations. The couplings provide a clear discrimination between presence of C3'-endo and C2'-endo conformations of the sugars and syn and anti bases arrangements

  9. Mongersen, an oral SMAD7 antisense oligonucleotide, and Crohn's disease.

    Science.gov (United States)

    Monteleone, Giovanni; Neurath, Markus F; Ardizzone, Sandro; Di Sabatino, Antonio; Fantini, Massimo C; Castiglione, Fabiana; Scribano, Maria L; Armuzzi, Alessandro; Caprioli, Flavio; Sturniolo, Giacomo C; Rogai, Francesca; Vecchi, Maurizio; Atreya, Raja; Bossa, Fabrizio; Onali, Sara; Fichera, Maria; Corazza, Gino R; Biancone, Livia; Savarino, Vincenzo; Pica, Roberta; Orlando, Ambrogio; Pallone, Francesco

    2015-03-19

    Crohn's disease-related inflammation is characterized by reduced activity of the immunosuppressive cytokine transforming growth factor β1 (TGF-β1) due to high levels of SMAD7, an inhibitor of TGF-β1 signaling. Preclinical studies and a phase 1 study have shown that an oral SMAD7 antisense oligonucleotide, mongersen, targets ileal and colonic SMAD7. In a double-blind, placebo-controlled, phase 2 trial, we evaluated the efficacy of mongersen for the treatment of persons with active Crohn's disease. Patients were randomly assigned to receive 10, 40, or 160 mg of mongersen or placebo per day for 2 weeks. The primary outcomes were clinical remission at day 15, defined as a Crohn's Disease Activity Index (CDAI) score of less than 150, with maintenance of remission for at least 2 weeks, and the safety of mongersen treatment. A secondary outcome was clinical response (defined as a reduction of 100 points or more in the CDAI score) at day 28. The proportions of patients who reached the primary end point were 55% and 65% for the 40-mg and 160-mg mongersen groups, respectively, as compared with 10% for the placebo group (P<0.001). There was no significant difference in the percentage of participants reaching clinical remission between the 10-mg group (12%) and the placebo group. The rate of clinical response was significantly greater among patients receiving 10 mg (37%), 40 mg (58%), or 160 mg (72%) of mongersen than among those receiving placebo (17%) (P=0.04, P<0.001, and P<0.001, respectively). Most adverse events were related to complications and symptoms of Crohn's disease. We found that study participants with Crohn's disease who received mongersen had significantly higher rates of remission and clinical response than those who received placebo. (Funded by Giuliani; EudraCT number, 2011-002640-27.).

  10. Application of heteronuclear couplings to conformational analysis of oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Zhu, G. [Univ. of Maryland, College Park, MD (United States); Live, D. [Memorial Sloan-Kettering Cancer Center, New York, NY (United States); Bax, A. [NIDDK National Institutes of Health, Bethesda, MD (United States)

    1994-12-01

    The value of vicinal coupling constants extracted from NMR spectra in deducing torsion angles for conformational analysis is well recognized. Due to the abundance of protons, their couplings have been mostly widely used. In many instances, couplings between protons and other nuclei may be a valuable complement to proton-proton couplings or, in some instances, may be the only coupling available to characterize the torsion angle about a bond. Recently, heteronuclear couplings have been used to great benefit in studies of isotopically enriched proteins, and this general approach has been extended to peptides at natural abundance. The possibility of using this approach to study oligonucleotides is also attractive but has not as yet been widely exploited. With the development of strategies for labeling such molecules, particularly RNAs, this may become an important component in conformational analysis. For DNA, labeling is less accessible, but sufficient quantities of unlabeled material are readily available for measuring these couplings at natural abundance. We chose several DNA systems to explore the usefulness of heteronuclear couplings in addressing the sugar conformation and the glycosidic torsion angle. Intensities of cross peaks in long-range HMQC experiments can be related to the couplings. Crosspeaks involving H1{prime} and C1{prime} atoms have been emphasized because of the superior shift dispersion at these positions between sugar protons and carbon atoms. Results will be shown for the self-complementary Dickerson duplex dodecamer sequence d(CGCGAATTCGCG) and for d(GGTCGG), which dimerizes to form a G-tetrad structure incorporating both syn and anti base orientations. The couplings provide a clear discrimination between presence of C3{prime}-endo and C2{prime}-endo conformations of the sugars and syn and anti bases arrangements.

  11. Degradation product characterization of therapeutic oligonucleotides using liquid chromatography mass spectrometry.

    Science.gov (United States)

    Elzahar, N M; Magdy, N; El-Kosasy, Amira M; Bartlett, Michael G

    2018-05-01

    Synthetic antisense phosphorothioate oligonucleotides (PS) have undergone rapid development as novel therapeutic agents. The increasing significance of this class of drugs requires significant investment in the development of quality control methods. The determination of the many degradation pathways of such complex molecules presents a significant challenge. However, an understanding of the potential impurities that may arise is necessary to continue to advance these powerful new therapeutics. In this study, four different antisense oligonucleotides representing several generations of oligonucleotide therapeutic agents were evaluated under various stress conditions (pH, thermal, and oxidative stress) using ion-pairing reversed-phase liquid chromatography tandem mass spectrometry (IP-RPLC-MS/MS) to provide in-depth characterization and identification of the degradation products. The oligonucleotide samples were stressed under different pH values at 45 and 90 °C. The main degradation products were observed to be losses of nucleotide moieties from the 3'- and 5'-terminus, depurination, formation of terminal phosphorothioates, and production of ribose, ribophosphorothioates (Rp), and phosphoribophosphorothioates (pRp). Moreover, the effects of different concentrations of hydrogen peroxide were studied resulting in primarily extensive desulfurization and subsequent oxidation of the phosphorothioate linkage to produce the corresponding phosphodiester. The reaction kinetics for the degradation of the oligonucleotides under the different stress conditions were studied and were found to follow pseudo-first-order kinetics. Differences in rates exist even for oligonucleotides of similar length but consisting of different sequences. Graphical abstract Identification of degradation products across several generations of oligonucleotide therapeutics using LC-MS.

  12. NMR studies concerning base-base interactions in oligonucleotides

    International Nuclear Information System (INIS)

    Hoogen, Y.T. van den.

    1988-01-01

    Two main subjects are treated in the present thesis. The firsst part principally deals with the base-base interactions in single-stranded oligoribonucleotides. The second part presents NMR and model-building studies of DNA and RNA duplexes containing an unpaired base. (author). 242 refs.; 26 figs.; 24 tabs

  13. Programmable RNA recognition and cleavage by CRISPR/Cas9.

    Science.gov (United States)

    O'Connell, Mitchell R; Oakes, Benjamin L; Sternberg, Samuel H; East-Seletsky, Alexandra; Kaplan, Matias; Doudna, Jennifer A

    2014-12-11

    The CRISPR-associated protein Cas9 is an RNA-guided DNA endonuclease that uses RNA-DNA complementarity to identify target sites for sequence-specific double-stranded DNA (dsDNA) cleavage. In its native context, Cas9 acts on DNA substrates exclusively because both binding and catalysis require recognition of a short DNA sequence, known as the protospacer adjacent motif (PAM), next to and on the strand opposite the twenty-nucleotide target site in dsDNA. Cas9 has proven to be a versatile tool for genome engineering and gene regulation in a large range of prokaryotic and eukaryotic cell types, and in whole organisms, but it has been thought to be incapable of targeting RNA. Here we show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. Using specially designed PAMmers, Cas9 can be specifically directed to bind or cut RNA targets while avoiding corresponding DNA sequences, and we demonstrate that this strategy enables the isolation of a specific endogenous messenger RNA from cells. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.

  14. Surface modification of plasmonic nanostructured materials with thiolated oligonucleotides in 10 seconds using selective microwave heating

    International Nuclear Information System (INIS)

    Abel, B.; Aslan, K.

    2012-01-01

    This study demonstrates the proof-of-principle of rapid surface modification of plasmonic nanostructured materials with oligonucleotides using low power microwave heating. Due to their interesting optical and electronic properties, silver nanoparticle films (SNFs, 2 nm thick) deposited onto glass slides were used as the model plasmonic nanostructured materials. Rapid surface modification of SNFs with oligonucleotides was carried out using two strategies (1) Strategy 1: for ss-oligonucleotides, surface hybridization and (2) Strategy 2: for ds-oligonucleotides, solution hybridization, where the samples were exposed to 10, 15, 30 and 60 seconds microwave heating. To assess the efficacy of our new rapid surface modification technique, identical experiments carried out without the microwave heating (i.e., conventional method), which requires 24 hours for the completion of the identical steps. It was found that SNFs can be modified with ss- and ds-oligonucleotides in 10 seconds, which typically requires several hours of incubation time for the chemisorption of thiol groups on to the planar metal surface using conventional techniques. (Copyright copyright 2012 WILEY-VCH Verlag GmbH and Co. KGaA, Weinheim)

  15. Development, characterization and experimental validation of a cultivated sunflower (Helianthus annuus L.) gene expression oligonucleotide microarray.

    Science.gov (United States)

    Fernandez, Paula; Soria, Marcelo; Blesa, David; DiRienzo, Julio; Moschen, Sebastian; Rivarola, Maximo; Clavijo, Bernardo Jose; Gonzalez, Sergio; Peluffo, Lucila; Príncipi, Dario; Dosio, Guillermo; Aguirrezabal, Luis; García-García, Francisco; Conesa, Ana; Hopp, Esteban; Dopazo, Joaquín; Heinz, Ruth Amelia; Paniego, Norma

    2012-01-01

    Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (psunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.

  16. Detection and quantitative analysis of actin mRNA by in situ hybridization with an oligodeoxynucleotide probe

    International Nuclear Information System (INIS)

    Taneja, K.; Singer, R.

    1987-01-01

    In situ hybridization is a useful method for localizing specific nucleic acid sequences intracellularly and for studying regulation of gene expression. Recently synthetic oligonucleotides have been successfully used as probes in this technique. Since they can be made easily to specific nucleic acid regions, they may be the best approach for analysis of a gene family of highly conserved sequences. They have analyzed these probes for the development of an in situ hybridization method. Oligonucleotides were made to different regions of chick beta-actin mRNA and used for detection of these sequences in a culture of chicken fibroblasts and myoblasts. They found that synthetic DNAs have different efficiencies of hybridization, indicating that not all target sequences are equivalent. They have investigated in detail a particular probe to the actin mRNA coding region and have optimized hybridization parameters. When hybridization was quantitated it was found that an oligonucleotide end labelled with 35 S or 32 P was capable of detecting several thousand messages per cell with a signal-to-noise ratio of 10:1. In situ hybridization confirmed the specificity of the hybridization as well as the background level. Increase in the number of oligonucleotides used should increase the signal-to-noise ratio-proportionately. Under particular circumstances the specificity of oligonucleotides make them an important reagent for in situ hybridization

  17. Comparison of gene coverage of mouse oligonucleotide microarray platforms

    Directory of Open Access Journals (Sweden)

    Medrano Juan F

    2006-03-01

    Full Text Available Abstract Background The increasing use of DNA microarrays for genetical genomics studies generates a need for platforms with complete coverage of the genome. We have compared the effective gene coverage in the mouse genome of different commercial and noncommercial oligonucleotide microarray platforms by performing an in-house gene annotation of probes. We only used information about probes that is available from vendors and followed a process that any researcher may take to find the gene targeted by a given probe. In order to make consistent comparisons between platforms, probes in each microarray were annotated with an Entrez Gene id and the chromosomal position for each gene was obtained from the UCSC Genome Browser Database. Gene coverage was estimated as the percentage of Entrez Genes with a unique position in the UCSC Genome database that is tested by a given microarray platform. Results A MySQL relational database was created to store the mapping information for 25,416 mouse genes and for the probes in five microarray platforms (gene coverage level in parenthesis: Affymetrix430 2.0 (75.6%, ABI Genome Survey (81.24%, Agilent (79.33%, Codelink (78.09%, Sentrix (90.47%; and four array-ready oligosets: Sigma (47.95%, Operon v.3 (69.89%, Operon v.4 (84.03%, and MEEBO (84.03%. The differences in coverage between platforms were highly conserved across chromosomes. Differences in the number of redundant and unspecific probes were also found among arrays. The database can be queried to compare specific genomic regions using a web interface. The software used to create, update and query the database is freely available as a toolbox named ArrayGene. Conclusion The software developed here allows researchers to create updated custom databases by using public or proprietary information on genes for any organisms. ArrayGene allows easy comparisons of gene coverage between microarray platforms for any region of the genome. The comparison presented here

  18. A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Chen Feng

    2010-10-01

    Full Text Available Abstract Background Microarrays are invaluable tools for genome interrogation, SNP detection, and expression analysis, among other applications. Such broad capabilities would be of value to many pathogen research communities, although the development and use of genome-scale microarrays is often a costly undertaking. Therefore, effective methods for reducing unnecessary probes while maintaining or expanding functionality would be relevant to many investigators. Results Taking advantage of available genome sequences and annotation for Toxoplasma gondii (a pathogenic parasite responsible for illness in immunocompromised individuals and Plasmodium falciparum (a related parasite responsible for severe human malaria, we designed a single oligonucleotide microarray capable of supporting a wide range of applications at relatively low cost, including genome-wide expression profiling for Toxoplasma, and single-nucleotide polymorphism (SNP-based genotyping of both T. gondii and P. falciparum. Expression profiling of the three clonotypic lineages dominating T. gondii populations in North America and Europe provides a first comprehensive view of the parasite transcriptome, revealing that ~49% of all annotated genes are expressed in parasite tachyzoites (the acutely lytic stage responsible for pathogenesis and 26% of genes are differentially expressed among strains. A novel design utilizing few probes provided high confidence genotyping, used here to resolve recombination points in the clonal progeny of sexual crosses. Recent sequencing of additional T. gondii isolates identifies >620 K new SNPs, including ~11 K that intersect with expression profiling probes, yielding additional markers for genotyping studies, and further validating the utility of a combined expression profiling/genotyping array design. Additional applications facilitating SNP and transcript discovery, alternative statistical methods for quantifying gene expression, etc. are also pursued at

  19. Efficient oligonucleotide probe selection for pan-genomic tiling arrays

    Directory of Open Access Journals (Sweden)

    Zhang Wei

    2009-09-01

    Full Text Available Abstract Background Array comparative genomic hybridization is a fast and cost-effective method for detecting, genotyping, and comparing the genomic sequence of unknown bacterial isolates. This method, as with all microarray applications, requires adequate coverage of probes targeting the regions of interest. An unbiased tiling of probes across the entire length of the genome is the most flexible design approach. However, such a whole-genome tiling requires that the genome sequence is known in advance. For the accurate analysis of uncharacterized bacteria, an array must query a fully representative set of sequences from the species' pan-genome. Prior microarrays have included only a single strain per array or the conserved sequences of gene families. These arrays omit potentially important genes and sequence variants from the pan-genome. Results This paper presents a new probe selection algorithm (PanArray that can tile multiple whole genomes using a minimal number of probes. Unlike arrays built on clustered gene families, PanArray uses an unbiased, probe-centric approach that does not rely on annotations, gene clustering, or multi-alignments. Instead, probes are evenly tiled across all sequences of the pan-genome at a consistent level of coverage. To minimize the required number of probes, probes conserved across multiple strains in the pan-genome are selected first, and additional probes are used only where necessary to span polymorphic regions of the genome. The viability of the algorithm is demonstrated by array designs for seven different bacterial pan-genomes and, in particular, the design of a 385,000 probe array that fully tiles the genomes of 20 different Listeria monocytogenes strains with overlapping probes at greater than twofold coverage. Conclusion PanArray is an oligonucleotide probe selection algorithm for tiling multiple genome sequences using a minimal number of probes. It is capable of fully tiling all genomes of a species on

  20. Detection of genomic deletions in rice using oligonucleotide microarrays

    Directory of Open Access Journals (Sweden)

    Bordeos Alicia

    2009-03-01

    Full Text Available Abstract Background The induction of genomic deletions by physical- or chemical- agents is an easy and inexpensive means to generate a genome-saturating collection of mutations. Different mutagens can be selected to ensure a mutant collection with a range of deletion sizes. This would allow identification of mutations in single genes or, alternatively, a deleted group of genes that might collectively govern a trait (e.g., quantitative trait loci, QTL. However, deletion mutants have not been widely used in functional genomics, because the mutated genes are not tagged and therefore, difficult to identify. Here, we present a microarray-based approach to identify deleted genomic regions in rice mutants selected from a large collection generated by gamma ray or fast neutron treatment. Our study focuses not only on the utility of this method for forward genetics, but also its potential as a reverse genetics tool through accumulation of hybridization data for a collection of deletion mutants harboring multiple genetic lesions. Results We demonstrate that hybridization of labeled genomic DNA directly onto the Affymetrix Rice GeneChip® allows rapid localization of deleted regions in rice mutants. Deletions ranged in size from one gene model to ~500 kb and were predicted on all 12 rice chromosomes. The utility of the technique as a tool in forward genetics was demonstrated in combination with an allelic series of mutants to rapidly narrow the genomic region, and eventually identify a candidate gene responsible for a lesion mimic phenotype. Finally, the positions of mutations in 14 mutants were aligned onto the rice pseudomolecules in a user-friendly genome browser to allow for rapid identification of untagged mutations http://irfgc.irri.org/cgi-bin/gbrowse/IR64_deletion_mutants/. Conclusion We demonstrate the utility of oligonucleotide arrays to discover deleted genes in rice. The density and distribution of deletions suggests the feasibility of a

  1. Hydration-dependent dynamics of human telomeric oligonucleotides in the picosecond timescale: A neutron scattering study

    Energy Technology Data Exchange (ETDEWEB)

    Sebastiani, F.; Comez, L.; Sacchetti, F. [Dipartimento di Fisica e Geologia, Università degli Studi di Perugia, Via A. Pascoli, 06123 Perugia (Italy); CNR, Istituto Officina dei Materiali, Unità di Perugia, c/o Dipartimento di Fisica e Geologia, Università di Perugia, 06123 Perugia (Italy); Longo, M. [Dipartimento di Fisica e Geologia, Università degli Studi di Perugia, Via A. Pascoli, 06123 Perugia (Italy); Elettra—Sincrotrone Trieste, 34149 Basovizza, Trieste (Italy); Orecchini, A.; Petrillo, C.; Paciaroni, A., E-mail: alessandro.paciaroni@fisica.unipg.it [Dipartimento di Fisica e Geologia, Università degli Studi di Perugia, Via A. Pascoli, 06123 Perugia (Italy); De Francesco, A. [CNR-IOM OGG c/o Institut Laue-Langevin, 71 Avenue des Martyrs, CS20156, 38042 Grenoble Cedex 9 (France); Muthmann, M. [Jülich Centre for Neutron Science, Forschungszentrum Jülich GmbH, Outstation at Heinz Maier-Leibnitz Zentrum, Lichtenbergstrasse 1, 85747 Garching (Germany); Teixeira, S. C. M. [EPSAM, Keele University, Staffordshire ST5 5BG (United Kingdom); Institut Laue–Langevin, 71 Avenue des Martyrs, CS20156, 38042 Grenoble Cedex 9 (France)

    2015-07-07

    The dynamics of the human oligonucleotide AG{sub 3}(T{sub 2}AG{sub 3}){sub 3} has been investigated by incoherent neutron scattering in the sub-nanosecond timescale. A hydration-dependent dynamical activation of thermal fluctuations in weakly hydrated samples was found, similar to that of protein powders. The amplitudes of such thermal fluctuations were evaluated in two different exchanged wave-vector ranges, so as to single out the different contributions from intra- and inter-nucleotide dynamics. The activation energy was calculated from the temperature-dependent characteristic times of the corresponding dynamical processes. The trends of both amplitudes and activation energies support a picture where oligonucleotides possess a larger conformational flexibility than long DNA sequences. This additional flexibility, which likely results from a significant relative chain-end contribution to the average chain dynamics, could be related to the strong structural polymorphism of the investigated oligonucleotides.

  2. Streptavidin-coated gold nanoparticles: critical role of oligonucleotides on stability and fractal aggregation

    Directory of Open Access Journals (Sweden)

    Roberta D'Agata

    2017-01-01

    Full Text Available Gold nanoparticles (AuNPs exhibit unique properties that can be modulated through a tailored surface functionalization, enabling their targeted use in biochemical sensing and medical diagnostics. In particular, streptavidin-modified AuNPs are increasingly used for biosensing purposes. We report here a study of AuNPs surface-functionalized with streptavidin-biotinylated oligonucleotide, focussing on the role played by the oligonucleotide probes in the stabilization/destabilization of the functionalized nanoparticle dispersion. The behaviour of the modified AuNP dispersion as a consequence of the competitive displacement of the biotinylated oligonucleotide has been investigated and the critical role of displaced oligonucletides in triggering the quasi one-dimensional aggregation of nanoparticles is demonstrated for the first time. The thorough understanding of the fundamental properties of bioconjugated AuNPs is of great importance for the design of highly sensitive and reliable functionalized AuNP-based assays.

  3. Reliability and applications of statistical methods based on oligonucleotide frequencies in bacterial and archaeal genomes

    DEFF Research Database (Denmark)

    Bohlin, J; Skjerve, E; Ussery, David

    2008-01-01

    with here are mainly used to examine similarities between archaeal and bacterial DNA from different genomes. These methods compare observed genomic frequencies of fixed-sized oligonucleotides with expected values, which can be determined by genomic nucleotide content, smaller oligonucleotide frequencies......, or be based on specific statistical distributions. Advantages with these statistical methods include measurements of phylogenetic relationship with relatively small pieces of DNA sampled from almost anywhere within genomes, detection of foreign/conserved DNA, and homology searches. Our aim was to explore...... the reliability and best suited applications for some popular methods, which include relative oligonucleotide frequencies (ROF), di- to hexanucleotide zero'th order Markov methods (ZOM) and 2.order Markov chain Method (MCM). Tests were performed on distant homology searches with large DNA sequences, detection...

  4. Detection of DNA oligonucleotides with base mutations by terahertz spectroscopy and microstructures.

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    Mingjie Tang

    Full Text Available DNA oligonucleotides with a 5-base mutation at the 3'-terminus were investigated by terahertz (THz spectroscopy in a marker-free manner. The four single-stranded oligonucleotides with 17nt have been detected with specificity on a microfluidic chip, and corroborated by spectral measurements with split-ring resonators. The number of hydrogen bonds formed between the oligonucleotide and its surrounding water molecules, deemed a key contribution to the THz absorption of biological solutions, was explored by molecular dynamics simulations to explain the experimental findings. Our work underlies the feasibility of THz spectroscopy combined with microstructures for marker-free detection of DNA, which may form the basis of a prospective diagnostic tool for studying genic mutation.

  5. Sensing miRNA: Signal Amplification by Cognate RISC for Intracellular Detection of miRNA in Live Cells.

    Science.gov (United States)

    Kavishwar, Amol; Medarova, Zdravka

    2016-01-01

    The ability to detect miRNA expression in live cells would leave these cells available for further manipulation or culture. Here, we describe the design of a miRNA sensor oligonucleotide whose sequence mimics the target mRNA. The sensor has a fluorescent label on one end of the oligo and a quencher on the other. When inside the cell, the sensor is recognized by its cognate miRNA-RISC and gets cleaved, setting the fluorophore free from its quencher. This results in fluorescence "turn on." Since cleavage by the RISC complex is an enzymatic process, the described approach has a very high level of sensitivity (nM). The rate of nonspecific cleavage of the sensor is very slow permitting the collection of meaningful signal over a long period of time.

  6. Polynucleotides. XXXII. Further studies on the synthesis of oligonucleotides containing 8,2'-S-cycloadenosine.

    Science.gov (United States)

    Ikehara, M; Tezuka, T

    1975-01-01

    A dinucleoside monophosphate, 8,2'-anhydro-8-mercapto-9-beta-D-arabinofuranosyladenine phosphoryl-(3'-5')-inosine (AspI) was synthesized by the condensation of protected 8-mercapto-adenosine 2',3'-cyclic phosphate and 2',3'-isopropylideneinosine with diphenylphosphorochloridate. 8-Mercaptoadenosine 2',3'-cyclic phosphate was polymerized by using tetraphenyl pyrophosphate as the condensing reagent. As oligonucleotides, thus obtained, contained some uncyclized 8-mercaptoadenosine residues and were cleaved at these sites with 0.3N KOH. As 5'-phosphate was synthesized and polymerized with DCC to give oligonucleotides with chain lengths 2 to 9. PMID:170595

  7. Genome-wide identification of specific oligonucleotides using artificial neural network and computational genomic analysis

    Directory of Open Access Journals (Sweden)

    Chen Jiun-Ching

    2007-05-01

    Full Text Available Abstract Background Genome-wide identification of specific oligonucleotides (oligos is a computationally-intensive task and is a requirement for designing microarray probes, primers, and siRNAs. An artificial neural network (ANN is a machine learning technique that can effectively process complex and high noise data. Here, ANNs are applied to process the unique subsequence distribution for prediction of specific oligos. Results We present a novel and efficient algorithm, named the integration of ANN and BLAST (IAB algorithm, to identify specific oligos. We establish the unique marker database for human and rat gene index databases using the hash table algorithm. We then create the input vectors, via the unique marker database, to train and test the ANN. The trained ANN predicted the specific oligos with high efficiency, and these oligos were subsequently verified by BLAST. To improve the prediction performance, the ANN over-fitting issue was avoided by early stopping with the best observed error and a k-fold validation was also applied. The performance of the IAB algorithm was about 5.2, 7.1, and 6.7 times faster than the BLAST search without ANN for experimental results of 70-mer, 50-mer, and 25-mer specific oligos, respectively. In addition, the results of polymerase chain reactions showed that the primers predicted by the IAB algorithm could specifically amplify the corresponding genes. The IAB algorithm has been integrated into a previously published comprehensive web server to support microarray analysis and genome-wide iterative enrichment analysis, through which users can identify a group of desired genes and then discover the specific oligos of these genes. Conclusion The IAB algorithm has been developed to construct SpecificDB, a web server that provides a specific and valid oligo database of the probe, siRNA, and primer design for the human genome. We also demonstrate the ability of the IAB algorithm to predict specific oligos through

  8. Design, validation and annotation of transcriptome-wide oligonucleotide probes for the oligochaete annelid Eisenia fetida.

    Directory of Open Access Journals (Sweden)

    Ping Gong

    Full Text Available High density oligonucleotide probe arrays have increasingly become an important tool in genomics studies. In organisms with incomplete genome sequence, one strategy for oligo probe design is to reduce the number of unique probes that target every non-redundant transcript through bioinformatic analysis and experimental testing. Here we adopted this strategy in making oligo probes for the earthworm Eisenia fetida, a species for which we have sequenced transcriptome-scale expressed sequence tags (ESTs. Our objectives were to identify unique transcripts as targets, to select an optimal and non-redundant oligo probe for each of these target ESTs, and to annotate the selected target sequences. We developed a streamlined and easy-to-follow approach to the design, validation and annotation of species-specific array probes. Four 244K-formatted oligo arrays were designed using eArray and were hybridized to a pooled E. fetida cRNA sample. We identified 63,541 probes with unsaturated signal intensities consistently above the background level. Target transcripts of these probes were annotated using several sequence alignment algorithms. Significant hits were obtained for 37,439 (59% probed targets. We validated and made publicly available 63.5K oligo probes so the earthworm research community can use them to pursue ecological, toxicological, and other functional genomics questions. Our approach is efficient, cost-effective and robust because it (1 does not require a major genomics core facility; (2 allows new probes to be easily added and old probes modified or eliminated when new sequence information becomes available, (3 is not bioinformatics-intensive upfront but does provide opportunities for more in-depth annotation of biological functions for target genes; and (4 if desired, EST orthologs to the UniGene clusters of a reference genome can be identified and selected in order to improve the target gene specificity of designed probes. This approach is

  9. Regulation of the activity of the promoter of RNA-induced silencing, C3PO.

    Science.gov (United States)

    Sahu, Shriya; Williams, Leo; Perez, Alberto; Philip, Finly; Caso, Giuseppe; Zurawsky, Walter; Scarlata, Suzanne

    2017-09-01

    RNA-induced silencing is a process which allows cells to regulate the synthesis of specific proteins. RNA silencing is promoted by the protein C3PO (component 3 of RISC). We have previously found that phospholipase Cβ, which increases intracellular calcium levels in response to specific G protein signals, inhibits C3PO activity towards certain genes. Understanding the parameters that control C3PO activity and which genes are impacted by G protein activation would help predict which genes are more vulnerable to downregulation. Here, using a library of 10 18 oligonucleotides, we show that C3PO binds oligonucleotides with structural specificity but little sequence specificity. Alternately, C3PO hydrolyzes oligonucleotides with a rate that is sensitive to substrate stability. Importantly, we find that oligonucleotides with higher Tm values are inhibited by bound PLCβ. This finding is supported by microarray analysis in cells over-expressing PLCβ1. Taken together, this study allows predictions of the genes whose post-transcriptional regulation is responsive to the G protein/phospholipase Cβ/calcium signaling pathway. © 2017 The Protein Society.

  10. Oligonucleotide-based pharmaceuticals: Non-clinical and clinical safety signals and non-clinical testing strategies.

    Science.gov (United States)

    Mustonen, Enni-Kaisa; Palomäki, Tiina; Pasanen, Markku

    2017-11-01

    Antisense oligonucleotides, short interfering RNAs (siRNAs) and aptamers are oligonucleotide-based pharmaceuticals with a promising role in targeted therapies. Currently, five oligonucleotide-based pharmaceuticals have achieved marketing authorization in Europe or USA and many more are undergoing clinical testing. However, several safety concerns have been raised in non-clinical and clinical studies. Oligonucleotides share properties with both chemical and biological pharmaceuticals and therefore they pose challenges also from the regulatory point of view. We have analyzed the safety data of oligonucleotides and evaluated the applicability of current non-clinical toxicological guidelines for assessing the safety of oligonucleotide-based pharmaceuticals. Oligonucleotide-based pharmaceuticals display a similar toxicological profile, exerting adverse effects on liver and kidney, evoking hematological alterations, as well as causing immunostimulation and prolonging the coagulation time. It is possible to extrapolate some of these effects from non-clinical studies to humans. However, evaluation strategies for genotoxicity testing of "non-natural" oligonucleotides should be revised. Additionally, the selective use of surrogates and prediction of clinical endpoints for non-clinically observed immunostimulation is complicated by its multiple potential manifestations, demanding improvements in the testing strategies. Utilizing more relevant and mechanistic-based approaches and taking better account of species differences, could possibly improve the prediction of relevant immunological/proinflammatory effects in humans. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Synthesis and Biophysical Investigations of Oligonucleotides Containing Galactose-Modified DNA, LNA and 2'-Amino-LNA Monomers

    DEFF Research Database (Denmark)

    Ries, Annika; Kumar, Rajesh; Lou, Chenguang

    2016-01-01

    Galactose-modified thymidine, LNA-T and 2'-amino-LNA-T nucleosides were synthesized, converted into the corresponding phosphoramidite derivatives and introduced into short oligonucleotides. Compared to the unmodified control strands, the galactose-modified oligonucleotides in general, and the N2'...

  12. RNA synthesis is modulated by G-quadruplex formation in Hepatitis C virus negative RNA strand.

    Science.gov (United States)

    Chloé, Jaubert; Amina, Bedrat; Laura, Bartolucci; Carmelo, Di Primo; Michel, Ventura; Jean-Louis, Mergny; Samir, Amrane; Marie-Line, Andreola

    2018-05-25

    DNA and RNA guanine-rich oligonucleotides can form non-canonical structures called G-quadruplexes or "G4" that are based on the stacking of G-quartets. The role of DNA and RNA G4 is documented in eukaryotic cells and in pathogens such as viruses. Yet, G4 have been identified only in a few RNA viruses, including the Flaviviridae family. In this study, we analysed the last 157 nucleotides at the 3'end of the HCV (-) strand. This sequence is known to be the minimal sequence required for an efficient RNA replication. Using bioinformatics and biophysics, we identified a highly conserved G4-prone sequence located in the stem-loop IIy' of the negative strand. We also showed that the formation of this G-quadruplex inhibits the in vitro RNA synthesis by the RdRp. Furthermore, Phen-DC3, a specific G-quadruplex binder, is able to inhibit HCV viral replication in cells in conditions where no cytotoxicity was measured. Considering that this domain of the negative RNA strand is well conserved among HCV genotypes, G4 ligands could be of interest for new antiviral therapies.

  13. Quantitative Analysis of Survivin Protein Expression and Its Therapeutic Depletion by an Antisense Oligonucleotide in Human Lung Tumors

    Directory of Open Access Journals (Sweden)

    Anna L Olsen

    2012-01-01

    Full Text Available RNA-directed antisense and interference therapeutics are a promising treatment option for cancer. The demonstration of depletion of target proteins within human tumors in vivo using validated methodology will be a key to the application of this technology. Here, we present a flow cytometric-based approach to quantitatively determine protein levels in solid tumor material derived by fiber optic brushing (FOB of non-small cell lung cancer (NSCLC patients. Focusing upon the survivin protein, and its depletion by an antisense oligonucleotide (ASO (LY2181308, we show that we can robustly identify a subpopulation of survivin positive tumor cells in FOB samples, and, moreover, detect survivin depletion in tumor samples from a patient treated with LY2181308. Survivin depletion appears to be a result of treatment with this ASO, because a tumor treated with conventional cytotoxic chemotherapy did not exhibit a decreased percentage of survivin positive cells. Our approach is likely to be broadly applicable to, and useful for, the quantification of protein levels in tumor samples obtained as part of clinical trials and studies, facilitating the proof-of-principle testing of novel targeted therapies.

  14. Preclinical Studies on Intestinal Administration of Antisense Oligonucleotides as a Model for Oral Delivery for Treatment of Duchenne Muscular Dystrophy

    Directory of Open Access Journals (Sweden)

    Maaike van Putten

    2014-01-01

    Full Text Available Antisense oligonucleotides (AONs used to reframe dystrophin mRNA transcripts for Duchenne muscular dystrophy (DMD patients are tested in clinical trials. Here, AONs are administered subcutaneously and intravenously, while the less invasive oral route would be preferred. Oral delivery of encapsulated AONs supplemented with a permeation enhancer, sodium caprate, has been successfully used to target tumor necrosis factor (TNF-α expression in liver. To test the feasibility of orally delivered AONs for DMD, we applied 2′-O-methyl phosphorothioate AONs (with or without sodium caprate supplementation directly to the intestine of mdx mice and compared pharmacokinetics and -dynamics with intravenous, intraperitoneal, and subcutaneous delivery. Intestinally infused AONs were taken up, but resulted in lower plasma levels compared to other delivery routes, although bioavailability could be largely improved by supplementation of sodium caprate. After intestinal infusion, AON levels in all tissues were lower than for other administration routes, as were the ratios of target versus nontarget organ levels, except for diaphragm and heart where comparable levels and ratios were observed. For each administration route, low levels of exon skipping in triceps was observed 3 hours post-AON administration. These data suggest that oral administration of naked 2′-O-methyl phosphorothioate AONs may be feasible, but only when high AON concentrations are used in combination with sodium caprate.

  15. Development and application of an oligonucleotide microarray and real-time quantitative PCR for detection of wastewater bacterial pathogens

    Energy Technology Data Exchange (ETDEWEB)

    Lee, Dae-Young [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada)], E-mail: daeyoung.lee@ec.gc.ca; Lauder, Heather; Cruwys, Heather; Falletta, Patricia [National Water Research Institute, Environment Canada, 867 Lakeshore Road, Burlington, Ontario, L7R 4A6 (Canada); Beaudette, Lee A. [Environmental Science and Technology Centre, Environment Canada, 335 River Road South, Ottawa, Ontario, K1A 0H3 (Canada)], E-mail: lee.beaudette@ec.gc.ca

    2008-07-15

    Conventional microbial water quality test methods are well known for their technical limitations, such as lack of direct pathogen detection capacity and low throughput capability. The microarray assay has recently emerged as a promising alternative for environmental pathogen monitoring. In this study, bacterial pathogens were detected in municipal wastewater using a microarray equipped with short oligonucleotide probes targeting 16S rRNA sequences. To date, 62 probes have been designed against 38 species, 4 genera, and 1 family of pathogens. The detection sensitivity of the microarray for a waterborne pathogen Aeromonas hydrophila was determined to be approximately 1.0% of the total DNA, or approximately 10{sup 3}A. hydrophila cells per sample. The efficacy of the DNA microarray was verified in a parallel study where pathogen genes and E. coli cells were enumerated using real-time quantitative PCR (qPCR) and standard membrane filter techniques, respectively. The microarray and qPCR successfully detected multiple wastewater pathogen species at different stages of the disinfection process (i.e. secondary effluents vs. disinfected final effluents) and at two treatment plants employing different disinfection methods (i.e. chlorination vs. UV irradiation). This result demonstrates the effectiveness of the DNA microarray as a semi-quantitative, high throughput pathogen monitoring tool for municipal wastewater.

  16. Optimization of Peptide Nucleic Acid Antisense Oligonucleotides for Local and Systemic Dystrophin Splice Correction in the mdx Mouse

    Science.gov (United States)

    Yin, HaiFang; Betts, Corinne; Saleh, Amer F; Ivanova, Gabriela D; Lee, Hyunil; Seow, Yiqi; Kim, Dalsoo; Gait, Michael J; Wood, Matthew JA

    2010-01-01

    Antisense oligonucleotides (AOs) have the capacity to alter the processing of pre-mRNA transcripts in order to correct the function of aberrant disease-related genes. Duchenne muscular dystrophy (DMD) is a fatal X-linked muscle degenerative disease that arises from mutations in the DMD gene leading to an absence of dystrophin protein. AOs have been shown to restore the expression of functional dystrophin via splice correction by intramuscular and systemic delivery in animal models of DMD and in DMD patients via intramuscular administration. Major challenges in developing this splice correction therapy are to optimize AO chemistry and to develop more effective systemic AO delivery. Peptide nucleic acid (PNA) AOs are an alternative AO chemistry with favorable in vivo biochemical properties and splice correcting abilities. Here, we show long-term splice correction of the DMD gene in mdx mice following intramuscular PNA delivery and effective splice correction in aged mdx mice. Further, we report detailed optimization of systemic PNA delivery dose regimens and PNA AO lengths to yield splice correction, with 25-mer PNA AOs providing the greatest splice correcting efficacy, restoring dystrophin protein in multiple peripheral muscle groups. PNA AOs therefore provide an attractive candidate AO chemistry for DMD exon skipping therapy. PMID:20068555

  17. MicroRNA silencing in primates: towards development of novel therapeutics

    DEFF Research Database (Denmark)

    Petri, Andreas; Lindow, Morten; Kauppinen, Sakari

    2009-01-01

    MicroRNAs (miRNA) comprise an abundant class of small noncoding RNAs that act as important posttranscriptional regulators of gene expression. Accumulating evidence showing that aberrantly expressed miRNAs play important roles in human cancers underscores them as potential targets for therapeutic ...... intervention. Recent reports on efficient miRNA silencing in rodents and nonhuman primates using high-affinity targeting by chemically modified antisense oligonucleotides highlight the utility of such compounds in the development of miRNA-based cancer therapeutics....

  18. Eukaryotic RNA polymerase subunit RPB8 is a new relative of the OB family.

    Science.gov (United States)

    Krapp, S; Kelly, G; Reischl, J; Weinzierl, R O; Matthews, S

    1998-02-01

    RNA polymerase II subunit RPB8 is an essential subunit that is highly conserved throughout eukaryotic evolution and is present in all three types of nuclear RNA polymerases. We report the first high resolution structural insight into eukaryotic RNA polymerase architecture with the solution structure of RPB8 from Saccharomyces cerevisiae. It consists of an eight stranded, antiparallel beta-barrel, four short helical regions and a large, unstructured omega-loop. The strands are connected in classic Greek-key fashion. The overall topology is unusual and contains a striking C2 rotational symmetry. Furthermore, it is most likely a novel associate of the oligonucleotide/oligosaccharide (OB) binding protein class.

  19. Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe

    Science.gov (United States)

    Xue, Yong; Wilkes, Jon G.; Moskal, Ted J.; Williams, Anna J.; Cooper, Willie M.; Nayak, Rajesh; Rafii, Fatemeh; Buzatu, Dan A.

    2016-01-01

    Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR) and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC) assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts. PMID:26913737

  20. Development of a Flow Cytometry-Based Method for Rapid Detection of Escherichia coli and Shigella Spp. Using an Oligonucleotide Probe.

    Directory of Open Access Journals (Sweden)

    Yong Xue

    Full Text Available Standard methods to detect Escherichia coli contamination in food use the polymerase chain reaction (PCR and agar culture plates. These methods require multiple incubation steps and take a long time to results. An improved rapid flow-cytometry based detection method was developed, using a fluorescence-labeled oligonucleotide probe specifically binding a16S rRNA sequence. The method positively detected 51 E. coli isolates as well as 4 Shigella species. All 27 non-E. coli strains tested gave negative results. Comparison of the new genetic assay with a total plate count (TPC assay and agar plate counting indicated similar sensitivity, agreement between cytometry cell and colony counts. This method can detect a small number of E.coli cells in the presence of large numbers of other bacteria. This method can be used for rapid, economical, and stable detection of E. coli and Shigella contamination in the food industry and other contexts.

  1. A hyaluronic acid-based hydrogel enabling CD44-mediated chondrocyte binding and gapmer oligonucleotide release for modulation of gene expression in osteoarthritis

    DEFF Research Database (Denmark)

    Cai, Yunpeng; López-Ruiz, Elena; Wengel, Jesper

    2017-01-01

    Hyaluronic acid (HA) is an attractive biomaterial for osteoarthritis (OA) treatment due to inherent functional and compatibility properties as an endogenous knee joint component. In this work, we describe a HA-based hydrogel with the dual functionality of increased CD44-dependent chondrocyte......:3) for identifying designs displaying optimal engagement of OA patient-derived CD44-expressing chondrocytes. Correlation was found between cell binding and CD44 expression, with maximal binding exhibited at a HA/chitosan ratio of 7:3, that was 181% higher than CD44-negative MCF-7 cell control cells. Transfection...... agent-free uptake into OA chondrocytes of fluorescent 13-mer DNA oligonucleotides with a flanked locked nucleic acid (LNA) gapmer design, in contrast to naked siRNA, was demonstrated by confocal and flow cytometric analysis. A sustained and complete release over 5days was found with the 7:3 hydrogel...

  2. Mass spectrometric detection of siRNA in plasma samples for doping control purposes.

    Science.gov (United States)

    Kohler, Maxie; Thomas, Andreas; Walpurgis, Katja; Schänzer, Wilhelm; Thevis, Mario

    2010-10-01

    Small interfering ribonucleic acid (siRNA) molecules can effect the expression of any gene by inducing the degradation of mRNA. Therefore, these molecules can be of interest for illicit performance enhancement in sports by affecting different metabolic pathways. An example of an efficient performance-enhancing gene knockdown is the myostatin gene that regulates muscle growth. This study was carried out to provide a tool for the mass spectrometric detection of modified and unmodified siRNA from plasma samples. The oligonucleotides are purified by centrifugal filtration and the use of an miRNA purification kit, followed by flow-injection analysis using an Exactive mass spectrometer to yield the accurate masses of the sense and antisense strands. Although chromatography and sensitive mass spectrometric analysis of oligonucleotides are still challenging, a method was developed and validated that has adequate sensitivity (limit of detection 0.25-1 nmol mL(-1)) and performance (precision 11-21%, recovery 23-67%) for typical antisense oligonucleotides currently used in clinical studies.

  3. Polyethylene glycol and polyvinylpirrolidone effect on bacterial rRNA extraction and hybridization from cells exposed to tannins.

    OpenAIRE

    ARCURI, P.B.; THONNEY, M.L.; SCHOFIELD, P.; PELL, A.N.

    2003-01-01

    In order to detect fluctuations in ruminal microbial populations due to forage tannins using 16S ribosomal RNA (rRNA) probes, recovery of intact rRNA is required. The objective of this work was to evaluate the effect of polyethylene glycol (PEG) and polyvinylpirrolidone (PVP) on extraction of bacterial rRNA, in the presence of tannins from tropical legume forages and other sources, that hybridize with oligonucleotide probes. Ruminococcus albus 8 cells were exposed to 8 g/L tannic acid or 1 g/...

  4. Application of hierarchical oligonucleotide primer extension (HOPE) to assess relative abundances of ammonia- and nitrite-oxidizing bacteria

    KAUST Repository

    Scarascia, Giantommaso

    2017-04-04

    Background: Establishing an optimal proportion of nitrifying microbial populations, including ammonia-oxidizing bacteria (AOB), nitrite-oxidizing bacteria (NOB), complete nitrite oxidizers (comammox) and ammonia-oxidizing archaea (AOA), is important for ensuring the efficiency of nitrification in water treatment systems. Hierarchical oligonucleotide primer extension (HOPE), previously developed to rapidly quantify relative abundances of specific microbial groups of interest, was applied in this study to track the abundances of the important nitrifying bacterial populations. Results: The method was tested against biomass obtained from a laboratory-scale biofilm-based trickling reactor, and the findings were validated against those obtained by 16S rRNA gene-based amplicon sequencing. Our findings indicated a good correlation between the relative abundance of nitrifying bacterial populations obtained using both HOPE and amplicon sequencing. HOPE showed a significant increase in the relative abundance of AOB, specifically Nitrosomonas, with increasing ammonium content and shock loading (p < 0.001). In contrast, Nitrosospira remained stable in its relative abundance against the total community throughout the operational phases. There was a corresponding significant decrease in the relative abundance of NOB, specifically Nitrospira and those affiliated to comammox, during the shock loading. Based on the relative abundance of AOB and NOB (including commamox) obtained from HOPE, it was determined that the optimal ratio of AOB against NOB ranged from 0.2 to 2.5 during stable reactor performance. Conclusions: Overall, the HOPE method was developed and validated against 16S rRNA gene-based amplicon sequencing for the purpose of performing simultaneous monitoring of relative abundance of nitrifying populations. Quantitative measurements of these nitrifying populations obtained via HOPE would be indicative of reactor performance and nitrification functionality.

  5. Sets of RNA repeated tags and hybridization-sensitive fluorescent probes for distinct images of RNA in a living cell.

    Directory of Open Access Journals (Sweden)

    Takeshi Kubota

    Full Text Available BACKGROUND: Imaging the behavior of RNA in a living cell is a powerful means for understanding RNA functions and acquiring spatiotemporal information in a single cell. For more distinct RNA imaging in a living cell, a more effective chemical method to fluorescently label RNA is now required. In addition, development of the technology labeling with different colors for different RNA would make it easier to analyze plural RNA strands expressing in a cell. METHODOLOGY/PRINCIPAL FINDINGS: Tag technology for RNA imaging in a living cell has been developed based on the unique chemical functions of exciton-controlled hybridization-sensitive oligonucleotide (ECHO probes. Repetitions of selected 18-nucleotide RNA tags were incorporated into the mRNA 3'-UTR. Pairs with complementary ECHO probes exhibited hybridization-sensitive fluorescence emission for the mRNA expressed in a living cell. The mRNA in a nucleus was detected clearly as fluorescent puncta, and the images of the expression of two mRNAs were obtained independently and simultaneously with two orthogonal tag-probe pairs. CONCLUSIONS/SIGNIFICANCE: A compact and repeated label has been developed for RNA imaging in a living cell, based on the photochemistry of ECHO probes. The pairs of an 18-nt RNA tag and the complementary ECHO probes are highly thermostable, sequence-specifically emissive, and orthogonal to each other. The nucleotide length necessary for one tag sequence is much shorter compared with conventional tag technologies, resulting in easy preparation of the tag sequences with a larger number of repeats for more distinct RNA imaging.

  6. Translational inhibition of CTX M extended spectrum β-lactamase in clinical strains of Escherichia coli by synthetic antisense oligonucleotides partially restores sensitivity to cefotaxime.

    Directory of Open Access Journals (Sweden)

    John Benedict Readman

    2016-03-01

    Full Text Available Synthetic antisense oligomers are DNA mimics that can specifically inhibit gene expression at the translational level by ribosomal steric hindrance. They bind to their mRNA targets by Watson Crick base pairing and are resistant to degradation by both nucleases and proteases. A 25 mer phosphorodiamidate morpholino oligomer (PMO and a 13 mer polyamide (peptide nucleic acid (PNA were designed to target mRNA (positions -4 to +21, and –17 to –5 respectively close to the translational initiation site of the extended spectrum β lactamase resistance genes of CTX M group 1. These antisense oligonucleotides were found to inhibit β lactamase activity by up to 96% in a cell free translation transcription coupled system using an expression vector carrying a blaCTX-M-15 gene cloned from a clinical isolate. Despite evidence for up regulation of CTX-M gene expression, they were both found to significantly restore sensitivity to cefotaxime in E. coli AS19, an atypical cell wall permeable mutant, in a dose dependant manner (0 - 40 nM. The PMO and PNA were covalently bound to the cell penetrating peptide (KFF3K and both significantly (P<0.05 increased sensitivity to cefotaxime in a dose dependent manner (0 - 40 nM in field isolates harbouring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine Dalgarno region of blaCTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates.

  7. An oral oligonucleotide delivery system based on a thiolated polymer: Development and in vitro evaluation.

    Science.gov (United States)

    Martien, Ronny; Hoyer, Herbert; Perera, Glen; Schnürch, Andreas Bernkop

    2011-08-01

    The purpose of this study was to develop and evaluate an oral oligonucleotide delivery system based on a thiolated polymer/reduced glutathione (GSH) system providing a protective effect toward nucleases and permeation enhancement. A polycarbophil-cysteine conjugate (PCP-Cys) was synthesized. Enzymatic degradation of a model oligonucleotide by DNase I and within freshly collected intestinal fluid was investigated in the absence and presence of PCP-Cys. Permeation studies with PCP-Cys/GSH versus control were performed in vitro on Caco-2 cell monolayers and ex vivo on rat intestinal mucosa. PCP-Cys displayed 223 ± 13.8 μmol thiol groups per gram polymer. After 4h, 61% of the free oligonucleotides were degraded by DNase I and 80% within intestinal fluid. In contrast, less than 41% (DNase I) and 60% (intestinal fluid) were degraded in the presence of 0.02% (m/v) PCP-Cys. Permeation studies revealed an 8-fold (Caco-2) and 10-fold (intestinal mucosa) increase in apparent permeability compared to buffer control. Hence, this PCP-Cys/GSH system might be a promising tool for the oral administration of oligonucleotides as it allows a significant protection toward degrading enzymes and facilitates their transport across intestinal membranes. Copyright © 2011 Elsevier B.V. All rights reserved.

  8. N-Branched acyclic nucleoside phosphonates as monomers for the synthesis of modified oligonucleotides

    Czech Academy of Sciences Publication Activity Database

    Hocková, Dana; Rosenbergová, Šárka; Ménová, Petra; Páv, Ondřej; Pohl, Radek; Novák, Pavel; Rosenberg, Ivan

    2015-01-01

    Roč. 13, č. 15 (2015), s. 4449-4458 ISSN 1477-0520 R&D Projects: GA ČR GAP207/11/0108; GA ČR GA13-26526S Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * oligonucleotides * solid phase synthesis Subject RIV: CC - Organic Chemistry Impact factor: 3.559, year: 2015

  9. A new achiral reagent for the incorporation of multiple amino groups into oligonucleotides

    DEFF Research Database (Denmark)

    Behrens, Carsten; Petersen, Kenneth H.; Egholm, Michael

    1995-01-01

    The synthesis of a new functionalized achiral linker reagent (10) for the incorporation of multiple primary amino groups into oligonucleotides is described. The linker reagent is compatible with conventional DNA-synthesis following the phosphoramidite methodology, and the linker can be incorporated...

  10. Short Oligonucleotides Aligned in Stretched Humid Matrix: Secondary DNA Structure in Poly(vinyl alcohol) Environment

    KAUST Repository

    Hanczyc, Piotr; Å kerman, Bjö rn; Nordé n, Bengt

    2012-01-01

    ) spectroscopy. Oligonucleotides of lengths varying between 10 (3.4 nm) and 60 bases (20.4 nm) were investigated with respect to structural properties in the gel-like polymer environment. The DNA conformation as a function of relative humidity reveals a strong

  11. Review on investigations of antisense oligonucleotides with the use of mass spectrometry.

    Science.gov (United States)

    Studzińska, Sylwia

    2018-01-01

    Antisense oligonucleotides have been investigated as potential drugs for years. They inhibit target gene or protein expression. The present review summarizes their modifications, modes of action, and applications of liquid chromatography coupled with mass spectrometry for qualitative and quantitative analysis of these compounds. The most recent reports on a given topic were given prominence, while some early studies were reviewed in order to provide a theoretical background. The present review covers the issues of using ion-exchange chromatography, ion-pair reversed-phase high performance liquid chromatography and hydrophilic interaction chromatography for the separation of antisense oligonucleotides. The application of mass spectrometry was described with regard to the ionization type used for the determination of these potential therapeutics. Moreover, the current approaches and applications of mass spectrometry for quantitative analysis of antisense oligonucleotides and their metabolites as well as their impurities during in vitro and in vivo studies were discussed. Finally, certain conclusions and perspectives on the determination of therapeutic oligonucleotides in various samples were briefly described. Copyright © 2017 Elsevier B.V. All rights reserved.

  12. Splice-correcting oligonucleotides restore BTK function in X-linked agammaglobulinemia model

    DEFF Research Database (Denmark)

    Bestas, Burcu; Moreno, Pedro M D; Blomberg, K Emelie M

    2014-01-01

    , splice-correcting oligonucleotides (SCOs) targeting mutated BTK transcripts for treating XLA. Both the SCO structural design and chemical properties were optimized using 2'-O-methyl, locked nucleic acid, or phosphorodiamidate morpholino backbones. In order to have access to an animal model of XLA, we...

  13. The illusion of specific capture: surface and solution studies of suboptimal oligonucleotide hybridization

    Science.gov (United States)

    2013-01-01

    Background Hybridization based assays and capture systems depend on the specificity of hybridization between a probe and its intended target. A common guideline in the construction of DNA microarrays, for instance, is that avoiding complementary stretches of more than 15 nucleic acids in a 50 or 60-mer probe will eliminate sequence specific cross-hybridization reactions. Here we present a study of the behavior of partially matched oligonucleotide pairs with complementary stretches starting well below this threshold complementarity length – in silico, in solution, and at the microarray surface. The modeled behavior of pairs of oligonucleotide probes and their targets suggests that even a complementary stretch of sequence 12 nt in length would give rise to specific cross-hybridization. We designed a set of binding partners to a 50-mer oligonucleotide containing complementary stretches from 6 nt to 21 nt in length. Results Solution melting experiments demonstrate that stable partial duplexes can form when only 12 bp of complementary sequence are present; surface hybridization experiments confirm that a signal close in magnitude to full-strength signal can be obtained from hybridization of a 12 bp duplex within a 50mer oligonucleotide. Conclusions Microarray and other molecular capture strategies that rely on a 15 nt lower complementarity bound for eliminating specific cross-hybridization may not be sufficiently conservative. PMID:23445545

  14. Direct Mutagenesis of Thousands of Genomic Targets using Microarray-derived Oligonucleotides

    DEFF Research Database (Denmark)

    Bonde, Mads; Kosuri, Sriram; Genee, Hans Jasper

    2015-01-01

    Multiplex Automated Genome Engineering (MAGE) allows simultaneous mutagenesis of multiple target sites in bacterial genomes using short oligonucleotides. However, large-scale mutagenesis requires hundreds to thousands of unique oligos, which are costly to synthesize and impossible to scale-up by ...

  15. Synthesis and Excellent Duplex Stability of Oligonucleotides Containing 2'-Amino-LNA Functionalized with Galactose Units

    DEFF Research Database (Denmark)

    Kumar, Rajesh; Ries, Annika; Wengel, Jesper

    2017-01-01

    A convenient method for the preparation of oligonucleotides containing internally-attached galactose and triantennary galactose units has been developed based on click chemistry between 2'-N-alkyne 2'-amino-LNA nucleosides and azido-functionalized galactosyl building blocks. The synthesized...

  16. Effective intracellular delivery of oligonucleotides in order to make sense of antisense

    NARCIS (Netherlands)

    Shi, FX; Hoekstra, D

    2004-01-01

    For more than two decades, antisense oligonucleotides (ODNs) have been used to modulate gene expression for the purpose of applications in cell biology and for development of novel sophisticated medical therapeutics. Conceptually, the antisense approach represents an elegant strategy, involving the

  17. Design and analysis of effects of triplet repeat oligonucleotides in cell models for myotonic dystrophy

    NARCIS (Netherlands)

    Gonzalez-Barriga, A.; Mulders, S.A.M.; Giessen, J. van der; Hooijer, J.D.; Bijl, S.; Kessel, I.D.G. van; Beers, J. van; Deutekom, J.C. van; Fransen, J.A.M.; Wieringa, B.; Wansink, D.G.

    2013-01-01

    Myotonic dystrophy type 1 (DM1) is caused by DM protein kinase (DMPK) transcripts containing an expanded (CUG)n repeat. Antisense oligonucleotide (AON)-mediated suppression of these mutant RNAs is considered a promising therapeutic strategy for this severe disorder. Earlier, we identified a

  18. Synthetic Nucleic Acid Analogues in Gene Therapy: An Update for Peptide–Oligonucleotide Conjugates

    DEFF Research Database (Denmark)

    Taskova, Maria; Mantsiou, Anna; Astakhova, Kira

    2017-01-01

    The main objective of this work is to provide an update on synthetic nucleic acid analogues and nanoassemblies as tools in gene therapy. In particular, the synthesis and properties of peptide–oligonucleotide conjugates (POCs), which have high potential in research and as therapeutics, are described...

  19. Quantitation of ultraviolet-induced single-strand breaks using oligonucleotide chip

    International Nuclear Information System (INIS)

    Pal, Sukdeb; Kim, Min Jung; Choo, Jaebum; Kang, Seong Ho; Lee, Kyeong-Hee; Song, Joon Myong

    2008-01-01

    A simple, accurate and robust methodology was established for the direct quantification of ultraviolet (UV)-induced single-strand break (SSB) using oligonucleotide chip. Oligonucleotide chips were fabricated by covalently anchoring the fluorescent-labeled ssDNAs onto silicon dioxide chip surfaces. Assuming that the possibility of more than one UV-induced SSB to be generated in a small oligonucleotide is extremely low, SSB formation was investigated quantifying the endpoint probe density by fluorescence measurement upon UV irradiation. The SSB yields obtained based on the highly sensitive laser-induced fluorometric determination of fluorophore-labeled oligonucleotides were found to coincide well with that predicted from a theoretical extrapolation of the results obtained for plasmid DNAs using conventional agarose gel electrophoresis. The developed method has the potential to serve as a high throughput, sample-thrifty, and time saving tool to realize more realistic, and direct quantification of radiation and chemical-induced strand breaks. It will be especially useful for determining the frequency of SSBs or lesions convertible to SSBs by specific cleaving reagents or enzymes

  20. Pd0-Catalyzed Methyl Transfer on Nucleosides and Oligonucleotides, Envisaged as a PET Tracer

    Directory of Open Access Journals (Sweden)

    Eric Fouquet

    2013-11-01

    Full Text Available The methyl transfer reaction from activated monomethyltin, via a modified Stille coupling reaction, was studied under “ligandless” conditions on fully deprotected 5'-modified nucleosides and one dinucleotide. The reaction was optimized to proceed in a few minutes and quantitative yield, even under dilute conditions, thus affording a rapid and efficient new method for oligonucleotide labelling with carbon-11.

  1. High-frequency genome editing using ssDNA oligonucleotides with zinc-finger nucleases

    DEFF Research Database (Denmark)

    Chen, Fuqiang; Pruett-Miller, Shondra M; Huang, Yuping

    2011-01-01

    Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point...

  2. Labelling of nucleosides and oligonucleotides by solvatochromic 4-aminophthalimide fluorophore for studying DNA–protein interactions

    Czech Academy of Sciences Publication Activity Database

    Riedl, Jan; Pohl, Radek; Ernsting, N. P.; Orság, Petr; Fojta, Miroslav; Hocek, Michal

    2012-01-01

    Roč. 3, č. 9 (2012), s. 2797-2806 ISSN 2041-6520 R&D Projects: GA ČR GA203/09/0317; GA ČR GBP206/12/G151 Institutional support: RVO:61388963 ; RVO:68081707 Keywords : DNA * oligonucleotides * polymerase * phthalimide * nucleotides * fluorescent labeling Subject RIV: CC - Organic Chemistry Impact factor: 8.314, year: 2012

  3. High-density rhesus macaque oligonucleotide microarray design using early-stage rhesus genome sequence information and human genome annotations

    Directory of Open Access Journals (Sweden)

    Magness Charles L

    2007-01-01

    Full Text Available Abstract Background Until recently, few genomic reagents specific for non-human primate research have been available. To address this need, we have constructed a macaque-specific high-density oligonucleotide microarray by using highly fragmented low-pass sequence contigs from the rhesus genome project together with the detailed sequence and exon structure of the human genome. Using this method, we designed oligonucleotide probes to over 17,000 distinct rhesus/human gene orthologs and increased by four-fold the number of available genes relative to our first-generation expressed sequence tag (EST-derived array. Results We constructed a database containing 248,000 exon sequences from 23,000 human RefSeq genes and compared each human exon with its best matching sequence in the January 2005 version of the rhesus genome project list of 486,000 DNA contigs. Best matching rhesus exon sequences for each of the 23,000 human genes were then concatenated in the proper order and orientation to produce a rhesus "virtual transcriptome." Microarray probes were designed, one per gene, to the region closest to the 3' untranslated region (UTR of each rhesus virtual transcript. Each probe was compared to a composite rhesus/human transcript database to test for cross-hybridization potential yielding a final probe set representing 18,296 rhesus/human gene orthologs, including transcript variants, and over 17,000 distinct genes. We hybridized mRNA from rhesus brain and spleen to both the EST- and genome-derived microarrays. Besides four-fold greater gene coverage, the genome-derived array also showed greater mean signal intensities for genes present on both arrays. Genome-derived probes showed 99.4% identity when compared to 4,767 rhesus GenBank sequence tag site (STS sequences indicating that early stage low-pass versions of complex genomes are of sufficient quality to yield valuable functional genomic information when combined with finished genome information from

  4. Preparation of oligonucleotide microarray for radiation-associated gene expression detection and its application in lung cancer cell lines

    International Nuclear Information System (INIS)

    Guo Wanfeng; Lin Ruxian; Huang Jian; Guo Guozhen; Wang Shengqi

    2005-01-01

    Objective: The response of tumor cell to radiation is accompanied by complex change in patterns of gene expression. It is highly probable that a better understanding of molecular and genetic changes can help to sensitize the radioresistant tumor cells. Methods: Oligonucleotide microarray provides a powerful tool for high-throughput identifying a wider range of genes involved in the radioresistance. Therefore, the authors designed one oligonucleotide microarray according to the biological effect of IR. By using different radiosensitive lung cancer cell lines, the authors identified genes showing altered expression in lung cancer cell lines. To provide independent confirmation of microarray data, semi-quantitative RT-PCR was performed on a selection of genes. Results: In radioresistant A549 cell lines, a total of 18 genes were selected as having significant fold-changes compared to NCI-H446, 8 genes were up-regulated and 10 genes were down-regulated. Subsequently, A549 and NCI-H446 cells were delivered by ionizing radiation. In A549 cell line, we found 22 (19 up-regulated and 3 down-regulated) and 26 (8 up-regulated and 18 down-regulated) differentially expressed genes at 6h and 24h after ionizing radiation. In NCI-H446 cell line, we identified 17 (9 up-regulated and 8 down-regulated) and 18 (6 up-regulated and 12 down-regulated) differentially expressed genes at 6 h and 24 h after ionizing radiation. The authors tested seven genes (MDM2, p53, XRCC5, Bcl-2, PIM2, NFKBIA and Cyclin B1) for RT-PCR, and found that the results were in good agreement with those from the microarray data except for NFKBIA gene, even though the value for each mRNA level might be different between the two measurements. In present study, the authors identified some genes with cell proliferation and anti-apoptosis, such as MdM2, BCL-2, PKCz and PIM2 expression levels increased in A549 cells and decreased in NCI-H446 cells after radiation, and other genes with DNA repair, such as XRCC5, ERCC5

  5. Short Hairpin RNA (shRNA): Design, Delivery, and Assessment of Gene Knockdown

    Science.gov (United States)

    Moore, Chris B.; Guthrie, Elizabeth H.; Huang, Max Tze-Han; Taxman, Debra J.

    2013-01-01

    Shortly after the cellular mechanism of RNA interference (RNAi) was first described, scientists began using this powerful technique to study gene function. This included designing better methods for the successful delivery of small interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs) into mammalian cells. While the simplest method for RNAi is the cytosolic delivery of siRNA oligonucleotides, this technique is limited to cells capable of transfection and is primarily utilized during transient in vitro studies. The introduction of shRNA into mammalian cells through infection with viral vectors allows for stable integration of shRNA and long-term knockdown of the targeted gene; however, several challenges exist with the implementation of this technology. Here we describe some well-tested protocols which should increase the chances of successful design, delivery, and assessment of gene knockdown by shRNA. We provide suggestions for designing shRNA targets and controls, a protocol for sequencing through the secondary structure of the shRNA hairpin structure, and protocols for packaging and delivery of shRNA lentiviral particles. Using real-time PCR and functional assays we demonstrate the successful knockdown of ASC, an inflammatory adaptor molecule. These studies demonstrate the practicality of including two shRNAs with different efficacies of knockdown to provide an additional level of control and to verify dose dependency of functional effects. Along with the methods described here, as new techniques and algorithms are designed in the future, shRNA is likely to include further promising application and continue to be a critical component of gene discovery. PMID:20387148

  6. Nucleic acid extraction, oligonucleotide probes and PCR methods

    International Nuclear Information System (INIS)

    Zhongtang Yu; Forster, R.J.

    2005-01-01

    analysis, and hybridization-based methods, such as RNA-targeted hybridization, fluorescence in situ hybridization (FISH), and microarray, have been employed. The application of these molecular techniques has been changing our perspectives about ruminal and GI microbiota. Except for FISH, all these methods analyse DNA or RNA extracted from samples collected from rumen or GI tracts. Therefore, reliable and efficient DNA/RNA extraction is the pre-requisite of molecular ecological studies of ruminal microbiota

  7. RNA oxidation

    DEFF Research Database (Denmark)

    Kjaer, L. K.; Cejvanovic, V.; Henriken, T.

    2015-01-01

    .9 significant hazard ratio for death compared with the quartile with the lowest 8oxoGuo excretion when adjusted for age, sex, BMI, smoker status, s-HbA1c, urine protein excretion and s-cholesterol. We conclude that it is now established that RNA oxidation is an independent risk factor for death in type 2...

  8. Phage annealing proteins promote oligonucleotide-directed mutagenesis in Escherichia coli and mouse ES cells

    Directory of Open Access Journals (Sweden)

    Muyrers Joep PP

    2003-01-01

    Full Text Available Abstract Background The phage protein pairs, RecE/RecT from Rac or Redα/Redβ from λ, initiate efficient double strand break repair (DSBR in Escherichia coli that has proven very useful for DNA engineering. These phage pairs initiate DSBR either by annealing or by another mechanism that is not defined. Results Here we report that these proteins also mediate single strand oligonucleotide repair (ssOR at high efficiencies. The ssOR activity, unlike DSBR, does not require a phage exonuclease (RecE or Redα but only requires a phage annealing protein (RecT or Redβ. Notably, the P22 phage annealing protein Erf, which does not mediate the same DSBR reactions, also delivers ssOR activity. By altering aspects of the oligonucleotides, we document length and design parameters that affect ssOR efficiency to show a simple relationship to homologies either side of the repair site. Notably, ssOR shows strand bias. Oligonucleotides that can prime lagging strand replication deliver more ssOR than their leading complements. This suggests a model in which the annealing proteins hybridize the oligonucleotides to single stranded regions near the replication fork. We also show that ssOR is a highly efficient way to engineer BACs and can be detected in a eukaryotic cell upon expression of a phage annealing protein. Conclusion Phage annealing proteins can initiate the recombination of single stranded oligonucleotides into endogenous targets in Escherichia coli at very high efficiencies. This expands the repertoire of useful DNA engineering strategies, shows promise for applications in eukaryotic cells, and has implications for the unanswered questions regarding DSBR mediated by RecE/RecT and Redα/Redβ.

  9. Development, characterization and experimental validation of a cultivated sunflower (Helianthus annuus L. gene expression oligonucleotide microarray.

    Directory of Open Access Journals (Sweden)

    Paula Fernandez

    Full Text Available Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de. The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons. The resulting Sunflower Unigen Resource (SUR version 1.0 was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01 allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement.

  10. Drop drying on surfaces determines chemical reactivity - the specific case of immobilization of oligonucleotides on microarrays

    Science.gov (United States)

    2013-01-01

    Background Drop drying is a key factor in a wide range of technical applications, including spotted microarrays. The applied nL liquid volume provides specific reaction conditions for the immobilization of probe molecules to a chemically modified surface. Results We investigated the influence of nL and μL liquid drop volumes on the process of probe immobilization and compare the results obtained to the situation in liquid solution. In our data, we observe a strong relationship between drop drying effects on immobilization and surface chemistry. In this work, we present results on the immobilization of dye labeled 20mer oligonucleotides with and without an activating 5′-aminoheptyl linker onto a 2D epoxysilane and a 3D NHS activated hydrogel surface. Conclusions Our experiments identified two basic processes determining immobilization. First, the rate of drop drying that depends on the drop volume and the ambient relative humidity. Oligonucleotides in a dried spot react unspecifically with the surface and long reaction times are needed. 3D hydrogel surfaces allow for immobilization in a liquid environment under diffusive conditions. Here, oligonucleotide immobilization is much faster and a specific reaction with the reactive linker group is observed. Second, the effect of increasing probe concentration as a result of drop drying. On a 3D hydrogel, the increasing concentration of probe molecules in nL spotting volumes accelerates immobilization dramatically. In case of μL volumes, immobilization depends on whether the drop is allowed to dry completely. At non-drying conditions, very limited immobilization is observed due to the low oligonucleotide concentration used in microarray spotting solutions. The results of our study provide a general guideline for microarray assay development. They allow for the initial definition and further optimization of reaction conditions for the immobilization of oligonucleotides and other probe molecule classes to different

  11. Short G-rich oligonucleotides as a potential therapeutic for Huntington's Disease

    Directory of Open Access Journals (Sweden)

    Parekh-Olmedo Hetal

    2006-10-01

    Full Text Available Abstract Background Huntington's Disease (HD is an inherited autosomal dominant genetic disorder in which neuronal tissue degenerates. The pathogenesis of the disease appears to center on the development of protein aggregates that arise initially from the misfolding of the mutant HD protein. Mutant huntingtin (Htt is produced by HD genes that contain an increased number of glutamine codons within the first exon and this expansion leads to the production of a protein that misfolds. Recent studies suggest that mutant Htt can nucleate protein aggregation and interfere with a multitude of normal cellular functions. Results As such, efforts to find a therapy for HD have focused on agents that disrupt or block the mutant Htt aggregation pathway. Here, we report that short guanosine monotonic oligonucleotides capable of adopting a G-quartet structure, are effective inhibitors of aggregation. By utilizing a biochemical/immunoblotting assay as an initial screen, we identified a 20-mer, all G-oligonucleotide (HDG as an active molecule. Subsequent testing in a cell-based assay revealed that HDG was an effective inhibitor of aggregation of a fusion protein, comprised of a mutant Htt fragment and green fluorescent protein (eGFP. Taken together, our results suggest that a monotonic G-oligonucleotide, capable of adopting a G-quartet conformation is an effective inhibitor of aggregation. This oligonucleotide can also enable cell survival in PC12 cells overexpressing a mutant Htt fragment fusion gene. Conclusion Single-stranded DNA oligonucleotides capable of forming stable G-quartets can inhibit aggregation of the mutant Htt fragment protein. This activity maybe an important part of the pathogenecity of Huntington's Disease. Our results reveal a new class of agents that could be developed as a therapeutic approach for Huntington's Disease.

  12. Covalent Chemical 5'-Functionalization of RNA with Diazo Reagents.

    Science.gov (United States)

    Gampe, Christian M; Hollis-Symynkywicz, Micah; Zécri, Frédéric

    2016-08-22

    Functionalization of RNA at the 5'-terminus is important for analytical and therapeutic purposes. Currently, these RNAs are synthesized de novo starting with a chemically functionalized 5'-nucleotide, which is incorporated into RNA using chemical synthesis or biochemical techniques. Methods for direct chemical modification of native RNA would provide an attractive alternative but are currently underexplored. Herein, we report that diazo compounds can be used to selectively alkylate the 5'-phosphate of ribo(oligo)nucleotides to give RNA labelled through a native phosphate ester bond. We applied this method to functionalize oligonucleotides with biotin and an orthosteric inhibitor of the eukaryotic initiation factor 4E (eIF4E), an enzyme involved in mRNA recognition. The modified RNA binds to eIF4E, demonstrating the utility of this labelling technique to modulate biological activity of RNA. This method complements existing techniques and may be used to chemically introduce a broad range of functional handles at the 5'-end of RNA. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Quantifying filamentous microorganisms in activated sludge before, during, and after an incident of foaming by oligonucleotide probe hybridizations and antibody staining.

    Science.gov (United States)

    Oerther, D B; de los Reyes, F L; de los Reyes, M F; Raskin, L

    2001-10-01

    Quantitative oligonucleotide probe hybridizations, immunostaining, and a simple foaming potential test were used to follow an incident of seasonal filamentous foaming at the Urbana-Champaign Sanitary District, Northeast Wastewater Treatment Plant. A positive correlation was observed between an increase in foaming potential and the appearance of foam on the surfaces of aeration basins and secondary clarifiers. In addition, during the occurrence of foaming, the mass and activity of Gordonia spp. increased as measured by fluorescence in situ hybridization, antibody staining, and quantitative membrane hybridization of RNA extracts. An increase in Gordonia spp. rRNA levels from 0.25 to 1.4% of total rRNA was observed using quantitative membrane hybridizations, whereas during the same period, the fraction of mixed liquor volatile suspended solids attributed to Gordonia spp. increased from 4% to more than 32% of the total mixed liquor volatile suspended solids. These results indicate that both the activity and biomass level of Gordonia spp. in activated sludge increased relative to the activity aid the biomass level of the complete microbial community during a seasonal occurrence of filamentous foaming. Thus, Gordonia spp. may represent a numerically dominant but metabolically limited fraction of the total biomass, and the role of Gordonia spp. in filamentous foaming may be linked more tightly to the physical presence of filamentous microorganisms than to the metabolic activity of the cells.

  14. Crystal-Structure-Guided Design of Self-Assembling RNA Nanotriangles.

    Science.gov (United States)

    Boerneke, Mark A; Dibrov, Sergey M; Hermann, Thomas

    2016-03-14

    RNA nanotechnology uses RNA structural motifs to build nanosized architectures that assemble through selective base-pair interactions. Herein, we report the crystal-structure-guided design of highly stable RNA nanotriangles that self-assemble cooperatively from short oligonucleotides. The crystal structure of an 81 nucleotide nanotriangle determined at 2.6 Å resolution reveals the so-far smallest circularly closed nanoobject made entirely of double-stranded RNA. The assembly of the nanotriangle architecture involved RNA corner motifs that were derived from ligand-responsive RNA switches, which offer the opportunity to control self-assembly and dissociation. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  15. 3' RNA ligase mediated rapid amplification of cDNA ends for validating viroid induced cleavage at the 3' extremity of the host mRNA.

    Science.gov (United States)

    Adkar-Purushothama, Charith Raj; Bru, Pierrick; Perreault, Jean-Pierre

    2017-12-01

    5' RNA ligase-mediated rapid amplification of cDNA ends (5' RLM-RACE) is a widely-accepted method for the validation of direct cleavage of a target gene by a microRNA (miRNA) and viroid-derived small RNA (vd-sRNA). However, this method cannot be used if cleavage takes place in the 3' extremity of the target RNA, as this gives insufficient sequence length to design nested PCR primers for 5' RLM RACE. To overcome this hurdle, we have developed 3' RNA ligase-mediated rapid amplification of cDNA ends (3' RLM RACE). In this method, an oligonucleotide adapter having 5' adenylated and 3' blocked is ligated to the 3' end of the cleaved RNA followed by PCR amplification using gene specific primers. In other words, in 3' RLM RACE, 3' end is mapped using 5' fragment instead of small 3' fragment. The method developed here was verified by examining the bioinformatics predicted and parallel analysis of RNA ends (PARE) proved cleavage sites of chloride channel protein CLC-b-like mRNA in Potato spindle tuber viroid infected tomato plants. The 3' RLM RACE developed in this study has the potential to validate the miRNA and vd-sRNA mediated cleavage of mRNAs at its 3' untranslated region (3' UTR). Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Fluorescence In Situ Hybridization for MicroRNA Detection in Archived Oral Cancer Tissues

    Directory of Open Access Journals (Sweden)

    Zonggao Shi

    2012-01-01

    Full Text Available The noncoding RNA designated as microRNA (miRNA is a large group of small single-stranded regulatory RNA and has generated wide-spread interest in human disease studies. To facilitate delineating the role of microRNAs in cancer pathology, we sought to explore the feasibility of detecting microRNA expression in formalin-fixed paraffin-embedded (FFPE tissues. Using FFPE materials, we have compared fluorescent in situ hybridization (FISH procedures to detect miR-146a with (a different synthetic probes: regular custom DNA oligonucleotides versus locked nucleic acid (LNA incorporated DNA oligonucleotides; (b different reporters for the probes: biotin versus digoxigenin (DIG; (c different visualization: traditional versus tyramide signal amplification (TSA system; (d different blocking reagents for endogenous peroxidase. Finally, we performed miR-146a FISH on a commercially available oral cancer tissue microarray, which contains 40 cases of oral squamous cell carcinoma (OSCC and 10 cases of normal epithelia from the human oral cavity. A sample FISH protocol for detecting miR-146a is provided. In summary, we have established reliable in situ hybridization procedures for detecting the expression of microRNA in FFPE oral cancer tissues. This method is an important tool for studies on the involvement of microRNA in oral cancer pathology and may have potential prognostic or diagnostic value.

  17. The Dynamics of Compound, Transcript, and Protein Effects After Treatment With 2OMePS Antisense Oligonucleotides in mdx Mice

    Directory of Open Access Journals (Sweden)

    Ingrid E C Verhaart

    2014-01-01

    Full Text Available Antisense-mediated exon skipping is currently in clinical development for Duchenne muscular dystrophy (DMD to amend the consequences of the underlying genetic defect and restore dystrophin expression. Due to turnover of compound, transcript, and protein, chronic treatment with effector molecules (antisense oligonucleotides will be required. To investigate the dynamics and persistence of antisense 2′-O-methyl phosphorothioate oligonucleotides, exon skipping, and dystrophin expression after dosing was concluded, mdx mice were treated subcutaneously for 8 weeks with 100 mg/kg oligonucleotides twice weekly. Thereafter, mice were sacrificed at different time points after the final injection (36 hours–24 weeks. Oligonucleotide half-life was longer in heart (~65 days compared with that in skeletal muscle, liver, and kidney (~35 days. Exon skipping half-lives varied between 33 and 53 days, whereas dystrophin protein showed a long half-life (>100 days. Oligonucleotide and exon-skipping levels peaked in the first week and declined thereafter. By contrast, dystrophin expression peaked after 3–8 weeks and then slowly declined, remaining detectable after 24 weeks. Concordance between levels of oligonucleotides, exon skipping, and proteins was observed, except in heart, wherein high oligonucleotide levels but low exon skipping and dystrophin expression were seen. Overall, these results enhance our understanding of the pharmacokinetics and pharmacodynamics of 2′-O-methyl phosphorothioate oligos used for the treatment of DMD.

  18. Identifying modifications in RNA by MALDI mass spectrometry

    DEFF Research Database (Denmark)

    Douthwaite, Stephen; Kirpekar, Finn

    2007-01-01

    as RNA modifications added in cell-free in vitro systems. MALDI-MS is particularly useful in cases in which other techniques such as those involving primer extension or chromatographic analyses are not practicable. To date, MALDI-MS has been used to localize rRNA modifications that are involved......Posttranscriptional modifications on the base or sugar of ribonucleosides generally result in mass increases that can be measured by mass spectrometry. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a direct and accurate means of determining the masses of RNAs. Mass...... spectra produced by MALDI are relatively straightforward to interpret, because they are dominated by singly charged ions, making it possible to analyze complex mixtures of RNA oligonucleotides ranging from trinucleotides up to 20-mers. Analysis of modifications within much longer RNAs, such as ribosomal...

  19. General method for labeling siRNA by click chemistry with fluorine-18 for the purpose of PET imaging.

    Science.gov (United States)

    Mercier, Frédéric; Paris, Jérôme; Kaisin, Geoffroy; Thonon, David; Flagothier, Jessica; Teller, Nathalie; Lemaire, Christian; Luxen, André

    2011-01-19

    The alkyne-azide Cu(I)-catalyzed Huisgen cycloaddition, a click-type reaction, was used to label a double-stranded oligonucleotide (siRNA) with fluorine-18. An alkyne solid support CPG for the preparation of monostranded oligonucleotides functionalized with alkyne has been developed. Two complementary azide labeling agents (1-(azidomethyl)-4-[(18)F]fluorobenzene) and 1-azido-4-(3-[(18)F]fluoropropoxy)benzene have been produced with 41% and 35% radiochemical yields (decay-corrected), respectively. After annealing with the complementary strand, the siRNA was directly labeled by click chemistry with [(18)F]fluoroazide to produce the [(18)F]-radiolabeled siRNA with excellent radiochemical yield and purity.

  20. Controllable molecular motors engineered from myosin and RNA

    Science.gov (United States)

    Omabegho, Tosan; Gurel, Pinar S.; Cheng, Clarence Y.; Kim, Laura Y.; Ruijgrok, Paul V.; Das, Rhiju; Alushin, Gregory M.; Bryant, Zev

    2018-01-01

    Engineering biomolecular motors can provide direct tests of structure-function relationships and customized components for controlling molecular transport in artificial systems1 or in living cells2. Previously, synthetic nucleic acid motors3-5 and modified natural protein motors6-10 have been developed in separate complementary strategies to achieve tunable and controllable motor function. Integrating protein and nucleic-acid components to form engineered nucleoprotein motors may enable additional sophisticated functionalities. However, this potential has only begun to be explored in pioneering work harnessing DNA scaffolds to dictate the spacing, number and composition of tethered protein motors11-15. Here, we describe myosin motors that incorporate RNA lever arms, forming hybrid assemblies in which conformational changes in the protein motor domain are amplified and redirected by nucleic acid structures. The RNA lever arm geometry determines the speed and direction of motor transport and can be dynamically controlled using programmed transitions in the lever arm structure7,9. We have characterized the hybrid motors using in vitro motility assays, single-molecule tracking, cryo-electron microscopy and structural probing16. Our designs include nucleoprotein motors that reversibly change direction in response to oligonucleotides that drive strand-displacement17 reactions. In multimeric assemblies, the controllable motors walk processively along actin filaments at speeds of 10-20 nm s-1. Finally, to illustrate the potential for multiplexed addressable control, we demonstrate sequence-specific responses of RNA variants to oligonucleotide signals.

  1. A short autocomplementary sequence plays an essential role in avian sarcoma-leukosis virus RNA dimerization.

    Science.gov (United States)

    Fossé, P; Motté, N; Roumier, A; Gabus, C; Muriaux, D; Darlix, J L; Paoletti, J

    1996-12-24

    Retroviral genomes consist of two identical RNA molecules joined noncovalently near their 5'-ends. Recently, two models have been proposed for RNA dimer formation on the basis of results obtained in vitro with human immunodeficiency virus type 1 RNA and Moloney murine leukemia virus RNA. It was first proposed that viral RNA dimerizes by forming an interstrand quadruple helix with purine tetrads. The second model postulates that RNA dimerization is initiated by a loop-loop interaction between the two RNA molecules. In order to better characterize the dimerization process of retroviral genomic RNA, we analyzed the in vitro dimerization of avian sarcoma-leukosis virus (ASLV) RNA using different transcripts. We determined the requirements for heterodimer formation, the thermal dissociation of RNA dimers, and the influence of antisense DNA oligonucleotides on dimer formation. Our results strongly suggest that purine tetrads are not involved in dimer formation. Data show that an autocomplementary sequence located upstream from the splice donor site and within a major packaging signal plays a crucial role in ASLV RNA dimer formation in vitro. This sequence is able to form a stem-loop structure, and phylogenetic analysis reveals that it is conserved in 28 different avian sarcoma and leukosis viruses. These results suggest that dimerization of ASLV RNA is initiated by a loop-loop interaction between two RNA molecules and provide an additional argument for the ubiquity of the dimerization process via loop-loop interaction.

  2. Cancer-targeting siRNA delivery from porous silicon nanoparticles.

    Science.gov (United States)

    Wan, Yuan; Apostolou, Sinoula; Dronov, Roman; Kuss, Bryone; Voelcker, Nicolas H

    2014-10-01

    Porous silicon nanoparticles (pSiNPs) with tunable pore size are biocompatible and biodegradable, suggesting that they are suitable biomaterials as vehicles for drug delivery. Loading of small interfering RNA (siRNA) into the pores of pSiNPs can protect siRNA from degradation as well as improve the cellular uptake. We aimed to deliver MRP1 siRNA loaded into pSiNPs to glioblastoma cells, and to demonstrate downregulation of MRP1 at the mRNA and protein levels. 50-220 nm pSiNPs with an average pore size of 26 nm were prepared, followed by electrostatic adsorption of siRNA into pores. Oligonucleotide loading and release profiles were investigated; MRP1 mRNA and protein expression, cell viability and cell apoptosis were studied. Approximately 7.7 µg of siRNA was loaded per mg of pSiNPs. Cells readily took up nanoparticles after 30 min incubation. siRNA-loaded pSiNPs were able to effectively downregulate target mRNA (~40%) and protein expression (31%), and induced cell apoptosis and necrosis (33%). siRNA loaded pSiNPs downregulated mRNA and protein expression and induced cell death. This novel siRNA delivery system may pave the way towards developing more effective tumor therapies.

  3. A locked nucleic acid antisense oligonucleotide (LNA silences PCSK9 and enhances LDLR expression in vitro and in vivo.

    Directory of Open Access Journals (Sweden)

    Nidhi Gupta

    2010-05-01

    Full Text Available The proprotein convertase subtilisin/kexin type 9 (PCSK9 is an important factor in the etiology of familial hypercholesterolemia (FH and is also an attractive therapeutic target to reduce low density lipoprotein (LDL cholesterol. PCSK9 accelerates the degradation of hepatic low density lipoprotein receptor (LDLR and low levels of hepatic PCSK9 activity are associated with reduced levels of circulating LDL-cholesterol.The present study presents the first evidence for the efficacy of a locked nucleic acid (LNA antisense oligonucleotide (LNA ASO that targets both human and mouse PCSK9. We employed human hepatocytes derived cell lines HepG2 and HuH7 and a pancreatic mouse beta-TC3 cell line known to express high endogenous levels of PCSK9. LNA ASO efficiently reduced the mRNA and protein levels of PCSK9 with a concomitant increase in LDLR protein levels after transfection in these cells. In vivo efficacy of LNA ASO was further investigated in mice by tail vein intravenous administration of LNA ASO in saline solution. The level of PCSK9 mRNA was reduced by approximately 60%, an effect lasting more than 16 days. Hepatic LDLR protein levels were significantly up-regulated by 2.5-3 folds for at least 8 days and approximately 2 fold for 16 days. Finally, measurement of liver alanine aminotransferase (ALT levels revealed that long term LNA ASO treatment (7 weeks does not cause hepatotoxicity.LNA-mediated PCSK9 mRNA inhibition displayed potent reduction of PCSK9 in cell lines and mouse liver. Our data clearly revealed the efficacy and safety of LNA ASO in reducing PCSK9 levels, an approach that is now ready for testing in primates. The major significance and take home message of this work is the development of a novel and promising approach for human therapeutic intervention of the PCSK9 pathway and hence for reducing some of the cardiovascular risk factors associated with the metabolic syndrome.

  4. Nucleolin inhibits G4 oligonucleotide unwinding by Werner helicase.

    Directory of Open Access Journals (Sweden)

    Fred E Indig

    Full Text Available The Werner protein (WRNp, a member of the RecQ helicase family, is strongly associated with the nucleolus, as is nucleolin (NCL, an important nucleolar constituent protein. Both WRNp and NCL respond to the effects of DNA damaging agents. Therefore, we have investigated if these nuclear proteins interact and if this interaction has a possible functional significance in DNA damage repair.Here we report that WRNp interacts with the RNA-binding protein, NCL, based on immunoprecipitation, immunofluorescent co-localization in live and fixed cells, and direct binding of purified WRNp to nucleolin. We also map the binding region to the C-terminal domains of both proteins. Furthermore, treatment of U2OS cells with 15 µM of the Topoisomerase I inhibitor, camptothecin, causes the dissociation of the nucleolin-Werner complex in the nucleolus, followed by partial re-association in the nucleoplasm. Other DNA damaging agents, such as hydroxyurea, Mitomycin C, and aphidicolin do not have these effects. Nucleolin or its C-terminal fragment affected the helicase, but not the exonuclease activity of WRNp, by inhibiting WRN unwinding of G4 tetraplex DNA structures, as seen in activity assays and electrophoretic mobility shift assays (EMSA.These data suggest that nucleolin may regulate G4 DNA unwinding by WRNp, possibly in response to certain DNA damaging agents. We postulate that the NCL-WRNp complex may contain an inactive form of WRNp, which is released from the nucleolus upon DNA damage. Then, when required, WRNp is released from inhibition and can participate in the DNA repair processes.

  5. MicroRNA-targeted therapeutics for lung cancer treatment.

    Science.gov (United States)

    Xue, Jing; Yang, Jiali; Luo, Meihui; Cho, William C; Liu, Xiaoming

    2017-02-01

    Lung cancer is one of the leading causes of cancer-related mortality worldwide. MicroRNAs (miRNAs) are endogenous non-coding small RNAs that repress the expression of a broad array of target genes. Many efforts have been made to therapeutically target miRNAs in cancer treatments using miRNA mimics and miRNA antagonists. Areas covered: This article summarizes the recent findings with the role of miRNAs in lung cancer, and discusses the potential and challenges of developing miRNA-targeted therapeutics in this dreadful disease. Expert opinion: The development of miRNA-targeted therapeutics has become an important anti-cancer strategy. Results from both preclinical and clinical trials of microRNA replacement therapy have shown some promise in cancer treatment. However, some obstacles, including drug delivery, specificity, off-target effect, toxicity mediation, immunological activation and dosage determination should be addressed. Several delivery strategies have been employed, including naked oligonucleotides, liposomes, aptamer-conjugates, nanoparticles and viral vectors. However, delivery remains a main challenge in miRNA-targeting therapeutics. Furthermore, immune-related serious adverse events are also a concern, which indicates the complexity of miRNA-based therapy in clinical settings.

  6. RNA detection in situ with FISH-STICs.

    Science.gov (United States)

    Sinnamon, John R; Czaplinski, Kevin

    2014-02-01

    The ability to detect RNA molecules in situ has long had important applications for molecular biological studies. Enzyme or dye-labeled antisense in vitro runoff transcripts and synthetic oligodeoxynucleotides (ODN) both have a proven track record of success, but each of these also has scientific and practical drawbacks and limitations to its use. We devised a means to use commercially synthesized oligonucleotides as RNA-FISH probes without further modification and show that such probes work well for detection of RNA in cultured cells. This approach can bind a high concentration of fluorescent ODN to a short stretch of an RNA using commercial DNA synthesis outlets available to any laboratory. We call this approach for creating in situ hybridization probes Fluorescence In Situ Hybridization with Sequential Tethered and Intertwined ODN Complexes (FISH-STICs). We demonstrate that one FISH-STIC probe can detect mRNA molecules in culture, and that probe detection can be improved by the addition of multiple probes that can be easily adapted for robust mRNA quantification. Using FISH-STICs, we demonstrate a nonoverlapping distribution for β-actin and γ-actin mRNA in cultured fibroblasts, and the detection of neuron-specific transcripts within cultured primary hippocampal neurons.

  7. G-Quadruplexes influence pri-microRNA processing.

    Science.gov (United States)

    Rouleau, Samuel G; Garant, Jean-Michel; Bolduc, François; Bisaillon, Martin; Perreault, Jean-Pierre

    2018-02-01

    RNA G-Quadruplexes (G4) have been shown to possess many biological functions, including the regulation of microRNA (miRNA) biogenesis and function. However, their impact on pri-miRNA processing remains unknown. We identified G4 located near the Drosha cleavage site in three distinct pri-miRNAs: pri-mir200c, pri-mir451a, and pri-mir497. The folding of the potential G4 motifs was determined in solution. Subsequently, mutations disrupting G4 folding led to important changes in the mature miRNAs levels in cells. Moreover, using small antisense oligonucleotides binding to the pri-miRNA, it was possible to modulate, either positively or negatively, the mature miRNA levels. Together, these data demonstrate that G4 motifs could contribute to the regulation of pri-mRNA processing, a novel role for G4. Considering that bio-informatics screening indicates that between 9% and 50% of all pri-miRNAs contain a putative G4, these structures possess interesting potential as future therapeutic targets.

  8. RNAstructure: software for RNA secondary structure prediction and analysis.

    Science.gov (United States)

    Reuter, Jessica S; Mathews, David H

    2010-03-15

    To understand an RNA sequence's mechanism of action, the structure must be known. Furthermore, target RNA structure is an important consideration in the design of small interfering RNAs and antisense DNA oligonucleotides. RNA secondary structure prediction, using thermodynamics, can be used to develop hypotheses about the structure of an RNA sequence. RNAstructure is a software package for RNA secondary structure prediction and analysis. It uses thermodynamics and utilizes the most recent set of nearest neighbor parameters from the Turner group. It includes methods for secondary structure prediction (using several algorithms), prediction of base pair probabilities, bimolecular structure prediction, and prediction of a structure common to two sequences. This contribution describes new extensions to the package, including a library of C++ classes for incorporation into other programs, a user-friendly graphical user interface written in JAVA, and new Unix-style text interfaces. The original graphical user interface for Microsoft Windows is still maintained. The extensions to RNAstructure serve to make RNA secondary structure prediction user-friendly. The package is available for download from the Mathews lab homepage at http://rna.urmc.rochester.edu/RNAstructure.html.

  9. Effects of CD49d-targeted antisense-oligonucleotide on α4 integrin expression and function of acute lymphoblastic leukemia cells: Results of in vitro and in vivo studies.

    Directory of Open Access Journals (Sweden)

    Yann Duchartre

    Full Text Available We recently demonstrated the effectiveness of blocking CD49d with anti-functional antibodies or small molecule inhibitors as a rational targeted approach to the treatment of acute leukemia in combination with chemotherapy. Antisense oligonucleotide promises to be no less specific than antibodies and inhibitors, but more interesting for pharmacokinetics and pharmacodynamics. We addressed this using the published CD49d antisense drug ATL1102. In vitro, we incubated/nucleofected the ALL cell line Kasumi-2 with ATL1102. In vivo, immunodeficient hosts were engrafted with primary ALL cells and treated with ATL1102. Changes in expression of CD49d mRNA and CD49d protein, and of cooperating gene products, including ß1 integrin and CXCR4, as well as survival in the mouse experiments were quantified. We observed dose-dependent down-regulation of CD49d mRNA and protein levels and its partner integrin ß1 cell surface protein level and, up-regulation of CXCR4 surface expression. The suppression was more pronounced after nucleofection than after incubation, where down-regulation was significant only at the higher doses. In vivo effects of ATL1102 were not sufficient to translate into "clinical" benefit in the leukemia model. In summary, antisense oligonucleotides are successful tools for specifically modulating gene expression but sufficient delivery to down-regulate CD49d in vivo may be difficult to achieve.

  10. Application of hierarchical oligonucleotide primer extension (HOPE) to assess relative abundances of ammonia- and nitrite-oxidizing bacteria

    KAUST Repository

    Scarascia, Giantommaso; Cheng, Hong; Harb, Moustapha; Hong, Pei-Ying

    2017-01-01

    for ensuring the efficiency of nitrification in water treatment systems. Hierarchical oligonucleotide primer extension (HOPE), previously developed to rapidly quantify relative abundances of specific microbial groups of interest, was applied in this study

  11. Interfacing click chemistry with automated oligonucleotide synthesis for the preparation of fluorescent DNA probes containing internal xanthene and cyanine dyes

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Wengel, Jesper

    2013-01-01

    Double-labeled oligonucleotide probes containing fluorophores interacting by energy-transfer mechanisms are essential for modern bioanalysis, molecular diagnostics, and in vivo imaging techniques. Although bright xanthene and cyanine dyes are gaining increased prominence within these fields, little...

  12. Detection and identification of intestinal pathogenic bacteria by hybridization to oligonucleotide microarrays

    Science.gov (United States)

    Jin, Lian-Qun; Li, Jun-Wen; Wang, Sheng-Qi; Chao, Fu-Huan; Wang, Xin-Wei; Yuan, Zheng-Quan

    2005-01-01

    AIM: To detect the common intestinal pathogenic bacteria quickly and accurately. METHODS: A rapid (<3 h) experimental procedure was set up based upon the gene chip technology. Target genes were amplified and hybridized by oligonucleotide microarrays. RESULTS: One hundred and seventy strains of bacteria in pure culture belonging to 11 genera were successfully discriminated under comparatively same conditions, and a series of specific hybridization maps corresponding to each kind of bacteria were obtained. When this method was applied to 26 divided cultures, 25 (96.2%) were identified. CONCLUSION: Salmonella sp., Escherichia coli, Shigella sp., Listeria monocytogenes, Vibrio parahaemolyticus, Staphylococcus aureus, Proteus sp., Bacillus cereus, Vibrio cholerae, Enterococcus faecalis, Yersinia enterocolitica, and Campylobacter jejuni can be detected and identified by our microarrays. The accuracy, range, and discrimination power of this assay can be continually improved by adding further oligonucleotides to the arrays without any significant increase of complexity or cost. PMID:16437687

  13. Heated oligonucleotide ligation assay (HOLA): an affordable single nucleotide polymorphism assay.

    Science.gov (United States)

    Black, W C; Gorrochotegui-Escalante, N; Duteau, N M

    2006-03-01

    Most single nucleotide polymorphism (SNP) detection requires expensive equipment and reagents. The oligonucleotide ligation assay (OLA) is an inexpensive SNP assay that detects ligation between a biotinylated "allele-specific detector" and a 3' fluorescein-labeled "reporter" oligonucleotide. No ligation occurs unless the 3' detector nucleotide is complementary to the SNP nucleotide. The original OLA used chemical denaturation and neutralization. Heated OLA (HOLA) instead uses a thermal stable ligase and cycles of denaturing and hybridization for ligation and SNP detection. The cost per genotype is approximately US$1.25 with two-allele SNPs or approximately US$1.75 with three-allele SNPs. We illustrate the development of HOLA for SNP detection in the Early Trypsin and Abundant Trypsin loci in the mosquito Aedes aegypti (L.) and at the a-glycerophosphate dehydrogenase locus in the mosquito Anopheles gambiae s.s.

  14. Dose-Dependent Lowering of Mutant Huntingtin Using Antisense Oligonucleotides in Huntington Disease Patients.

    Science.gov (United States)

    van Roon-Mom, Willeke M C; Roos, Raymund A C; de Bot, Susanne T

    2018-04-01

    On December 11 of 2017, Ionis Pharmaceuticals published a press release announcing dose-dependent reductions of mutant huntingtin protein in their HTTRx Phase 1/2a study in Huntington disease (HD) patients. The results from this Ionis trial have gained much attention from the patient community and the oligonucleotide therapeutics field, since it is the first trial targeting the cause of HD, namely the mutant huntingtin protein, using antisense oligonucleotides (ASOs). The press release also states that the primary endpoints of the study (safety and tolerability) were met, but does not contain data. This news follows the approval of another therapeutic ASO nusinersen (trade name Spinraza) for a neurological disease, spinal muscular atrophy, by the U.S. Food and Drug Administration and European Medicines Agency, in 2016 and 2017, respectively. Combined, this offers hope for the development of the HTTRx therapy for HD patients.

  15. Single-Labeled Oligonucleotides Showing Fluorescence Changes upon Hybridization with Target Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Gil Tae Hwang

    2018-01-01

    Full Text Available Sequence-specific detection of nucleic acids has been intensively studied in the field of molecular diagnostics. In particular, the detection and analysis of single-nucleotide polymorphisms (SNPs is crucial for the identification of disease-causing genes and diagnosis of diseases. Sequence-specific hybridization probes, such as molecular beacons bearing the fluorophore and quencher at both ends of the stem, have been developed to enable DNA mutation detection. Interestingly, DNA mutations can be detected using fluorescently labeled oligonucleotide probes with only one fluorophore. This review summarizes recent research on single-labeled oligonucleotide probes that exhibit fluorescence changes after encountering target nucleic acids, such as guanine-quenching probes, cyanine-containing probes, probes containing a fluorophore-labeled base, and microenvironment-sensitive probes.

  16. Oligonucleotide fishing for STAT6: cross-talk between IL-4 and chemokines

    DEFF Research Database (Denmark)

    Eriksen, K W; Nielsen, M; Kaltoft, K

    2001-01-01

    Signal transducer and activator of transcription 6 (STAT6) is essential for the biological activities of interleukin-4 (IL-4) and the development of allergic responses in mice. Here we report on a sensitive and specific assay for STAT6 activation in response to IL-4. We took advantage of double-s...... activation, whereas other chemokines and cytokines do not. In conclusion, our data show that oligonucleotide fishing is a supplementary tool for studying cytokine cross-talk at a genomic level....

  17. Intrinsically Labeled Fluorescent Oligonucleotide Probes on Quantum Dots for Transduction of Nucleic Acid Hybridization.

    Science.gov (United States)

    Shahmuradyan, Anna; Krull, Ulrich J

    2016-03-15

    Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.

  18. Genome dynamics of short oligonucleotides: the example of bacterial DNA uptake enhancing sequences.

    Directory of Open Access Journals (Sweden)

    Mohammed Bakkali

    Full Text Available Among the many bacteria naturally competent for transformation by DNA uptake-a phenomenon with significant clinical and financial implications- Pasteurellaceae and Neisseriaceae species preferentially take up DNA containing specific short sequences. The genomic overrepresentation of these DNA uptake enhancing sequences (DUES causes preferential uptake of conspecific DNA, but the function(s behind this overrepresentation and its evolution are still a matter for discovery. Here I analyze DUES genome dynamics and evolution and test the validity of the results to other selectively constrained oligonucleotides. I use statistical methods and computer simulations to examine DUESs accumulation in Haemophilus influenzae and Neisseria gonorrhoeae genomes. I analyze DUESs sequence and nucleotide frequencies, as well as those of all their mismatched forms, and prove the dependence of DUESs genomic overrepresentation on their preferential uptake by quantifying and correlating both characteristics. I then argue that mutation, uptake bias, and weak selection against DUESs in less constrained parts of the genome combined are sufficient enough to cause DUESs accumulation in susceptible parts of the genome with no need for other DUES function. The distribution of overrepresentation values across sequences with different mismatch loads compared to the DUES suggests a gradual yet not linear molecular drive of DNA sequences depending on their similarity to the DUES. Other genomically overrepresented sequences, both pro- and eukaryotic, show similar distribution of frequencies suggesting that the molecular drive reported above applies to other frequent oligonucleotides. Rare oligonucleotides, however, seem to be gradually drawn to genomic underrepresentation, thus, suggesting a molecular drag. To my knowledge this work provides the first clear evidence of the gradual evolution of selectively constrained oligonucleotides, including repeated, palindromic and protein

  19. Selective Detection of Peptide-Oligonucleotide Heteroconjugates Utilizing Capillary HPLC-ICPMS

    Science.gov (United States)

    Catron, Brittany; Caruso, Joseph A.; Limbach, Patrick A.

    2012-06-01

    A method for the selective detection and quantification of peptide:oligonucleotide heteroconjugates, such as those generated by protein:nucleic acid cross-links, using capillary reversed-phase high performance liquid chromatography (cap-RPHPLC) coupled with inductively coupled plasma mass spectrometry detection (ICPMS) is described. The selective detection of phosphorus as 31P+, the only natural isotope, in peptide-oligonucleotide heteroconjugates is enabled by the elemental detection capabilities of the ICPMS. Mobile phase conditions that allow separation of heteroconjugates while maintaining ICPMS compatibility were investigated. We found that trifluoroacetic acid (TFA) mobile phases, used in conventional peptide separations, and hexafluoroisopropanol/triethylamine (HFIP/TEA) mobile phases, used in conventional oligonucleotide separations, both are compatible with ICPMS and enable heteroconjugate separation. The TFA-based separations yielded limits of detection (LOD) of ~40 ppb phosphorus, which is nearly seven times lower than the LOD for HFIP/TEA-based separations. Using the TFA mobile phase, 1-2 pmol of a model heteroconjugate were routinely separated and detected by this optimized capLC-ICPMS method.

  20. Optical properties and electronic transitions of DNA oligonucleotides as a function of composition and stacking sequence.

    Science.gov (United States)

    Schimelman, Jacob B; Dryden, Daniel M; Poudel, Lokendra; Krawiec, Katherine E; Ma, Yingfang; Podgornik, Rudolf; Parsegian, V Adrian; Denoyer, Linda K; Ching, Wai-Yim; Steinmetz, Nicole F; French, Roger H

    2015-02-14

    The role of base pair composition and stacking sequence in the optical properties and electronic transitions of DNA is of fundamental interest. We present and compare the optical properties of DNA oligonucleotides (AT)10, (AT)5(GC)5, and (AT-GC)5 using both ab initio methods and UV-vis molar absorbance measurements. Our data indicate a strong dependence of both the position and intensity of UV absorbance features on oligonucleotide composition and stacking sequence. The partial densities of states for each oligonucleotide indicate that the valence band edge arises from a feature associated with the PO4(3-) complex anion, and the conduction band edge arises from anti-bonding states in DNA base pairs. The results show a strong correspondence between the ab initio and experimentally determined optical properties. These results highlight the benefit of full spectral analysis of DNA, as opposed to reductive methods that consider only the 260 nm absorbance (A260) or simple purity ratios, such as A260/A230 or A260/A280, and suggest that the slope of the absorption edge onset may provide a useful metric for the degree of base pair stacking in DNA. These insights may prove useful for applications in biology, bioelectronics, and mesoscale self-assembly.

  1. Diamond cubic phase of monoolein and water as an amphiphilic matrix for electrophoresis of oligonucleotides.

    Science.gov (United States)

    Carlsson, Nils; Winge, Ann-Sofie; Engström, Sven; Akerman, Björn

    2005-10-06

    We used a cubic liquid crystal formed by the nonionic monoglyceride monoolein and water as a porous matrix for the electrophoresis of oligonucleotides. The diamond cubic phase is thermodynamically stable when in contact with a water-rich phase, which we exploit to run the electrophoresis in the useful submarine mode. Oligonucleotides are separated according to size and secondary structure by migration through the space-filling aqueous nanometer pores of the regular liquid crystal, but the comparatively slow migration means the cubic phase will not be a replacement for the conventional DNA gels. However, our demonstration that the cubic phase can be used in submarine electrophoresis opens up the possibility for a new matrix for electrophoresis of amphiphilic molecules. From this perspective, the results on the oligonucleotides show that water-soluble particles of nanometer size, typical for the hydrophilic parts of membrane-bound proteins, may be a useful separation motif. A charged contamination in the commercial sample of monoolein, most likely oleic acid that arises from its hydrolysis, restricts useful buffer conditions to a pH below 5.6.

  2. 18F-labelling of oligonucleotides using succinimido 4-[18F]fluorobenzoat

    International Nuclear Information System (INIS)

    Hedberg, Elisabeth; Laangstroem, Bengt

    1998-01-01

    A general method for the labelling of oligodeoxynucleotide and oligonucleoside phosphorothioates in the 5'-position with the positron-emitting radionuclide 18 F (t 1/2 = 110 min) is described. The label was incorporated by the reaction of succinimido 4 -[ 18 F]fluorobenzoate 4 with oligonucleotides (18- and 20-mers) modified in the 5'-position with a hexylamine linker. Oligodeoxynucleotides 5'-GCT,AAG,CGA,TGC,CTC,CGT-3' (MTCa) and 5'-GAA,CCT,CTG,AGA,GTT,CAT,CT-3' (CROa) were labelled in 20±3 % (MTCa) and 13±3 % (CROa) radiochemical yields (non-isolated, decay-corrected and based on 4). Oligonucleoside phosphorotioates MTCa (S-MTCa) and CROa (S-CROa) were labelled in 9 and 7% isolated radiochemical yield, respectively (decay-corrected and based on 4). Labelled oligonucleotides and phosphorothioate analogues were separated from their unlabelled counterparts using reversed-phase perfusion chromatography. The molecular mass of a labelled oligonucleotide CROa was determined by ESI-MS after a mixed 18 F/ 19 F fluorobenzoate labelling experiment and corresponded with the expected structure. (au)

  3. Membrane-based oligonucleotide array developed from multiple markers for the detection of many Phytophthora species.

    Science.gov (United States)

    Chen, Wen; Djama, Zeinab Robleh; Coffey, Michael D; Martin, Frank N; Bilodeau, Guillaume J; Radmer, Lorien; Denton, Geoff; Lévesque, C André

    2013-01-01

    Most Phytophthora spp. are destructive plant pathogens; therefore, effective monitoring and accurate early detection are important means of preventing potential epidemics and outbreaks of diseases. In the current study, a membrane-based oligonucleotide array was developed that can detect Phytophthora spp. reliably using three DNA regions; namely, the internal transcribed spacer (ITS), the 5' end of cytochrome c oxidase 1 gene (cox1), and the intergenic region between cytochrome c oxidase 2 gene (cox2) and cox1 (cox2-1 spacer). Each sequence data set contained ≈250 sequences representing 98 described and 15 undescribed species of Phytophthora. The array was validated with 143 pure cultures and 35 field samples. Together, nonrejected oligonucleotides from all three markers have the ability to reliably detect 82 described and 8 undescribed Phytophthora spp., including several quarantine or regulated pathogens such as Phytophthora ramorum. Our results showed that a DNA array containing signature oligonucleotides designed from multiple genomic regions provided robustness and redundancy for the detection and differentiation of closely related taxon groups. This array has the potential to be used as a routine diagnostic tool for Phytophthora spp. from complex environmental samples without the need for extensive growth of cultures.

  4. Sequence-Dependent Mechanism of DNA Oligonucleotide Dehybridization Resolved through Infrared Spectroscopy.

    Science.gov (United States)

    Sanstead, Paul J; Stevenson, Paul; Tokmakoff, Andrei

    2016-09-14

    Despite its important role in biology and nanotechnology, many questions remain regarding the molecular mechanism and dynamics by which oligonucleotides recognize and hybridize to their complementary sequence. The thermodynamics and kinetics of DNA oligonucleotide hybridization and dehybridization are often assumed to involve an all-or-nothing two-state dissociation pathway, but deviations from this behavior can be considerable even for short sequences. We introduce a new strategy to characterize the base-pair-specific thermal dissociation mechanism of DNA oligonucleotides through steady-state and time-resolved infrared spectroscopy. Experiments are interpreted with a lattice model to provide a structure-specific interpretation. This method is applied to a model set of self-complementary 10-base-pair sequences in which the placement of GC base pairs is varied in an otherwise AT strand. Through a combination of Fourier transform infrared and two-dimensional infrared spectroscopy, experiments reveal varying degrees of deviation from simple two-state behavior. As the temperature is increased, duplexes dissociate through a path in which the terminal bases fray, without any significant contribution from loop configurations. Transient temperature jump experiments reveal time scales of 70-100 ns for fraying and 10-30 μs for complete dissociation near the melting temperature. Whether or not frayed states are metastable intermediates or short-lived configurations during the full dissociation of the duplex is dictated by the nucleobase sequence.

  5. i-Genome: A database to summarize oligonucleotide data in genomes

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    Chang Yu-Chung

    2004-10-01

    Full Text Available Abstract Background Information on the occurrence of sequence features in genomes is crucial to comparative genomics, evolutionary analysis, the analyses of regulatory sequences and the quantitative evaluation of sequences. Computing the frequencies and the occurrences of a pattern in complete genomes is time-consuming. Results The proposed database provides information about sequence features generated by exhaustively computing the sequences of the complete genome. The repetitive elements in the eukaryotic genomes, such as LINEs, SINEs, Alu and LTR, are obtained from Repbase. The database supports various complete genomes including human, yeast, worm, and 128 microbial genomes. Conclusions This investigation presents and implements an efficiently computational approach to accumulate the occurrences of the oligonucleotides or patterns in complete genomes. A database is established to maintain the information of the sequence features, including the distributions of oligonucleotide, the gene distribution, the distribution of repetitive elements in genomes and the occurrences of the oligonucleotides. The database can provide more effective and efficient way to access the repetitive features in genomes.

  6. Specificity tests of an oligonucleotide probe against food-outbreak salmonella for biosensor detection

    Science.gov (United States)

    Chen, I.-H.; Horikawa, S.; Xi, J.; Wikle, H. C.; Barbaree, J. M.; Chin, B. A.

    2017-05-01

    Phage based magneto-elastic (ME) biosensors have been shown to be able to rapidly detect Salmonella in various food systems to serve food pathogen monitoring purposes. In this ME biosensor platform, the free-standing strip-shaped magneto-elastic sensor is the transducer and the phage probe that recognizes Salmonella in food serves as the bio-recognition element. According to Sorokulova et al. at 2005, a developed oligonucleotide probe E2 was reported to have high specificity to Salmonella enterica Typhimurium. In the report, the specificity tests were focused in most of Enterobacterace groups outside of Salmonella family. Here, to understand the specificity of phage E2 to different Salmonella enterica serotypes within Salmonella Family, we further tested the specificity of the phage probe to thirty-two Salmonella serotypes that were present in the major foodborne outbreaks during the past ten years (according to Centers for Disease Control and Prevention). The tests were conducted through an Enzyme linked Immunosorbent Assay (ELISA) format. This assay can mimic probe immobilized conditions on the magnetoelastic biosensor platform and also enable to study the binding specificity of oligonucleotide probes toward different Salmonella while avoiding phage/ sensor lot variations. Test results confirmed that this oligonucleotide probe E2 was high specific to Salmonella Typhimurium cells but showed cross reactivity to Salmonella Tennessee and four other serotypes among the thirty-two tested Salmonella serotypes.

  7. Development of Multiexon Skipping Antisense Oligonucleotide Therapy for Duchenne Muscular Dystrophy

    Science.gov (United States)

    Yokota, Toshifumi; Wood, Matthew J. A.

    2013-01-01

    Duchenne muscular dystrophy (DMD) is an incurable, X-linked progressive muscle degenerative disorder that results from the absence of dystrophin protein and leads to premature death in affected individuals due to respiratory and/or cardiac failure typically by age of 30. Very recently the exciting prospect of an effective oligonucleotide therapy has emerged which restores dystrophin protein expression to affected tissues in DMD patients with highly promising data from a series of clinical trials. This therapeutic approach is highly mutation specific and thus is personalised. Therefore DMD has emerged as a model genetic disorder for understanding and overcoming of the challenges of developing personalised genetic medicines. One of the greatest weaknesses of the current oligonucleotide approach is that it is a mutation-specific therapy. To address this limitation, we have recently demonstrated that exons 45–55 skipping therapy has the potential to treat clusters of mutations that cause DMD, which could significantly reduce the number of compounds that would need to be developed in order to successfully treat all DMD patients. Here we discuss and review the latest preclinical work in this area as well as a variety of accompanying issues, including efficacy and potential toxicity of antisense oligonucleotides, prior to human clinical trials. PMID:23984357

  8. Structural properties of oligonucleotide monolayers on gold surfaces probed by fluorescence investigations.

    Science.gov (United States)

    Rant, Ulrich; Arinaga, Kenji; Fujita, Shozo; Yokoyama, Naoki; Abstreiter, Gerhard; Tornow, Marc

    2004-11-09

    We present optical investigations on the conformation of oligonucleotide layers on Au surfaces. Our studies concentrate on the effect of varying surface coverage densities on the structural properties of layers of 12- and 24mer single-stranded DNA, tethered to the Au surface at one end while being labeled with a fluorescent marker at the opposing end. The distance-dependent energy transfer from the marker dye to the metal surface, which causes quenching of the observed fluorescence, is used to provide information on the orientation of the DNA strands relative to the surface. Variations in the oligonucleotide coverage density, as determined from electrochemical quantification, over 2 orders of magnitude are achieved by employing different preparation conditions. The observed enhancement in fluorescence intensity with increasing DNA coverage can be related to a model involving mutual steric interactions of oligonucleotides on the surface, as well as fluorescence quenching theory. Finally, the applicability of the presented concepts for investigations of heterogeneous monolayers is demonstrated by means of studying the coadsorption of mercaptohexanol onto DNA-modified Au surfaces.

  9. Ultrasensitive Detection of Ebola Virus Oligonucleotide Based on Upconversion Nanoprobe/Nanoporous Membrane System.

    Science.gov (United States)

    Tsang, Ming-Kiu; Ye, WeiWei; Wang, Guojing; Li, Jingming; Yang, Mo; Hao, Jianhua

    2016-01-26

    Ebola outbreaks are currently of great concern, and therefore, development of effective diagnosis methods is urgently needed. The key for lethal virus detection is high sensitivity, since early-stage detection of virus may increase the probability of survival. Here, we propose a luminescence scheme of assay consisting of BaGdF5:Yb/Er upconversion nanoparticles (UCNPs) conjugated with oligonucleotide probe and gold nanoparticles (AuNPs) linked with target Ebola virus oligonucleotide. As a proof of concept, a homogeneous assay was fabricated and tested, yielding a detection limit at picomolar level. The luminescence resonance energy transfer is ascribed to the spectral overlapping of upconversion luminescence and the absorption characteristics of AuNPs. Moreover, we anchored the UCNPs and AuNPs on a nanoporous alumina (NAAO) membrane to form a heterogeneous assay. Importantly, the detection limit was greatly improved, exhibiting a remarkable value at the femtomolar level. The enhancement is attributed to the increased light-matter interaction throughout the nanopore walls of the NAAO membrane. The specificity test suggested that the nanoprobes were specific to Ebola virus oligonucleotides. The strategy combining UCNPs, AuNPs, and NAAO membrane provides new insight into low-cost, rapid, and ultrasensitive detection of different diseases. Furthermore, we explored the feasibility of clinical application by using inactivated Ebola virus samples. The detection results showed great potential of our heterogeneous design for practical application.

  10. Targeting eukaryotic translation in mesothelioma cells with an eIF4E-specific antisense oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Blake A Jacobson

    Full Text Available BACKGROUND: Aberrant cap-dependent translation is implicated in tumorigenesis in multiple tumor types including mesothelioma. In this study, disabling the eIF4F complex by targeting eIF4E with eIF4E-specific antisense oligonucleotide (4EASO is assessed as a therapy for mesothelioma. METHODS: Mesothelioma cells were transfected with 4EASO, designed to target eIF4E mRNA, or mismatch-ASO control. Cell survival was measured in mesothelioma treated with 4EASO alone or combined with either gemcitabine or pemetrexed. Levels of eIF4E, ODC, Bcl-2 and β-actin were assessed following treatment. Binding to a synthetic cap-analogue was used to study the strength of eIF4F complex activation following treatment. RESULTS: eIF4E level and the formation of eIF4F cap-complex decreased in response to 4EASO, but not mismatch control ASO, resulting in cleavage of PARP indicating apoptosis. 4EASO treatment resulted in dose dependent decrease in eIF4E levels, which corresponded to cytotoxicity of mesothelioma cells. 4EASO resulted in decreased levels of eIF4E in non-malignant LP9 cells, but this did not correspond to increased cytotoxicity. Proteins thought to be regulated by cap-dependent translation, Bcl-2 and ODC, were decreased upon treatment with 4EASO. Combination therapy of 4EASO with pemetrexed or gemcitabine further reduced cell number. CONCLUSION: 4EASO is a novel drug that causes apoptosis and selectively reduces eIF4E levels, eIF4F complex formation, and proliferation of mesothelioma cells. eIF4E knockdown results in decreased expression of anti-apoptotic and pro-growth proteins and enhances chemosensitivity.

  11. Antisense Oligonucleotide-based Splice Correction for USH2A-associated Retinal Degeneration Caused by a Frequent Deep-intronic Mutation

    Directory of Open Access Journals (Sweden)

    Radulfus WN Slijkerman

    2016-01-01

    Full Text Available Usher syndrome (USH is the most common cause of combined deaf-blindness in man. The hearing loss can be partly compensated by providing patients with hearing aids or cochlear implants, but the loss of vision is currently untreatable. In general, mutations in the USH2A gene are the most frequent cause of USH explaining up to 50% of all patients worldwide. The first deep-intronic mutation in the USH2A gene (c.7595-2144A>G was reported in 2012, leading to the insertion of a pseudoexon (PE40 into the mature USH2A transcript. When translated, this PE40-containing transcript is predicted to result in a truncated non-functional USH2A protein. In this study, we explored the potential of antisense oligonucleotides (AONs to prevent aberrant splicing of USH2A pre-mRNA as a consequence of the c.7595-2144A>G mutation. Engineered 2'-O-methylphosphorothioate AONs targeting the PE40 splice acceptor site and/or exonic splice enhancer regions displayed significant splice correction potential in both patient derived fibroblasts and a minigene splice assay for USH2A c.7595-2144A>G, whereas a non-binding sense oligonucleotide had no effect on splicing. Altogether, AON-based splice correction could be a promising approach for the development of a future treatment for USH2A-associated retinitis pigmentosa caused by the deep-intronic c.7595-2144A>G mutation.

  12. Sequence-specific RNA Photocleavage by Single-stranded DNA in Presence of Riboflavin

    Science.gov (United States)

    Zhao, Yongyun; Chen, Gangyi; Yuan, Yi; Li, Na; Dong, Juan; Huang, Xin; Cui, Xin; Tang, Zhuo

    2015-10-01

    Constant efforts have been made to develop new method to realize sequence-specific RNA degradation, which could cause inhibition of the expression of targeted gene. Herein, by using an unmodified short DNA oligonucleotide for sequence recognition and endogenic small molecue, vitamin B2 (riboflavin) as photosensitizer, we report a simple strategy to realize the sequence-specific photocleavage of targeted RNA. The DNA strand is complimentary to the target sequence to form DNA/RNA duplex containing a G•U wobble in the middle. The cleavage reaction goes through oxidative elimination mechanism at the nucleoside downstream of U of the G•U wobble in duplex to obtain unnatural RNA terminal, and the whole process is under tight control by using light as switch, which means the cleavage could be carried out according to specific spatial and temporal requirements. The biocompatibility of this method makes the DNA strand in combination with riboflavin a promising molecular tool for RNA manipulation.

  13. PLGA-PEG-PLGA microspheres as a delivery vehicle for antisense oligonucleotides to CTGF: Implications on post-surgical peritoneal adhesion prevention

    Science.gov (United States)

    Azeke, John Imuetinyan-Jesu, Jr.

    Abdominal adhesions are the aberrant result of peritoneal wound healing commonly associated with surgery and inflammation. A subject of a large number of studies since the first half of the last century, peritoneal adhesion prevention has, for the most part, evaded the scientific community and continues to cost Americans an estimated $2-4 billion annually. It is known that transforming growth factor-beta (TGF-beta) plays a key role in the wound healing cascade; however, suppression of this multifunctional growth factor's activity may have more harmful consequences than can be tolerated. As a result, much attention has fallen on connective tissue growth factor (CTGF), a downstream mediator of TGF-beta's fibrotic action. It has been demonstrated in several in vitro models, that the suppression of CTGF hinders fibroblast proliferation, a necessary condition for fibrosis. Furthermore, antisense oligonucleotides (antisense oligos, AO) to CTGF have been shown to knock down CTGF mRNA levels by specifically hindering the translation of CTGF protein. Antisense technologies have met with a great deal of excitement as a viable means of preventing diseases such as adhesions by hindering protein translation at the mRNA level. However, the great challenge associated with the use of these drugs lies in the short circulation time when administered "naked". Viral delivery systems, although excellent platforms in metabolic studies, are not ideal for diagnostic use because of the inherent danger associated with viral vectors. Microparticles made of biodegradable polymers have therefore presented themselves as a viable means of delivering these drugs to target cells over extended periods. Herein, we present two in vivo studies confirming the up-regulation of TGF-beta protein and CTGF mRNA following injury to the uterine tissues of female rats. We were able to selectively knockdown post-operative CTGF protein levels following surgery, however, our observations led us to conclude that

  14. Efficient construction of an inverted minimal H1 promoter driven siRNA expression cassette: facilitation of promoter and siRNA sequence exchange.

    Directory of Open Access Journals (Sweden)

    Hoorig Nassanian

    2007-08-01

    Full Text Available RNA interference (RNAi, mediated by small interfering RNA (siRNA, is an effective method used to silence gene expression at the post-transcriptional level. Upon introduction into target cells, siRNAs incorporate into the RNA-induced silencing complex (RISC. The antisense strand of the siRNA duplex then "guides" the RISC to the homologous mRNA, leading to target degradation and gene silencing. In recent years, various vector-based siRNA expression systems have been developed which utilize opposing polymerase III promoters to independently drive expression of the sense and antisense strands of the siRNA duplex from the same template.We show here the use of a ligase chain reaction (LCR to develop a new vector system called pInv-H1 in which a DNA sequence encoding a specific siRNA is placed between two inverted minimal human H1 promoters (approximately 100 bp each. Expression of functional siRNAs from this construct has led to efficient silencing of both reporter and endogenous genes. Furthermore, the inverted H1 promoter-siRNA expression cassette was used to generate a retrovirus vector capable of transducing and silencing expression of the targeted protein by>80% in target cells.The unique design of this construct allows for the efficient exchange of siRNA sequences by the directional cloning of short oligonucleotides via asymmetric restriction sites. This provides a convenient way to test the functionality of different siRNA sequences. Delivery of the siRNA cassette by retroviral transduction suggests that a single copy of the siRNA expression cassette efficiently knocks down gene expression at the protein level. We note that this vector system can potentially be used to generate a random siRNA library. The flexibility of the ligase chain reaction suggests that additional control elements can easily be introduced into this siRNA expression cassette.

  15. Antisense Oligonucleotides Promote Exon Inclusion and Correct the Common c.-32-13T>G GAA Splicing Variant in Pompe Disease

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    Erik van der Wal

    2017-06-01

    Full Text Available The most common variant causing Pompe disease is c.-32-13T>G (IVS1 in the acid α-glucosidase (GAA gene, which weakens the splice acceptor of GAA exon 2 and induces partial and complete exon 2 skipping. It also allows a low level of leaky wild-type splicing, leading to a childhood/adult phenotype. We hypothesized that cis-acting splicing motifs may exist that could be blocked using antisense oligonucleotides (AONs to promote exon inclusion. To test this, a screen was performed in patient-derived primary fibroblasts using a tiling array of U7 small nuclear RNA (snRNA-based AONs. This resulted in the identification of a splicing regulatory element in GAA intron 1. We designed phosphorodiamidate morpholino oligomer-based AONs to this element, and these promoted exon 2 inclusion and enhanced GAA enzyme activity to levels above the disease threshold. These results indicate that the common IVS1 GAA splicing variant in Pompe disease is subject to negative regulation, and inhibition of a splicing regulatory element using AONs is able to restore canonical GAA splicing and endogenous GAA enzyme activity.

  16. Pharmacomodulation of microRNA Expression in Neurocognitive Diseases: Obstacles and Future Opportunities.

    Science.gov (United States)

    Simion, Viorel; Nadim, Wissem Deraredj; Benedetti, Helene; Pichon, Chantal; Morisset-Lopez, Severine; Baril, Patrick

    2017-01-01

    Given the importance of microRNAs (miRNAs) in modulating brain functions and their implications in neurocognitive disorders there are currently significant efforts devoted in the field of miRNA-based therapeutics to correct and/or to treat these brain diseases. The observation that miRNA 29a/b-1 cluster, miRNA 10b and miRNA 7, for instance, are frequently deregulated in the brains of patients with neurocognitive diseases and in animal models of Alzheimer, Huntington's and Parkinson's diseases, suggest that correction of miRNA expression using agonist or antagonist miRNA oligonucleotides might be a promising approach to correct or even to cure such diseases. The encouraging results from recent clinical trials allow envisioning that pharmacological approaches based on miRNAs might, in a near future, reach the requirements for successful therapeutic outcomes and will improve the healthcare of patients with brain injuries or disorders. This review will focus on the current strategies used to modulate pharmacological function of miRNA using chemically modified oligonucleotides. We will then review the recent literature on strategies to improve nucleic acid delivery across the blood-brain barrier which remains a severe obstacle to the widespread application of miRNA therapeutics to treat brain diseases. Finally, we provide a state-of-art of current preclinical research performed in animal models for the treatment of neurocognitive disorders using miRNA as therapeutic agents and discuss future developments of miRNA therapeutics.

  17. Improved nucleic acid descriptors for siRNA efficacy prediction.

    Science.gov (United States)

    Sciabola, Simone; Cao, Qing; Orozco, Modesto; Faustino, Ignacio; Stanton, Robert V

    2013-02-01

    Although considerable progress has been made recently in understanding how gene silencing is mediated by the RNAi pathway, the rational design of effective sequences is still a challenging task. In this article, we demonstrate that including three-dimensional descriptors improved the discrimination between active and inactive small interfering RNAs (siRNAs) in a statistical model. Five descriptor types were used: (i) nucleotide position along the siRNA sequence, (ii) nucleotide composition in terms of presence/absence of specific combinations of di- and trinucleotides, (iii) nucleotide interactions by means of a modified auto- and cross-covariance function, (iv) nucleotide thermodynamic stability derived by the nearest neighbor model representation and (v) nucleic acid structure flexibility. The duplex flexibility descriptors are derived from extended molecular dynamics simulations, which are able to describe the sequence-dependent elastic properties of RNA duplexes, even for non-standard oligonucleotides. The matrix of descriptors was analysed using three statistical packages in R (partial least squares, random forest, and support vector machine), and the most predictive model was implemented in a modeling tool we have made publicly available through SourceForge. Our implementation of new RNA descriptors coupled with appropriate statistical algorithms resulted in improved model performance for the selection of siRNA candidates when compared with publicly available siRNA prediction tools and previously published test sets. Additional validation studies based on in-house RNA interference projects confirmed the robustness of the scoring procedure in prospective studies.

  18. Self-Amplifying Replicon RNA Vaccine Delivery to Dendritic Cells by Synthetic Nanoparticles

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    Kenneth C. McCullough

    2014-10-01

    Full Text Available Dendritic cells (DC play essential roles determining efficacy of vaccine delivery with respect to immune defence development and regulation. This renders DCs important targets for vaccine delivery, particularly RNA vaccines. While delivery of interfering RNA oligonucleotides to the appropriate intracellular sites for RNA-interference has proven successful, the methodologies are identical for RNA vaccines, which require delivery to RNA translation sites. Delivery of mRNA has benefitted from application of cationic entities; these offer value following endocytosis of RNA, when cationic or amphipathic properties can promote endocytic vesicle membrane perturbation to facilitate cytosolic translocation. The present review presents how such advances are being applied to the delivery of a new form of RNA vaccine, replicons (RepRNA carrying inserted foreign genes of interest encoding vaccine antigens. Approaches have been developed for delivery to DCs, leading to the translation of the RepRNA and encoded vaccine antigens both in vitro and in vivo. Potential mechanisms favouring efficient delivery leading to translation are discussed with respect to the DC endocytic machinery, showing the importance of cytosolic translocation from acidifying endocytic structures. The review relates the DC endocytic pathways to immune response induction, and the potential advantages for these self-replicating RNA vaccines in the near future.

  19. Detection of small interfering RNA (siRNA) by mass spectrometry procedures in doping controls.

    Science.gov (United States)

    Thomas, Andreas; Walpurgis, Katja; Delahaut, Philippe; Kohler, Maxie; Schänzer, Wilhelm; Thevis, Mario

    2013-01-01

    Uncovering manipulation of athletic performance via small interfering (si)RNA is an emerging field in sports drug testing. Due to the potential to principally knock down every target gene in the organism by means of the RNA interference pathway, this facet of gene doping has become a realistic scenario. In the present study, two distinct model siRNAs comprising 21 nucleotides were designed as double strands which were perfect counterparts to a sequence of the respective messenger RNA coding the muscle regulator myostatin of Rattus norvegicus. Several modified nucleotides were introduced in both the sense and the antisense strand comprising phosphothioates, 2'-O-methylation, 2'-fluoro-nucleotides, locked nucleic acids and a cholesterol tag at the 3'-end. The model siRNAs were applied to rats at 1 mg/kg (i.v.) and blood as well as urine samples were collected. After isolation of the RNA by means of a RNA purification kit, the target analytes were detected by liquid chromatography - high resolution/high accuracy mass spectrometry (LC-HRMS). Analytes were detected as modified nucleotides after alkaline hydrolysis, as intact oligonucleotide strands (top-down) and by means of denaturing SDS-PAGE analysis. The gel-separated siRNA was further subjected to in-gel hydrolysis with different RNases and subsequent identification of the fragments by untargeted LC-HRMS analysis (bottom-up, 'experimental RNomics'). Combining the results of all approaches, the identification of several 3'-truncated urinary metabolites was accomplished and target analytes were detected up to 24 h after a single administration. Simultaneously collected blood samples yielded no promising results. The methods were validated and found fit-for-purpose for doping controls. Copyright © 2013 John Wiley & Sons, Ltd.

  20. DETECTING LOW-LEVEL SYNTHESIS IMPURITIES IN MODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING LIQUID CHROMATOGRAPHY – HIGH RESOLUTION MASS SPECTROMETRY

    Science.gov (United States)

    Nikcevic, Irena; Wyrzykiewicz, Tadeusz K.; Limbach, Patrick A.

    2010-01-01

    Summary An LC-MS method based on the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS) for profiling oligonucleotides synthesis impurities is described. Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3′-terminal phosphate monoester and 3′-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N3 (2-cyanoethyl) adducts

  1. MicroRNA modulation combined with sunitinib as a novel therapeutic strategy for pancreatic cancer

    Directory of Open Access Journals (Sweden)

    Passadouro M

    2014-07-01

    Full Text Available Marta Passadouro,1,2 Maria C Pedroso de Lima,1,2 Henrique Faneca11Center for Neuroscience and Cell Biology, 2Department of Life Sciences, Faculty of Science and Technology, University of Coimbra, Coimbra, PortugalAbstract: Pancreatic ductal adenocarcinoma (PDAC is a highly aggressive and mortal cancer, characterized by a set of known mutations, invasive features, and aberrant microRNA expression that have been associated with hallmark malignant properties of PDAC. The lack of effective PDAC treatment options prompted us to investigate whether microRNAs would constitute promising therapeutic targets toward the generation of a gene therapy approach with clinical significance for this disease. In this work, we show that the developed human serum albumin–1-palmitoyl-2-oleoyl-sn-glycero-3-ethylphosphocholine:cholesterol/anti-microRNA oligonucleotides (+/– (4/1 nanosystem exhibits the ability to efficiently deliver anti-microRNA oligonucleotides targeting the overexpressed microRNAs miR-21, miR-221, miR-222, and miR-10 in PDCA cells, promoting an almost complete abolishment of microRNA expression. Silencing of these microRNAs resulted in a significant increase in the levels of their targets. Moreover, the combination of microRNA silencing, namely miR-21, with low amounts of the chemotherapeutic drug sunitinib resulted in a strong and synergistic antitumor effect, showing that this combined strategy could be of great importance for therapeutic application in PDAC. Keywords: pancreatic cancer gene therapy, anti-microRNAs oligonucleotides, delivery nanosystems, albumin-associated lipoplexes

  2. Polyethylene glycol and polyvinylpirrolidone effect on bacterial rRNA extraction and hybridization from cells exposed to tannins Efeito de polietilenoglicol e polivinilpirrolidona na extração e hibridização de rRNA bacteriano de células expostas a taninos

    OpenAIRE

    Pedro Braga Arcuri; Michael Larry Thonney; Peter Schofield; Alice Nelson Pell

    2003-01-01

    In order to detect fluctuations in ruminal microbial populations due to forage tannins using 16S ribosomal RNA (rRNA) probes, recovery of intact rRNA is required. The objective of this work was to evaluate the effect of polyethylene glycol (PEG) and polyvinylpirrolidone (PVP) on extraction of bacterial rRNA, in the presence of tannins from tropical legume forages and other sources, that hybridize with oligonucleotide probes. Ruminococcus albus 8 cells were exposed to 8 g/L tannic acid or 1 g/...

  3. Telomeric repeat-containing RNA/G-quadruplex-forming sequences cause genome-wide alteration of gene expression in human cancer cells in vivo.

    Science.gov (United States)

    Hirashima, Kyotaro; Seimiya, Hiroyuki

    2015-02-27

    Telomere erosion causes cell mortality, suggesting that longer telomeres enable more cell divisions. In telomerase-positive human cancer cells, however, telomeres are often kept shorter than those of surrounding normal tissues. Recently, we showed that cancer cell telomere elongation represses innate immune genes and promotes their differentiation in vivo. This implies that short telomeres contribute to cancer malignancy, but it is unclear how such genetic repression is caused by elongated telomeres. Here, we report that telomeric repeat-containing RNA (TERRA) induces a genome-wide alteration of gene expression in telomere-elongated cancer cells. Using three different cell lines, we found that telomere elongation up-regulates TERRA signal and down-regulates innate immune genes such as STAT1, ISG15 and OAS3 in vivo. Ectopic TERRA oligonucleotides repressed these genes even in cells with short telomeres under three-dimensional culture conditions. This appeared to occur from the action of G-quadruplexes (G4) in TERRA, because control oligonucleotides had no effect and a nontelomeric G4-forming oligonucleotide phenocopied the TERRA oligonucleotide. Telomere elongation and G4-forming oligonucleotides showed similar gene expression signatures. Most of the commonly suppressed genes were involved in the innate immune system and were up-regulated in various cancers. We propose that TERRA G4 counteracts cancer malignancy by suppressing innate immune genes. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  4. Extracellular RNA Communication (ExRNA)

    Data.gov (United States)

    Federal Laboratory Consortium — Until recently, scientists believed RNA worked mostly inside the cell that produced it. Some types of RNA help translate genes into proteins that are necessary for...

  5. New redox-active layer create via epoxy-amine reaction - The base of genosensor for the detection of specific DNA and RNA sequences of avian influenza virus H5N1.

    Science.gov (United States)

    Malecka, Kamila; Stachyra, Anna; Góra-Sochacka, Anna; Sirko, Agnieszka; Zagórski-Ostoja, Włodzimierz; Dehaen, Wim; Radecka, Hanna; Radecki, Jerzy

    2015-03-15

    This paper concerns the development of a redox-active monolayer and its application for the construction of an electrochemical genosensor designed for the detection of specific DNA and RNA oligonucleotide sequences related to the avian influenza virus (AIV) type H5N1. This new redox layer was created on a gold electrode surface step by step. Cyclic Voltammetry, Osteryoung Square-Wave Voltammetry and Differential Pulse Voltammetry were used for its characterization. This new redox-active layer was applied for the construction of the DNA biosensor. The NH2-NC3 probe (20-mer) was covalently attached to the gold electrode surface via a "click" reaction between the amine and an epoxide group. The hybridization process was monitored using the Osteryoung Square-Wave Voltammetry. The 20-mer DNA and ca. 280-mer RNA oligonucleotides were used as the targets. The constructed genosensor was capable to determine complementary oligonucleotide sequences with a detection limit in the pM range. It is able to distinguish the different position of the part RNA complementary to the DNA probe. The genosensor was very selective. The 20-mer DNA as well as the 280-mer RNA oligonucleotides without a complementary sequence generated a weak signal. Copyright © 2014 Elsevier B.V. All rights reserved.

  6. Detection of hepatitis C virus RNA: comparison of one-stage polymerase chain reaction (PCR) with nested-set PCR.

    OpenAIRE

    Gretch, D R; Wilson, J J; Carithers, R L; dela Rosa, C; Han, J H; Corey, L

    1993-01-01

    We evaluated a new hepatitis C virus RNA assay based on one-stage PCR followed by liquid hybridization with an oligonucleotide probe and compared it with nested-set PCR. The one-stage and nested-set PCR assays had identical sensitivities in analytical experiments and showed 100% concordance when clinical specimens were used. One-stage PCR may be less prone to contamination than nested-set PCR.

  7. Analysis of oligonucleotide array experiments with repeated measures using mixed models

    Directory of Open Access Journals (Sweden)

    Getchell Thomas V

    2004-12-01

    Full Text Available Abstract Background Two or more factor mixed factorial experiments are becoming increasingly common in microarray data analysis. In this case study, the two factors are presence (Patients with Alzheimer's disease or absence (Control of the disease, and brain regions including olfactory bulb (OB or cerebellum (CER. In the design considered in this manuscript, OB and CER are repeated measurements from the same subject and, hence, are correlated. It is critical to identify sources of variability in the analysis of oligonucleotide array experiments with repeated measures and correlations among data points have to be considered. In addition, multiple testing problems are more complicated in experiments with multi-level treatments or treatment combinations. Results In this study we adopted a linear mixed model to analyze oligonucleotide array experiments with repeated measures. We first construct a generalized F test to select differentially expressed genes. The Benjamini and Hochberg (BH procedure of controlling false discovery rate (FDR at 5% was applied to the P values of the generalized F test. For those genes with significant generalized F test, we then categorize them based on whether the interaction terms were significant or not at the α-level (αnew = 0.0033 determined by the FDR procedure. Since simple effects may be examined for the genes with significant interaction effect, we adopt the protected Fisher's least significant difference test (LSD procedure at the level of αnew to control the family-wise error rate (FWER for each gene examined. Conclusions A linear mixed model is appropriate for analysis of oligonucleotide array experiments with repeated measures. We constructed a generalized F test to select differentially expressed genes, and then applied a specific sequence of tests to identify factorial effects. This sequence of tests applied was designed to control for gene based FWER.

  8. Ultrasensitive electrochemical biosensor based on the oligonucleotide self-assembled monolayer-mediated immunosensing interface

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Dengyou; Luo, Qimei [Science College of Hunan Agricultural University, Changsha 410128 (China); Deng, Fawen [The Fourth Hospital of Chansha, Changsha 410006 (China); Li, Zhen [Science College of Hunan Agricultural University, Changsha 410128 (China); Li, Benxiang, E-mail: 172170960@qq.com [Science College of Hunan Agricultural University, Changsha 410128 (China); Shen, Zhifa, E-mail: shenzhifa@wmu.edu.cn [Key Laboratory of Laboratory Medicine, Ministry of Education, Zhejiang Provincial Key Laboratory of Medical Genetics, College of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035 (China)

    2017-06-08

    Highly sensitive and selective quantitation of a variety of proteins over a wide concentration range is highly desirable for increased accuracy of biomarker detection or for multidisease diagnostics. In the present contribution, using human immunoglobulin G (HIgG) as the model target protein, an electrochemical ultrasensitive immunosensing platform was developed based on the oligonucleotide self-assembled monolayer-mediated (OSAM) sensing interface. For this immunosensor, the “signal-on” signaling mechanism and enzymatic signal amplification effect were integrated into one sensing architecture. Moreover, the thiolated flexible single-stranded DNAs immobilized onto gold electrode surface not only performs the wobbling motion to facilitate the electron transfer between the electrode surface and biosensing layer but also fundamentally prohibiting the direct interaction of proteins with gold substrate. Thus, the electrochemical signal could be efficiently enhanced and the unspecific adsorption or cross-reaction might be eliminated. As a result, utilizing the newly-proposed immunosensor, the HIgG can be detected down to 0.5 ng/mL, and the high detection specificity is offered. The successful design of OSAM and the highly desirable detection capability of new immunosensor are expected to provide a perspective for fabricating new robust immunosensing platform and for promising potential of oligonucleotide probe in biological research and biomedical diagnosis. - Highlights: • An electrochemical ultrasensitive immunosensing platform was developed based on the oligonucleotide self-assembled monolayer (OASM). • OASM severs as a flexible monolayer to promote electron transfer and prohibits the direct interaction of proteins with gold substrate. • The electrochemical signal is efficiently enhanced and the unspecific adsorption or cross-reaction is eliminated. • Target protein can be detected down to 0.5 ng/mL, and the high detection specificity can be obtained.

  9. NAA-modified DNA oligonucleotides with zwitterionic backbones: stereoselective synthesis of A-T phosphoramidite building blocks.

    Science.gov (United States)

    Schmidtgall, Boris; Höbartner, Claudia; Ducho, Christian

    2015-01-01

    Modifications of the nucleic acid backbone are essential for the development of oligonucleotide-derived bioactive agents. The NAA-modification represents a novel artificial internucleotide linkage which enables the site-specific introduction of positive charges into the otherwise polyanionic backbone of DNA oligonucleotides. Following initial studies with the introduction of the NAA-linkage at T-T sites, it is now envisioned to prepare NAA-modified oligonucleotides bearing the modification at X-T motifs (X = A, C, G). We have therefore developed the efficient and stereoselective synthesis of NAA-linked 'dimeric' A-T phosphoramidite building blocks for automated DNA synthesis. Both the (S)- and the (R)-configured NAA-motifs were constructed with high diastereoselectivities to furnish two different phosphoramidite reagents, which were employed for the solid phase-supported automated synthesis of two NAA-modified DNA oligonucleotides. This represents a significant step to further establish the NAA-linkage as a useful addition to the existing 'toolbox' of backbone modifications for the design of bioactive oligonucleotide analogues.

  10. Evaluation of fluorine-18-labeled alkylating agents as potential synthons for the labeling of oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Vries, E.F.J. de E-mail: e.f.j.de.vries@pet.azg.nl; Vroegh, Joke; Elsinga, P.H.; Vaalburg, Willem

    2003-04-01

    Six fluorine-18-labeled alkylating agents were selected as potentially suitable synthons for the labeling of antisense oligonucleotides. The selected synthons were evaluated in a model reaction with the monomer adenosine 5'-O-thiomonophosphate. Of these synthons, {alpha}-bromo-{alpha}'-[{sup 18}F]fluoro-m-xylene and N-(4-[{sup 18}F]fluorobenzyl)-2-bromoacetamide were found to be the most promising. Labeling with the former synthon was less complicated and time consuming and gave higher uncorrected overall yields. The latter synthon required smaller amounts of the costly precursor to achieve acceptable labeling yields.

  11. A New Achiral Linker Reagent for the Incorporation of Multiple Amino Groups Into Oligonucleotides

    DEFF Research Database (Denmark)

    1997-01-01

    The present invention relates to a new functionalized achiral linker reagent for incorporating multiple primary amino groups or reporter groups into oligonucleotides following the phosphoramidite methodology. It is possible to substitute any ribodeoxynucleotide, deoxynucleotide, or nucleotide......-oxyl-2,2,5,5-tetramethylpyrrolidine), TEMPO (N-oxyl-2,2,6,6-tetramethylpiperidine), dinitrophenyl, texas red, tetramethyl rhodamine, 7-nitrobenzo-2-oxa-1-diazole (NBD), or pyrene. The present invention also relates to a solid phase support, e.g. a Controlled Pore Glass (CPG), immobilized linker reagent...

  12. Information Limited Oligonucleotide Amplification Assay for Affinity-Based, Parallel Detection Studies.

    Directory of Open Access Journals (Sweden)

    Harish Bokkasam

    Full Text Available Molecular communication systems encounter similar constraints as telecommunications. In either case, channel crosstalk at the receiver end will result in information loss that statistical analysis cannot compensate. This is because in any communication channel there is a physical limit to the amount of information that can be transmitted. We present a novel and simple modified end amplification (MEA technique to generate reduced and defined amounts of specific information in form of short fragments from an oligonucleotide source that also contains unrelated and redundant information. Our method can be a valuable tool to investigate information overflow and channel capacity in biomolecular recognition systems.

  13. Synthesis of modified oligonucleotides that contain purine and pyrimidine radio-induced base lesions

    International Nuclear Information System (INIS)

    Romieu, Anthony

    1999-01-01

    Different factors as oxidizing or carcinogenesis agents, UV and ionizing radiations,.... can generate a wide, spectrum of DNA base damages. In order to study the biochemical and structural features of these DNA damages, it is important to prepare short DNA fragments (20 to 50 bases long) bearing a single or several modifications at specific sites in their sequences. The chemical synthesis is a powerful tool to prepare such modified DNA fragments. This work focusses on the chemical incorporation of several modified nucleosides formed in DNA by ionizing radiations or by photo-sensitization. The first part of this study describes the preparation of a phosphoramidite synthon of 5-hydroxy-2'-deoxyuridine and its subsequent incorporation into synthetic oligonucleotides ranging from 14 to 33 bases long. In a second part (chapters Ill and IV), the synthesis and incorporation of original radiation-induced tandem lesions: the carbon-bridged cyclo-nucleosides are presented. Both (5'R)- and (5'S) diastereomers of 5',8-cyclo-2'-deoxyadenosine and 5',8-cyclo-2'-deoxyguanosine have been individually inserted into various different oligonucleotides (3 to 22 bases long) by using the standard phosphoramidite chemistry. The chemical incorporation of a pyrimidine analogue: (5'S, 6S)-5',6-cyclo-5,6-dihydro-thymidine has been also achieved. The loss of aromaticity of this modified nucleoside and its poor reactivity required the development of a synthetic strategy entirely different from that used for the preparation and subsequent incorporation of the phosphoramidite synthons of 5',8-cyclo-purine-2'-deoxyribo-nucleosides. The third part of this study deals with the synthesis of a phosphoramidite synthon of 4-hydroxy-8-oxo-4,8-dihydro-2'-deoxyguanosine. The two (4R)- and (4S)- diastereomers of this oxidized purine have been separated and individually inserted in several synthetic DNA fragments. No epimerization of C-4 position was observed during the solid-phase synthesis and during the

  14. Sequence characterization of 5S ribosomal RNA from eight gram positive procaryotes

    Science.gov (United States)

    Woese, C. R.; Luehrsen, K. R.; Pribula, C. D.; Fox, G. E.

    1976-01-01

    Complete nucleotide sequences are presented for 5S rRNA from Bacillus subtilis, B. firmus, B. pasteurii, B. brevis, Lactobacillus brevis, and Streptococcus faecalis, and 5S rRNA oligonucleotide catalogs and partial sequence data are given for B. cereus and Sporosarcina ureae. These data demonstrate a striking consistency of 5S rRNA primary and secondary structure within a given bacterial grouping. An exception is B. brevis, in which the 5S rRNA sequence varies significantly from that of other bacilli in the tuned helix and the procaryotic loop. The localization of these variations suggests that B. brevis occupies an ecological niche that selects such changes. It is noted that this organism produces antibiotics which affect ribosome function.

  15. Import of desired nucleic acid sequences using addressing motif of mitochondrial ribosomal 5S-rRNA for fluorescent in vivo hybridization of mitochondrial DNA and RNA.

    Science.gov (United States)

    Zelenka, Jaroslav; Alán, Lukáš; Jabůrek, Martin; Ježek, Petr

    2014-04-01

    Based on the matrix-addressing sequence of mitochondrial ribosomal 5S-rRNA (termed MAM), which is naturally imported into mitochondria, we have constructed an import system for in vivo targeting of mitochondrial DNA (mtDNA) or mt-mRNA, in order to provide fluorescence hybridization of the desired sequences. Thus DNA oligonucleotides were constructed, containing the 5'-flanked T7 RNA polymerase promoter. After in vitro transcription and fluorescent labeling with Alexa Fluor(®) 488 or 647 dye, we obtained the fluorescent "L-ND5 probe" containing MAM and exemplar cargo, i.e., annealing sequence to a short portion of ND5 mRNA and to the light-strand mtDNA complementary to the heavy strand nd5 mt gene (5'-end 21 base pair sequence). For mitochondrial in vivo fluorescent hybridization, HepG2 cells were treated with dequalinium micelles, containing the fluorescent probes, bringing the probes proximally to the mitochondrial outer membrane and to the natural import system. A verification of import into the mitochondrial matrix of cultured HepG2 cells was provided by confocal microscopy colocalizations. Transfections using lipofectamine or probes without 5S-rRNA addressing MAM sequence or with MAM only were ineffective. Alternatively, the same DNA oligonucleotides with 5'-CACC overhang (substituting T7 promoter) were transcribed from the tetracycline-inducible pENTRH1/TO vector in human embryonic kidney T-REx®-293 cells, while mitochondrial matrix localization after import of the resulting unlabeled RNA was detected by PCR. The MAM-containing probe was then enriched by three-order of magnitude over the natural ND5 mRNA in the mitochondrial matrix. In conclusion, we present a proof-of-principle for mitochondrial in vivo hybridization and mitochondrial nucleic acid import.

  16. Enzymatic synthesis of tRNA-peptide conjugates and spectroscopic studies of fluorine-modified RNA

    International Nuclear Information System (INIS)

    Graber, D.

    2010-01-01

    possess the naturally occurring nucleoside modifications. Hence, an alternative process for access to 5'-fragments containing these modifications was needed. Starting from wild-type tRNA, a DNA-enzyme mediated position-specific cleavage at the desired cleavage site was elaborated. For quantitative cleavage, the introduction of repeated temperature cycles was inevitable. Dephosphorylation of the so obtained 2',3'-cyclophosphate cleavage products had to be performed prior to ligating the wild-type 5'-fragment by T4 RNA ligase to the chimeric 3'-fragment yielding the fully modified tRNA-peptide conjugate. The broad applicability of that approach was demonstrated by successful ligation of various tRNA, and tRNA from different species. In the second part of this thesis fluorinated nucleic acids were applied to 19F NMR spectroscopic investigations. One subproject concerned fluorinated nucleic acids for probing secondary structures. For that reason, a 2,4-difluorotoluyl-ribofuranose phosphoramidite was synthesized and site-specifically incorporated into oligonucleotides. As a proof of principle, the differentiation between monomolecular and bimolecular melting transitions was demonstrated by monitoring the temperature dependent alterations in the chemical shift signatures. It was also shown that oligonucleotides of self-complementary sequences - which simultaneously adopt different secondary structures - can be analyzed in terms of quantification of the coexisting populations. Moreover, melting temperatures determined by 19F NMR spectroscopy were in excellent accordance with those found using traditional UV-techniques. In another subproject, the interaction of tRNA pseudouridine synthase (TruB) with its TΨC loop tRNA substrate was studied using 19F NMR spectroscopy. So far, published contributions have focused on 5-fluorouridine substrate/enzyme reactions which were expected to result in a stable covalently linked RNA-enzyme complex. However, the enzyme was capable of

  17. Use of bacterially expressed dsRNA to downregulate Entamoeba histolytica gene expression.

    Directory of Open Access Journals (Sweden)

    Carlos F Solis

    Full Text Available BACKGROUND: Modern RNA interference (RNAi methodologies using small interfering RNA (siRNA oligonucleotide duplexes or episomally synthesized hairpin RNA are valuable tools for the analysis of gene function in the protozoan parasite Entamoeba histolytica. However, these approaches still require time-consuming procedures including transfection and drug selection, or costly synthetic molecules. PRINCIPAL FINDINGS: Here we report an efficient and handy alternative for E. histolytica gene down-regulation mediated by bacterial double-stranded RNA (dsRNA targeting parasite genes. The Escherichia coli strain HT115 which is unable to degrade dsRNA, was genetically engineered to produce high quantities of long dsRNA segments targeting the genes that encode E. histolytica beta-tubulin and virulence factor KERP1. Trophozoites cultured in vitro were directly fed with dsRNA-expressing bacteria or soaked with purified dsRNA. Both dsRNA delivery methods resulted in significant reduction of protein expression. In vitro host cell-parasite assays showed that efficient downregulation of kerp1 gene expression mediated by bacterial dsRNA resulted in significant reduction of parasite adhesion and lytic capabilities, thus supporting a major role for KERP1 in the pathogenic process. Furthermore, treatment of trophozoites cultured in microtiter plates, with a repertoire of eighty-five distinct bacterial dsRNA segments targeting E. histolytica genes with unknown function, led to the identification of three genes potentially involved in the growth of the parasite. CONCLUSIONS: Our results showed that the use of bacterial dsRNA is a powerful method for the study of gene function in E. histolytica. This dsRNA delivery method is also technically suitable for the study of a large number of genes, thus opening interesting perspectives for the identification of novel drug and vaccine targets.

  18. Automated synthesis of an {sup 18}F-labelled pyridine-based alkylating agent for high yield oligonucleotide conjugation

    Energy Technology Data Exchange (ETDEWEB)

    Guggenberg, Elisabeth von; Sader, Jayden A.; Wilson, John S.; Shahhosseini, Soraya; Koslowsky, Ingrid; Wuest, Frank [Edmonton PET Centre, Division of Oncologic Imaging, Department of Oncology, Cross Cancer Institute, 11560 University Ave, Edmonton, AB, T6G 1Z2 (Canada); Mercer, John R. [Edmonton PET Centre, Division of Oncologic Imaging, Department of Oncology, Cross Cancer Institute, 11560 University Ave, Edmonton, AB, T6G 1Z2 (Canada)], E-mail: johnmerc@cancerboard.ab.ca

    2009-09-15

    Alkylating agents have been shown to be very promising for the radiolabelling of oligonucleotides with fluorine-18. In this report we describe the fully automated synthesis of 2-bromo-N-[3-(2-[{sup 18}F]fluoropyridin-3-yloxy)propyl]acetamide ([{sup 18}F]FPyBrA) utilizing a modular synthesis unit. Reaction conditions for the coupling of this pyridine-based alkylating agent at the 5' end of a fully phosphorothioated random 20-mer DNA sequence were optimized to achieve very high radiochemical yields (>90%) and a maximum specific activity of 5-6 GBq/{mu}moL. The potential for rapid purification by solid phase extraction without need of chromatographic isolation of the radiolabelled oligonucleotide presents an overall benefit for the application of oligonucleotides in preclinical studies and potential clinical applications.

  19. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    Science.gov (United States)

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

  20. Spectroscopic (UV/VIS, Raman and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    Directory of Open Access Journals (Sweden)

    Seyedeh Maryam Banihashemian

    Full Text Available Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100, is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT. As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field.

  1. Cy3 and Cy5 dyes attached to oligonucleotide terminus stabilize DNA duplexes: predictive thermodynamic model.

    Science.gov (United States)

    Moreira, Bernardo G; You, Yong; Owczarzy, Richard

    2015-03-01

    Cyanine dyes are important chemical modifications of oligonucleotides exhibiting intensive and stable fluorescence at visible light wavelengths. When Cy3 or Cy5 dye is attached to 5' end of a DNA duplex, the dye stacks on the terminal base pair and stabilizes the duplex. Using optical melting experiments, we have determined thermodynamic parameters that can predict the effects of the dyes on duplex stability quantitatively (ΔG°, Tm). Both Cy dyes enhance duplex formation by 1.2 kcal/mol on average, however, this Gibbs energy contribution is sequence-dependent. If the Cy5 is attached to a pyrimidine nucleotide of pyrimidine-purine base pair, the stabilization is larger compared to the attachment to a purine nucleotide. This is likely due to increased stacking interactions of the dye to the purine of the complementary strand. Dangling (unpaired) nucleotides at duplex terminus are also known to enhance duplex stability. Stabilization originated from the Cy dyes is significantly larger than the stabilization due to the presence of dangling nucleotides. If both the dangling base and Cy3 are present, their thermodynamic contributions are approximately additive. New thermodynamic parameters improve predictions of duplex folding, which will help design oligonucleotide sequences for biophysical, biological, engineering, and nanotechnology applications. Copyright © 2015. Published by Elsevier B.V.

  2. Reliable Assessment and Quantification of the Fluorescence-Labeled Antisense Oligonucleotides In Vivo

    Directory of Open Access Journals (Sweden)

    Maria Chiara Munisso

    2014-01-01

    Full Text Available The availability of fluorescent dyes and the advances in the optical systems for in vivo imaging have stimulated an increasing interest in developing new methodologies to study and quantify the biodistribution of labeled agents. However, despite these great achievements, we are facing significant challenges in determining if the observed fluorescence does correspond to the quantity of the dye in the tissues. In fact, although the far-red and near-infrared lights can propagate through several centimetres of tissue, they diffuse within a few millimetres as consequence of the elastic scattering of photons. In addition, when dye-labeled oligonucleotides form stable complex with cationic carriers, a large change in the fluorescence intensity of the dye is observed. Therefore, the measured fluorescence intensity is altered by the tissue heterogeneity and by the fluctuation of dye intensity. Hence, in this study a quantification strategy for fluorescence-labeled oligonucleotides was developed to solve these disadvantageous effects. Our results proved that upon efficient homogenization and dilution with chaotropic agents, such as guanidinium thiocyanate, it is possible to achieve a complete fluorescence intensity recovery. Furthermore, we demonstrated that this method has the advantage of good sensitivity and reproducibility, as well as easy handling of the tissue samples.

  3. Antiproliferation effects of an androgen receptor triple-helix forming oligonucleotide on prostate cancer cells

    International Nuclear Information System (INIS)

    Zhang Yong; Chen Weizhen; Xie Yao; Gao Jinhui

    2005-01-01

    Objective: To provide experimental basis for antigene radiation therapy through exploring the effects of antigene strategy on androgen receptor (AR) expression and proliferation of prostate cancer cells. Methods: The triple-helix forming oligonucleotide (TFO) targeting 2447-2461nt of AR cDNA was designed and transfected LNCaP prostate cancer cells with liposome. 24-72 h after transfection, the cellular proliferation was detected by 3 H-thymidine (TdR) incorporation test, the expression of AR gene was examined by reverse transcription-polymerase chain reaction (RT-PCR) and expression of AR protein was performed by radioligand binding assay. The results of TFO were compared with antisense oligonucleotide (ASON). Results: At all time points, the AR expression levels in TFO group were markedly lower than that of ASON group (P<0.05). The inhibitory rate of TFO for cellular proliferation was significantly higher than that of ASON (P<0.05). Conclusion: The TFO was a potent inhibitor for AR expression and cell proliferation of LNCaP cells , and could be used in antigene radiotherapy. (authors)

  4. The detection and differentiation of canine respiratory pathogens using oligonucleotide microarrays.

    Science.gov (United States)

    Wang, Lih-Chiann; Kuo, Ya-Ting; Chueh, Ling-Ling; Huang, Dean; Lin, Jiunn-Horng

    2017-05-01

    Canine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip. The detection limit is 10, 10, 100, 50 and 50 copy numbers for CDV, influenza virus, CHV, B. bronchiseptica and M. cynos, respectively. The clinical test results of nasal swab samples showed that the microarray had remarkably better efficacy than the multiplex PCR-agarose gel method. The positive detection rate of microarray and agarose gel was 59.0% (n=33) and 41.1% (n=23) among the 56 samples, respectively. CDV vaccine strain and pathogen co-infections were further demonstrated by the microarray but not by the multiplex PCR-agarose gel. The oligonucleotide microarray provides a highly efficient diagnosis alternative that could be applied to clinical usage, greatly assisting in disease therapy and control. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Pretreatment of Mice with Oligonucleotide prop5 Protects Them from Influenza Virus Infections

    Directory of Open Access Journals (Sweden)

    Kang Li

    2014-02-01

    Full Text Available Influenza A virus is a successful parasite and requires host factors to complete its life cycle. Prop5 is an antisense oligonucleotide, targeting programmed cell death protein 5 (PDCD5. In this study, we tested the antiviral activity of prop5 against mouse-adapted A/FM/1/47 strain of influenza A virus in a mouse model. Prop5 intranasally administered the mice at dosages of 10 and 20 mg/kg/d at 24 h and 30 min before infection, provided 80% and 100% survival rates and prolonged mean survival days in comparison with influenza virus-infected mice (both p < 0.01. Moreover, viral titres in mice pretreated with prop5, at dose of 10 and 20 mg/kg/d, had declined significantly on day two, four, and six post-infection compared with the yields in infected mice (p < 0.05 or p < 0.01; lung index in mice pretreated with prop5 (20 mg/kg/d had been inhibited on day six post-infection (p < 0.05. Western blotting and immunohistochemistry showed that prop5 could down-regulate the PDCD5 protein expression levels in lung tissues of infected mice. These data indicate that antisense oligonucleotide prop5 is a promising drug for prophylaxis and control influenza virus infections and provides an insight into the host-pathogen interaction.

  6. Template-Independent Enzymatic Oligonucleotide Synthesis (TiEOS): Its History, Prospects, and Challenges.

    Science.gov (United States)

    Jensen, Michael A; Davis, Ronald W

    2018-03-27

    There is a growing demand for sustainable methods in research and development, where instead of hazardous chemicals, an aqueous medium is chosen to perform biological reactions. In this Perspective, we examine the history and current methodology of using enzymes to generate artificial single-stranded DNA. By using traditional solid-phase phosphoramidite chemistry as a metric, we also explore criteria for the method of template-independent enzymatic oligonucleotide synthesis (TiEOS). As its key component, we delve into the biology of one of the most enigmatic enzymes, terminal deoxynucleotidyl transferase (TdT). As TdT is found to exponentially increase antigen receptor diversity in the vertebrate immune system by adding nucleotides in a template-free manner, researchers have exploited this function as an alternative to the phosphoramidite synthesis method. Though TdT is currently the preferred enzyme for TiEOS, its random nucleotide incorporation presents a barrier in synthesis automation. Taking a closer look at the TiEOS cycle, particularly the coupling step, we find it is comprised of additions > n+1 and deletions. By tapping into the physical and biochemical properties of TdT, we strive to further elucidate its mercurial behavior and offer ways to better optimize TiEOS for production-grade oligonucleotide synthesis.

  7. Molecular imaging of atherosclerotic plaques with technetium-99m-labelled antisense oligonucleotides

    International Nuclear Information System (INIS)

    Qin Guangming; Zhang Yongxue; Cao Wei; An Rui; Gao Zairong; Xu Wendai; Zhang Kaijun; Li Guiling; Li Shuren

    2005-01-01

    The purpose of this study was to visualise experimental atherosclerotic lesions using radiolabelled antisense oligonucleotides (ASONs). Atherosclerosis was induced in New Zealand White rabbits fed 1% cholesterol for approximately 60 days. In vivo and ex vivo imaging was performed in atherosclerotic rabbits and normal control rabbits after i.v. injection of 92.5±18.5 MBq 99m Tc-labelled ASON or 99m Tc-labelled sense oligonucleotides. Immediately after the in vivo imaging, the animals were sacrificed and ex vivo imaging of the aortic specimens was performed. Biodistribution of radiolabelled c-mycASON was evaluated in vivo in atherosclerotic rabbits. Planar imaging revealed accumulation of 99m Tc-labelled c-mycASON in atherosclerotic lesions along the artery wall. Ex vivo imaging further demonstrated that the area of activity accumulation matched the area of atherosclerotic lesions. In contrast, no atherosclerotic lesions were found in the vessel wall and no positive imaging results were obtained in animals of the control group. This molecular imaging approach has potential for non-invasive imaging of atherosclerotic plaques at an early stage. (orig.)

  8. Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray.

    Science.gov (United States)

    Wang, Zheng; Vora, Gary J; Stenger, David A

    2004-07-01

    Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.

  9. Short Oligonucleotides Aligned in Stretched Humid Matrix: Secondary DNA Structure in Poly(vinyl alcohol) Environment

    KAUST Repository

    Hanczyc, Piotr

    2012-04-24

    We report that short, synthetic, double- as well as single-stranded DNA can be aligned in stretched humid poly(vinyl alcohol) (PVA) matrix, and the secondary structure (nucleobase orientation) can be characterized with linear dichroism (LD) spectroscopy. Oligonucleotides of lengths varying between 10 (3.4 nm) and 60 bases (20.4 nm) were investigated with respect to structural properties in the gel-like polymer environment. The DNA conformation as a function of relative humidity reveals a strong dependence of helical structure of DNA on PVA hydration level, results of relevance for nanotechnical studies of DNA-based supramolecular systems. Also, the PVA gel could provide possibilities to test models for nucleic acid interactions and distribution in cell contexts, including structural stability of genetic material in the cell and PVA-packaging for gene delivery. A method by which duplex oligonucleotides, with sequences designed to provide specific binding sites, become amenable to polarized-light spectroscopy opens up new possibilities for studying structure in DNA complexes with small adduct molecules as well as proteins. © 2012 American Chemical Society.

  10. Efficacy and Safety Profile of Tricyclo-DNA Antisense Oligonucleotides in Duchenne Muscular Dystrophy Mouse Model

    Directory of Open Access Journals (Sweden)

    Karima Relizani

    2017-09-01

    Full Text Available Antisense oligonucleotides (AONs hold promise for therapeutic splice-switching correction in many genetic diseases. However, despite advances in AON chemistry and design, systemic use of AONs is limited due to poor tissue uptake and sufficient therapeutic efficacy is still difficult to achieve. A novel class of AONs made of tricyclo-DNA (tcDNA is considered very promising for the treatment of Duchenne muscular dystrophy (DMD, a neuromuscular disease typically caused by frameshifting deletions or nonsense mutations in the gene-encoding dystrophin and characterized by progressive muscle weakness, cardiomyopathy, and respiratory failure in addition to cognitive impairment. Herein, we report the efficacy and toxicology profile of a 13-mer tcDNA in mdx mice. We show that systemic delivery of 13-mer tcDNA allows restoration of dystrophin in skeletal muscles and to a lower extent in the brain, leading to muscle function improvement and correction of behavioral features linked to the emotional/cognitive deficiency. More importantly, tcDNA treatment was generally limited to minimal glomerular changes and few cell necroses in proximal tubules, with only slight variation in serum and urinary kidney toxicity biomarker levels. These results demonstrate an encouraging safety profile for tcDNA, albeit typical of phosphorothiate AONs, and confirm its therapeutic potential for the systemic treatment of DMD patients. Keywords: antisense oligonucleotides, Duchenne muscular dystrophy, preclinical, splice switching, tcDNA-AONs

  11. Design and kinetic analysis of hammerhead ribozyme and DNAzyme that specifically cleave TEL-AML1 chimeric mRNA

    International Nuclear Information System (INIS)

    Choi, Woo-Hyung; Choi, Bo-Ra; Kim, Jae Hyun; Yeo, Woon-Seok; Oh, Sangtaek; Kim, Dong-Eun

    2008-01-01

    In order to develop the oligonucleotides to abolish an expression of TEL-AML1 chimeric RNA, which is a genetic aberration that causes the acute lymphoblastic leukemia (ALL), hammerhead ribozymes and deoxyoligoribozymes that can specifically cleave TEL-AML1 fusion RNA were designed. Constructs of the deoxyribozyme with an asymmetric substrate binding arm (Dz26) and the hammerhead ribozyme with a 4 nt-bulged substrate binding arm in the stem III (buRz28) were able to cleave TEL-AML1 chimeric RNA specifically at sites close to the junction in vitro, without cleaving the normal TEL and AML1 RNA. Single-turnover kinetic analysis under enzyme-excess condition revealed that the buRz28 is superior to the Dz26 in terms of substrate binding and RNA-cleavage. In conjunction with current progress in a gene-delivery technology, the designed oligonucleotides that specifically cleave the TEL-AML1 chimeric mRNA are hoped to be applicable for the treatment of ALL in vivo

  12. RNA binding specificity of Ebola virus transcription factor VP30.

    Science.gov (United States)

    Schlereth, Julia; Grünweller, Arnold; Biedenkopf, Nadine; Becker, Stephan; Hartmann, Roland K

    2016-09-01

    The transcription factor VP30 of the non-segmented RNA negative strand Ebola virus balances viral transcription and replication. Here, we comprehensively studied RNA binding by VP30. Using a novel VP30:RNA electrophoretic mobility shift assay, we tested truncated variants of 2 potential natural RNA substrates of VP30 - the genomic Ebola viral 3'-leader region and its complementary antigenomic counterpart (each ∼155 nt in length) - and a series of other non-viral RNAs. Based on oligonucleotide interference, the major VP30 binding region on the genomic 3'-leader substrate was assigned to the internal expanded single-stranded region (∼ nt 125-80). Best binding to VP30 was obtained with ssRNAs of optimally ∼ 40 nt and mixed base composition; underrepresentation of purines or pyrimidines was tolerated, but homopolymeric sequences impaired binding. A stem-loop structure, particularly at the 3'-end or positioned internally, supports stable binding to VP30. In contrast, dsRNA or RNAs exposing large internal loops flanked by entirely helical arms on both sides are not bound. Introduction of a 5´-Cap(0) structure impaired VP30 binding. Also, ssDNAs bind substantially weaker than isosequential ssRNAs and heparin competes with RNA for binding to VP30, indicating that ribose 2'-hydroxyls and electrostatic contacts of the phosphate groups contribute to the formation of VP30:RNA complexes. Our results indicate a rather relaxed RNA binding specificity of filoviral VP30, which largely differs from that of the functionally related transcription factor of the Paramyxoviridae which binds to ssRNAs as short as 13 nt with a preference for oligo(A) sequences.

  13. RNA helicase A is not required for RISC activity.

    Science.gov (United States)

    Liang, Xue-Hai; Crooke, Stanley T

    2013-10-01

    It has been shown that siRNAs can compete with each other or with endogenous miRNAs for RISC components. This competition may complicate the interpretations of phenotypes observed through siRNA-mediated knockdown of genes, especially those genes implicated in the RISC pathway. In this study, we re-examined the function of RNA helicase A (RHA), which has been previously proposed to function in RISC loading based on siRNA-mediated knockdown studies. Here we show that reduced RISC activity or loading of siRNAs was observed only in cells depleted of RHA using siRNA, but not using RNaseH-dependent antisense oligonucleotides (ASOs), suggesting that the impaired RISC function stems from the competition between pre-existing and newly transfected siRNAs, but not from reduction of the RHA protein. This view is further supported by the findings that cells depleted of a control protein, NCL1, using siRNA, but not ASO, exhibited similar defects on the loading and activity of a subsequently transfected siRNA. Transfection of RHA or NCL1 siRNAs, but not ASOs, reduced the levels of endogenous miRNAs, suggesting a competition mechanism. As a positive control, we showed that reduction of MOV10 by either siRNA or ASO decreased siRNA activity, confirming its role in RISC function. Together, our results indicate that RHA is not required for RISC activity or loading, and suggest that proper controls are required when using siRNAs to functionalize genes to avoid competition effects. © 2013. Published by Elsevier B.V. All rights reserved.

  14. SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

    Science.gov (United States)

    Kota, Venkatesh; Sommer, Gunhild; Durette, Chantal; Thibault, Pierre; van Niekerk, Erna A; Twiss, Jeffery L; Heise, Tilman

    2016-01-01

    The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO), but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP) elements from the 5' untranslated regions (UTR) of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1) mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

  15. SUMO-Modification of the La Protein Facilitates Binding to mRNA In Vitro and in Cells.

    Directory of Open Access Journals (Sweden)

    Venkatesh Kota

    Full Text Available The RNA-binding protein La is involved in several aspects of RNA metabolism including the translational regulation of mRNAs and processing of pre-tRNAs. Besides its well-described phosphorylation by Casein kinase 2, the La protein is also posttranslationally modified by the Small Ubiquitin-like MOdifier (SUMO, but the functional outcome of this modification has not been defined. The objective of this study was to test whether sumoylation changes the RNA-binding activity of La. Therefore, we established an in vitro sumoylation assay for recombinant human La and analyzed its RNA-binding activity by electrophoretic mobility shift assays. We identified two novel SUMO-acceptor sites within the La protein located between the RNA recognition motif 1 and 2 and we demonstrate for the first time that sumoylation facilitates the RNA-binding of La to small RNA oligonucleotides representing the oligopyrimidine tract (TOP elements from the 5' untranslated regions (UTR of mRNAs encoding ribosomal protein L22 and L37 and to a longer RNA element from the 5' UTR of cyclin D1 (CCND1 mRNA in vitro. Furthermore, we show by RNA immunoprecipitation experiments that a La mutant deficient in sumoylation has impaired RNA-binding activity in cells. These data suggest that modulating the RNA-binding activity of La by sumoylation has important consequences on its functionality.

  16. Nucleoside-O-Methyl-(H)-Phosphinates: Novel Monomers for the Synthesis of Methylphosphonate Oligonucleotides Using H-Phosphonate Chemistry.

    Science.gov (United States)

    Kostov, Ondřej; Páv, Ondřej; Rosenberg, Ivan

    2017-09-18

    This unit comprises the straightforward synthesis of protected 2'-deoxyribonucleoside-O-methyl-(H)-phosphinates in both 3'- and 5'-series. These compounds represent a new class of monomers compatible with the solid-phase synthesis of oligonucleotides using H-phosphonate chemistry and are suitable for the preparation of both 3'- and 5'-O-methylphosphonate oligonucleotides. The synthesis of 4-toluenesulfonyloxymethyl-(H)-phosphinic acid as a new reagent for the preparation of O-methyl-(H)-phosphinic acid derivatives is described. © 2017 by John Wiley & Sons, Inc. Copyright © 2017 John Wiley & Sons, Inc.

  17. Different modes of interaction by TIAR and HuR with target RNA and DNA.

    Science.gov (United States)

    Kim, Henry S; Wilce, Matthew C J; Yoga, Yano M K; Pendini, Nicole R; Gunzburg, Menachem J; Cowieson, Nathan P; Wilson, Gerald M; Williams, Bryan R G; Gorospe, Myriam; Wilce, Jacqueline A

    2011-02-01

    TIAR and HuR are mRNA-binding proteins that play important roles in the regulation of translation. They both possess three RNA recognition motifs (RRMs) and bind to AU-rich elements (AREs), with seemingly overlapping specificity. Here we show using SPR that TIAR and HuR bind to both U-rich and AU-rich RNA in the nanomolar range, with higher overall affinity for U-rich RNA. However, the higher affinity for U-rich sequences is mainly due to faster association with U-rich RNA, which we propose is a reflection of the higher probability of association. Differences between TIAR and HuR are observed in their modes of binding to RNA. TIAR is able to bind deoxy-oligonucleotides with nanomolar affinity, whereas HuR affinity is reduced to a micromolar level. Studies with U-rich DNA reveal that TIAR binding depends less on the 2'-hydroxyl group of RNA than HuR binding. Finally we show that SAXS data, recorded for the first two domains of TIAR in complex with RNA, are more consistent with a flexible, elongated shape and not the compact shape that the first two domains of Hu proteins adopt upon binding to RNA. We thus propose that these triple-RRM proteins, which compete for the same binding sites in cells, interact with their targets in fundamentally different ways.

  18. DNA/RNA hybrid substrates modulate the catalytic activity of purified AID.

    Science.gov (United States)

    Abdouni, Hala S; King, Justin J; Ghorbani, Atefeh; Fifield, Heather; Berghuis, Lesley; Larijani, Mani

    2018-01-01

    Activation-induced cytidine deaminase (AID) converts cytidine to uridine at Immunoglobulin (Ig) loci, initiating somatic hypermutation and class switching of antibodies. In vitro, AID acts on single stranded DNA (ssDNA), but neither double-stranded DNA (dsDNA) oligonucleotides nor RNA, and it is believed that transcription is the in vivo generator of ssDNA targeted by AID. It is also known that the Ig loci, particularly the switch (S) regions targeted by AID are rich in transcription-generated DNA/RNA hybrids. Here, we examined the binding and catalytic behavior of purified AID on DNA/RNA hybrid substrates bearing either random sequences or GC-rich sequences simulating Ig S regions. If substrates were made up of a random sequence, AID preferred substrates composed entirely of DNA over DNA/RNA hybrids. In contrast, if substrates were composed of S region sequences, AID preferred to mutate DNA/RNA hybrids over substrates composed entirely of DNA. Accordingly, AID exhibited a significantly higher affinity for binding DNA/RNA hybrid substrates composed specifically of S region sequences, than any other substrates composed of DNA. Thus, in the absence of any other cellular processes or factors, AID itself favors binding and mutating DNA/RNA hybrids composed of S region sequences. AID:DNA/RNA complex formation and supporting mutational analyses suggest that recognition of DNA/RNA hybrids is an inherent structural property of AID. Copyright © 2017 Elsevier Ltd. All rights reserved.

  19. Carbohydrates/nucleosides/RNA-DNA-ligand interactions

    International Nuclear Information System (INIS)

    Kaptein, R.; McConnell, B.; Serianni, A.S.; Silks, L.A. III.

    1994-01-01

    Carbohydrate and nucleotide structural determination using modern spectroscopic techniques is dependent on our ability to label oligonucleotides and oligosaccharides with stable isotopes. Uniform Carbon 13 and Nitrogen 15 labeling of oligonucleotides is important to present-day efforts, which are focused on determining the structure of relatively small oligosaccharides and oligonucleotides, which form the elements of larger structures. Because of the relatively recent interest in three-dimensional structure, the development of techniques used to label them has lagged behind parallel techniques used to label peptides and proteins. Therefore, this group's discussion focused primarily on problems faced today in obtaining oligonucleotides labeled uniformly with carbon 13 and nitrogen 15

  20. Carbohydrates/nucleosides/RNA-DNA-ligand interactions

    Energy Technology Data Exchange (ETDEWEB)

    Kaptein, R.; McConnell, B.; Serianni, A.S.; Silks, L.A. III

    1994-12-01

    Carbohydrate and nucleotide structural determination using modern spectroscopic techniques is dependent on our ability to label oligonucleotides and oligosaccharides with stable isotopes. Uniform Carbon 13 and Nitrogen 15 labeling of oligonucleotides is important to present-day efforts, which are focused on determining the structure of relatively small oligosaccharides and oligonucleotides, which form the elements of larger structures. Because of the relatively recent interest in three-dimensional structure, the development of techniques used to label them has lagged behind parallel techniques used to label peptides and proteins. Therefore, this group`s discussion focused primarily on problems faced today in obtaining oligonucleotides labeled uniformly with carbon 13 and nitrogen 15.

  1. Community structure, cellular rRNA content, and activity of sulfate-reducing bacteria in marine Arctic sediments

    DEFF Research Database (Denmark)

    Ravenschlag, K.; Sahm, K.; Knoblauch, C.

    2000-01-01

    The community structure of sulfate-reducing bacteria (SRB) of a marine Arctic sediment (Smeerenburg-fjorden, Svalbard) a-as characterized by both fluorescence in situ hybridization (FISH) and rRNA slot blot hybridization by using group- and genus-specific 16S rRNA-targeted oligonucleotide probes...... that FISH and rRNA slot blot hybridization gave comparable results. Furthermore, a combination of the two methods allowed us to calculate specific cellular rRNA contents with respect to localization in the sediment profile. The rRNA contents of Desulfosarcina-Desulfococcus cells were highest in the first 5...... mm of the sediment (0.9 and 1.4 fg, respectively) and decreased steeply with depth, indicating that maximal metabolic activity occurred close to the surface, Based on SRB cell numbers, cellular sulfate reduction rates were calculated. The rates were highest in the surface layer (0.14 fmol cell(-1...

  2. The effect of tissue decalcification on mRNA retention within bone for in-situ hybridization studies.

    Science.gov (United States)

    Walsh, L; Freemont, A J; Hoyland, J A

    1993-06-01

    Tissue decalcification is a routine part of the preparation of bone tissue for histological studies. Although in-situ hybridization has been employed to localize mRNA of collagenous and non-collagenous bone related proteins in skeletal tissue, little is known regarding the effects of decalcifying agents on mRNA retention within tissue. In this study in-situ hybridization using an oligonucleotide probe (i.e. a poly d(T) probe) to detect total messenger RNA has been employed to investigate the effects of the decalcifying agents nitric acid, formic acid and EDTA on mRNA retention compared to undeacalcified tissue. The results show that formalin fixation and EDTA decalcification preserve substantial amounts of mRNA within the tissue. In particular, this study illustrates that it is possible to perform in-situ hybridization on formalin fixed decalcified paraffin embedded tissue.

  3. Scientific and Regulatory Policy Committee Points-to-consider Paper*: Drug-induced Vascular Injury Associated with Nonsmall Molecule Therapeutics in Preclinical Development: Part 2. Antisense Oligonucleotides.

    Science.gov (United States)

    Engelhardt, Jeffery A; Fant, Pierluigi; Guionaud, Silvia; Henry, Scott P; Leach, Michael W; Louden, Calvert; Scicchitano, Marshall S; Weaver, James L; Zabka, Tanja S; Frazier, Kendall S

    2015-10-01

    Drug-induced vascular injury (DIVI) is a recurrent challenge in the development of novel pharmaceutical agents. In recent years, DIVI has been occasionally observed in nonhuman primates given RNA-targeting therapeutics such as antisense oligonucleotide therapies (ASOs) during chronic toxicity studies. While DIVI in laboratory animal species has been well characterized for vasoactive small molecules, and immune-mediated responses against large molecule biotherapeutics have been well described, there is little published information regarding DIVI induced by ASOs to date. Preclinical DIVI findings in monkeys have caused considerable delays in development of promising new ASO therapies, because of the uncertainty about whether DIVI in preclinical studies is predictive of effects in humans, and the lack of robust biomarkers of DIVI. This review of DIVI discusses clinical and microscopic features of vasculitis in monkeys, their pathogenic mechanisms, and points to consider for the toxicologist and pathologist when confronted with ASO-related DIVI. Relevant examples of regulatory feedback are included to provide insight into risk assessment of ASO therapies. © 2015 by The Author(s).

  4. Replicate high-density rat genome oligonucleotide microarrays reveal hundreds of regulated genes in the dorsal root ganglion after peripheral nerve injury.

    Directory of Open Access Journals (Sweden)

    Mannion James W

    2002-10-01

    Full Text Available Abstract Background Rat oligonucleotide microarrays were used to detect changes in gene expression in the dorsal root ganglion (DRG 3 days following sciatic nerve transection (axotomy. Two comparisons were made using two sets of triplicate microarrays, naïve versus naïve and naïve versus axotomy. Results Microarray variability was assessed using the naïve versus naïve comparison. These results support use of a P 1.5-fold expression change and P 1.5-fold and P in situ hybridization verified the expression of 24 transcripts. These data showed an 83% concordance rate with the arrays; most mismatches represent genes with low expression levels reflecting limits of array sensitivity. A significant correlation was found between actual mRNA differences and relative changes between microarrays (r2 = 0.8567. Temporal patterns of individual genes regulation varied. Conclusions We identify parameters for microarray analysis which reduce error while identifying many putatively regulated genes. Functional classification of these genes suggest reorganization of cell structural components, activation of genes expressed by immune and inflammatory cells and down-regulation of genes involved in neurotransmission.

  5. Evolution of specific 3'-5'-linkages in RNA in pre-biotic soup: a new hypothesis.

    Science.gov (United States)

    Kumar, Vaijayanti A

    2016-11-02

    This article reviews the different possibilities towards progression of the formation of DNA/RNA in the chemical world, before life, in enzyme-free conditions. The advent of deoxyribo- and ribopentose-sugars, nucleosides, nucleotides and oligonucleotides in the prebiotic soup is briefly discussed. Further, the formation of early single stranded oligomers, base-pairing possibilities and information transfer based on the stability parameters of the derived duplexes is reviewed. Each theory has its own merits and demerits which we have elaborated upon. Lastly, using clues from this literature, a possible explanation for the specific 3'-5'-linkages in RNA is proposed.

  6. Differentiation of Actinobacillus pleuropneumoniae strains by sequence analysis of 16S rDNA and ribosomal intergenic regions, and development of a species specific oligonucleotide for in situ detection

    DEFF Research Database (Denmark)

    Fussing, Vivian; Paster, Bruce J.; Dewhirst, Floyd E.

    1998-01-01

    . The larger RIS's were different between the 3 species tested. The sequence of the 16S ribosomal gene was determined for 8 serotypes of A. pleuropneumoniae. These sequences showed only minor base differences, indicating a close genetic relatedness of these serotypes within the species. An oligonucleotide DNA...... probe designed from the 16S rRNA gene sequence of A. pleuropneumoniae was specific for all strains of the target species and did not cross react with A. lignieresii, the closest known relative of A. pleuropneumoniae. This species-specific DNA probe labeled with fluorescein was used for in situ......The aims of this study were to characterize and determine intraspecies and interspecies relatedness of Actinobacillus pleuropneumoniae to Actinobacillus lignieresii and Actinobacillus suis by sequence analysis of the ribosomal operon and to find a species-specific area for in situ detection of A...

  7. Combinatorics of RNA-RNA interaction

    DEFF Research Database (Denmark)

    Li, Thomas J X; Reidys, Christian

    2012-01-01

    RNA-RNA binding is an important phenomenon observed for many classes of non-coding RNAs and plays a crucial role in a number of regulatory processes. Recently several MFE folding algorithms for predicting the joint structure of two interacting RNA molecules have been proposed. Here joint structure...... means that in a diagram representation the intramolecular bonds of each partner are pseudoknot-free, that the intermolecular binding pairs are noncrossing, and that there is no so-called "zigzag" configuration. This paper presents the combinatorics of RNA interaction structures including...

  8. Simple methods for the 3' biotinylation of RNA.

    Science.gov (United States)

    Moritz, Bodo; Wahle, Elmar

    2014-03-01

    Biotinylation of RNA allows its tight coupling to streptavidin and is thus useful for many types of experiments, e.g., pull-downs. Here we describe three simple techniques for biotinylating the 3' ends of RNA molecules generated by chemical or enzymatic synthesis. First, extension with either the Schizosaccharomyces pombe noncanonical poly(A) polymerase Cid1 or Escherichia coli poly(A) polymerase and N6-biotin-ATP is simple, efficient, and generally applicable independently of the 3'-end sequences of the RNA molecule to be labeled. However, depending on the enzyme and the reaction conditions, several or many biotinylated nucleotides are incorporated. Second, conditions are reported under which splint-dependent ligation by T4 DNA ligase can be used to join biotinylated and, presumably, other chemically modified DNA oligonucleotides to RNA 3' ends even if these are heterogeneous as is typical for products of enzymatic synthesis. Third, we describe the use of 29 DNA polymerase for a template-directed fill-in reaction that uses biotin-dUTP and, thanks to the enzyme's proofreading activity, can cope with more extended 3' heterogeneities.

  9. Development of bis-locked nucleic acid (bisLNA) oligonucleotides for efficient invasion of supercoiled duplex DNA

    DEFF Research Database (Denmark)

    Moreno, Pedro M D; Geny, Sylvain; Pabon, Y Vladimir

    2013-01-01

    In spite of the many developments in synthetic oligonucleotide (ON) chemistry and design, invasion into double-stranded DNA (DSI) under physiological salt and pH conditions remains a challenge. In this work, we provide a new ON tool based on locked nucleic acids (LNAs), designed for strand invasi...

  10. Development of a general methodology for labelling peptide-morpholino oligonucleotide conjugates using alkyne-azide click chemistry.

    Science.gov (United States)

    Shabanpoor, Fazel; Gait, Michael J

    2013-11-11

    We describe a general methodology for fluorescent labelling of peptide conjugates of phosphorodiamidate morpholino oligonucleotides (PMOs) by alkyne functionalization of peptides, subsequent conjugation to PMOs and labelling with a fluorescent compound (Cy5-azide). Two peptide-PMO (PPMO) examples are shown. No detrimental effect of such labelled PMOs was seen in a biological assay.

  11. Peptide-oligonucleotide conjugates as nanoscale building blocks for assembly of an artificial three-helix protein mimic

    DEFF Research Database (Denmark)

    Lou, Chenguang; Martos-Maldonado, Manuel C.; Madsen, Charlotte Stahl

    2016-01-01

    Peptide-based structures can be designed to yield artificial proteins with specific folding patterns and functions. Template-based assembly of peptide units is one design option, but the use of two orthogonal self-assembly principles, oligonucleotide triple helix and a coiled coil protein domain ...

  12. Development of mercury (II) ion biosensors based on mercury-specific oligonucleotide probes.

    Science.gov (United States)

    Li, Lanying; Wen, Yanli; Xu, Li; Xu, Qin; Song, Shiping; Zuo, Xiaolei; Yan, Juan; Zhang, Weijia; Liu, Gang

    2016-01-15

    Mercury (II) ion (Hg(2+)) contamination can be accumulated along the food chain and cause serious threat to the public health. Plenty of research effort thus has been devoted to the development of fast, sensitive and selective biosensors for monitoring Hg(2+). Thymine was demonstrated to specifically combine with Hg(2+) and form a thymine-Hg(2+)-thymine (T-Hg(2+)-T) structure, with binding constant even higher than T-A Watson-Crick pair in DNA duplex. Recently, various novel Hg(2+) biosensors have been developed based on T-rich Mercury-Specific Oligonucleotide (MSO) probes, and exhibited advanced selectivity and excellent sensitivity for Hg(2+) detection. In this review, we explained recent development of MSO-based Hg(2+) biosensors mainly in 3 groups: fluorescent biosensors, colorimetric biosensors and electrochemical biosensors. Copyright © 2015 Elsevier B.V. All rights reserved.

  13. Two-dimensional condensation of pyrimidine oligonucleotides during their self-assemblies at mercury based surfaces

    Energy Technology Data Exchange (ETDEWEB)

    Hason, Stanislav [Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i. Kralovopolska 135, CZ-612 65 Brno (Czech Republic)], E-mail: hasons@ibp.cz; Vetterl, Vladimir [Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i. Kralovopolska 135, CZ-612 65 Brno (Czech Republic); Fojta, Miroslav [Institute of Biophysics, Academy of Sciences of the Czech Republic, v.v.i. Kralovopolska 135, CZ-612 65 Brno (Czech Republic)], E-mail: fojta@ibp.cz

    2008-02-15

    For the first time it is shown that homopyrimidine oligodeoxynucleotides (ODNs) adsorbed at mercury or amalgam electrode surface can condensate upon applying negative potentials (around -1.35 V vs. Ag/AgCl/3M KCl). This 2D condensation resulted in formation of capacitance pits on the C-E curves resembling those observed earlier with monomeric nucleic acid bases, nucleosides and nucleotides. Differences in behavior of the condensed layers of dT{sub 30} and dC{sub 30} ODNs, reflecting different physico-chemical and electrochemical properties of thymine and cytosine, were observed. Formation of the ODN condensed film involved reorientation of the oligonucleotide molecules firmly adsorbed at the electrode and took place even in the absence of any ODN in the bulk of solution. Homopurine ODNs did not form these two-dimensional (2D) condensed monolayers under the same conditions. A preliminary thermodynamic analysis of the condensed ODN layers is presented.

  14. Higher order structure of short immunostimulatory oligonucleotides studied by atomic force microscopy

    Energy Technology Data Exchange (ETDEWEB)

    Klein, Dionne C.G., E-mail: dionne.c.g.klein@ntnu.no [Department of Physics, Norwegian University of Science and Technology, N-7491, Trondheim (Norway); Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7489, Trondheim (Norway); Latz, Eicke [Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7489, Trondheim (Norway); Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605 (United States); Institute of Innate Immunity, University Hospitals, University of Bonn, Sigmund-Freud-Str. 25, 53127 Bonn (Germany); Espevik, Terje [Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, N-7489, Trondheim (Norway); Stokke, Bjorn T. [Department of Physics, Norwegian University of Science and Technology, N-7491, Trondheim (Norway)

    2010-05-15

    Immunostimulatory CpG-DNA activates the innate immune system by binding to Toll-like receptor 9. Structurally different CpG-containing oligonucleotides trigger a different type of immune response while activating the same receptor. We therefore investigated the higher order structure of two different classes of immunostimulatory CpG-DNA. Class A, which contains a partly self-complementary sequence and poly-G ends, forms duplexes and nanoparticles in salt solution, while class B, which does not contain these features and is purely linear, does not form a duplex or nanoparticles. Results obtained here by high-resolution atomic force microscopy of classes A and B CpG-DNA, reflect these differences in secondary structure. Detailed structural analysis of the atomic force microscopy topographs is presented for two different sample preparation methods.

  15. Higher order structure of short immunostimulatory oligonucleotides studied by atomic force microscopy

    International Nuclear Information System (INIS)

    Klein, Dionne C.G.; Latz, Eicke; Espevik, Terje; Stokke, Bjorn T.

    2010-01-01

    Immunostimulatory CpG-DNA activates the innate immune system by binding to Toll-like receptor 9. Structurally different CpG-containing oligonucleotides trigger a different type of immune response while activating the same receptor. We therefore investigated the higher order structure of two different classes of immunostimulatory CpG-DNA. Class A, which contains a partly self-complementary sequence and poly-G ends, forms duplexes and nanoparticles in salt solution, while class B, which does not contain these features and is purely linear, does not form a duplex or nanoparticles. Results obtained here by high-resolution atomic force microscopy of classes A and B CpG-DNA, reflect these differences in secondary structure. Detailed structural analysis of the atomic force microscopy topographs is presented for two different sample preparation methods.

  16. Oligonucleotides as probes for studying polymerization reactions in dilute aqueous solution

    Science.gov (United States)

    Kolb, V.; Orgel, L. E.; Miller, S. L. (Principal Investigator)

    1994-01-01

    We have prepared a [32P]-labled oligonucleotide probe carrying a free primary amine at its 3'-terminus. This probe is used to initiate polymerization of aziridine (ethyleneimine) in aqueous solution. The nature of the oligomeric products and the kinetics of their formation are then monitored by gel electrophoresis. Our results are generally consistent with those obtained using conventional techniques. We have also investigated the effect of polyanionic templates on the rate of oligomerization of aziridine. We find that water-soluble polyanions generally accelerate the polymerization. The sodium salt of polymethacrylic acid is the most effective of the templates that we studied. The methods introduced in this paper should be applicable to a variety of polymerization reactions in aqueous solution. They should greatly simplify the screening of potentially prebiotic polymerization reactions.

  17. Photochemical immobilization of anthraquinone conjugated oligonucleotides and PCR amplicons on solid surfaces

    DEFF Research Database (Denmark)

    Koch, T.; Jacobsen, N.; Fensholdt, J.

    2000-01-01

    Ligand immobilization on solid surfaces is an essential step in fields such as diagnostics, bio sensor manufacturing, and new material sciences in general. In this paper a photochemical approach based on anthraquinone as the chromophore is presented. Photochemical procedures offer special...... advantages as they are able to generate highly reactive species in an orientation specific manner. As presented here, anthraquinone (AQ) mediated covalent DNA immobilization appears to be superior to currently known procedures. A synthetic procedure providing AQ-phosphoramidites is presented. These reagents...... facilitate AQ conjugation during routine DNA synthesis, thus enabling the AQ-oligonucleotides to be immobilized in a very convenient and efficient manner. AQ-conjugated PCR primers can be used directly in PCR. When the PCR is performed in solution, the amplicons can be immobilized after the PCR. Moreover...

  18. Microsatellite and HLA class II oligonucleotide typing in a population of Yanomami Indians.

    Science.gov (United States)

    Roewer, L; Nagy, M; Schmidt, P; Epplen, J T; Herzog-Schröder, G

    1993-01-01

    We have used three different microsatellites (on chromosome 12 and Y) together with HLA class II oligonucleotide typing (DQA and DQB) to analyze families of Yanomami indians settling in villages in Southern Venezuela. There exist complex networks of biological relationship between villages as a result of wife exchange, village fissioning and changing patterns of alliances associated with inter-village warfare. Social status in this society is largely determined by the kinship system. Polygyny is common, especially among headmen, with additional wives, frequently being chosen among the sisters of the first wife. Our preliminary results mainly obtained from inhabitants of the village HAP show the expected allele distribution in populations with a high degree of consanguinity: (i) deficiency of observed heterozygotes at the autosomal loci and (ii) almost all men carry the same Y chromosomal allele. Nevertheless in the Yanomami village two thirds of the described autosomal microsatellite alleles were identified. Several paternities were clarified.

  19. An algorithm and program for finding sequence specific oligo-nucleotide probes for species identification

    Directory of Open Access Journals (Sweden)

    Tautz Diethard

    2002-03-01

    Full Text Available Abstract Background The identification of species or species groups with specific oligo-nucleotides as molecular signatures is becoming increasingly popular for bacterial samples. However, it shows also great promise for other small organisms that are taxonomically difficult to tract. Results We have devised here an algorithm that aims to find the optimal probes for any given set of sequences. The program requires only a crude alignment of these sequences as input and is optimized for performance to deal also with very large datasets. The algorithm is designed such that the position of mismatches in the probes influences the selection and makes provision of single nucleotide outloops. Program implementations are available for Linux and Windows.

  20. Merlin: Computer-Aided Oligonucleotide Design for Large Scale Genome Engineering with MAGE.

    Science.gov (United States)

    Quintin, Michael; Ma, Natalie J; Ahmed, Samir; Bhatia, Swapnil; Lewis, Aaron; Isaacs, Farren J; Densmore, Douglas

    2016-06-17

    Genome engineering technologies now enable precise manipulation of organism genotype, but can be limited in scalability by their design requirements. Here we describe Merlin ( http://merlincad.org ), an open-source web-based tool to assist biologists in designing experiments using multiplex automated genome engineering (MAGE). Merlin provides methods to generate pools of single-stranded DNA oligonucleotides (oligos) for MAGE experiments by performing free energy calculation and BLAST scoring on a sliding window spanning the targeted site. These oligos are designed not only to improve recombination efficiency, but also to minimize off-target interactions. The application further assists experiment planning by reporting predicted allelic replacement rates after multiple MAGE cycles, and enables rapid result validation by generating primer sequences for multiplexed allele-specific colony PCR. Here we describe the Merlin oligo and primer design procedures and validate their functionality compared to OptMAGE by eliminating seven AvrII restriction sites from the Escherichia coli genome.

  1. Plume characteristics and dynamics of UV and IR laser-desorbed oligonucleotides.

    Science.gov (United States)

    Merrigan, Tony L; Timson, David J; Hunniford, C Adam; Catney, Martin; McCullough, Robert W

    2012-05-01

    Laser desorption of dye-tagged oligonucleotides was studied using laser-induced fluorescence imaging. Desorption with ultra violet (UV) and infra-red (IR) lasers resulted in forward directed plumes of molecules. In the case of UV desorption, the initial shot desorbed approximately seven-fold more material than subsequent shots. In contrast, the initial shot in IR desorption resulted in the ejection of less material compared to subsequent shots and these plumes had a component directed along the path of the laser. Thermal equilibrium of the molecules in the plume was achieved after approximately 25 μs with a spread in molecular temperature which was described by a modified Maxwell-Boltzmann equation. Copyright © 2012 Elsevier B.V. All rights reserved.

  2. Staphylococcus aureus detection in blood samples by silica nanoparticle-oligonucleotides conjugates.

    Science.gov (United States)

    Borsa, Baris A; Tuna, Bilge G; Hernandez, Frank J; Hernandez, Luiza I; Bayramoglu, Gulay; Arica, M Yakup; Ozalp, V Cengiz

    2016-12-15

    A fast, specific and sensitive homogeneous assay for Staphylococcus aureus detection was developed by measuring the activity of secreted nuclease from the bacteria via a modified DNA oligonucleotide. As biosensor format, an effective system, Nanokeepers as previously reported, were used for triggered release of confined fluorophores, and hence specific detection of S. aureus on nuclease activity was obtained. The interference from blood components for fluorescent quantification was eliminated by a pre-purification by aptamer-functionalized silica magnetic nanoparticles. The reported assay system was exclusively formed by nucleic acid oligos and magnetic or mesoporous silica nanoparticles, that can be used on blood samples in a stepwise manner. The assay was successfully used as a sensing platform for the specific detection of S. aureus cells as low as 682 CFU in whole blood. Copyright © 2016 Elsevier B.V. All rights reserved.

  3. A PCR-Based Method to Construct Lentiviral Vector Expressing Double Tough Decoy for miRNA Inhibition.

    Directory of Open Access Journals (Sweden)

    Huiling Qiu

    Full Text Available DNA vector-encoded Tough Decoy (TuD miRNA inhibitor is attracting increased attention due to its high efficiency in miRNA suppression. The current methods used to construct TuD vectors are based on synthesizing long oligonucleotides (~90 mer, which have been costly and problematic because of mutations during synthesis. In this study, we report a PCR-based method for the generation of double Tough Decoy (dTuD vector in which only two sets of shorter oligonucleotides (< 60 mer were used. Different approaches were employed to test the inhibitory potency of dTuDs. We demonstrated that dTuD is the most efficient method in miRNA inhibition in vitro and in vivo. Using this method, a mini dTuD library against 88 human miRNAs was constructed and used for a high-throughput screening (HTS of AP-1 pathway-related miRNAs. Seven miRNAs (miR-18b-5p, -101-3p, -148b-3p, -130b-3p, -186-3p, -187-3p and -1324 were identified as candidates involved in AP-1 pathway regulation. This novel method allows for an accurate and cost-effective generation of dTuD miRNA inhibitor, providing a powerful tool for efficient miRNA suppression in vitro and in vivo.

  4. Molecular trade-offs in RNA ligases affected the modular emergence of complex ribozymes at the origin of life

    Science.gov (United States)

    Dhar, Nisha; Weinberg, Marc S.; Michod, Richard E.; Durand, Pierre M.

    2017-09-01

    In the RNA world hypothesis complex, self-replicating ribozymes were essential. For the emergence of an RNA world, less is known about the early processes that accounted for the formation of complex, long catalysts from small passively formed molecules. The functional role of small sequences has not been fully explored and, here, a possible role for smaller ligases is demonstrated. An established RNA polymerase model, the R18, was truncated from the 3' end to generate smaller molecules. All the molecules were investigated for self-ligation functions with a set of oligonucleotide substrates without predesigned base pairing. The smallest molecule that exhibited self-ligation activity was a 40-nucleotide RNA. It also demonstrated the greatest functional flexibility as it was more general in the kinds of substrates it ligated to itself although its catalytic efficiency was the lowest. The largest ribozyme (R18) ligated substrates more selectively and with greatest efficiency. With increase in size and predicted structural stability, self-ligation efficiency improved, while functional flexibility decreased. These findings reveal that molecular size could have increased from the activity of small ligases joining oligonucleotides to their own end. In addition, there is a size-associated molecular-level trade-off that could have impacted the evolution of RNA-based life.

  5. Molecular trade-offs in RNA ligases affected the modular emergence of complex ribozymes at the origin of life

    Science.gov (United States)

    Weinberg, Marc S.; Michod, Richard E.

    2017-01-01

    In the RNA world hypothesis complex, self-replicating ribozymes were essential. For the emergence of an RNA world, less is known about the early processes that accounted for the formation of complex, long catalysts from small passively formed molecules. The functional role of small sequences has not been fully explored and, here, a possible role for smaller ligases is demonstrated. An established RNA polymerase model, the R18, was truncated from the 3′ end to generate smaller molecules. All the molecules were investigated for self-ligation functions with a set of oligonucleotide substrates without predesigned base pairing. The smallest molecule that exhibited self-ligation activity was a 40-nucleotide RNA. It also demonstrated the greatest functional flexibility as it was more general in the kinds of substrates it ligated to itself although its catalytic efficiency was the lowest. The largest ribozyme (R18) ligated substrates more selectively and with greatest efficiency. With increase in size and predicted structural stability, self-ligation efficiency improved, while functional flexibility decreased. These findings reveal that molecular size could have increased from the activity of small ligases joining oligonucleotides to their own end. In addition, there is a size-associated molecular-level trade-off that could have impacted the evolution of RNA-based life. PMID:28989747

  6. The respiratory syncytial virus polymerase has multiple RNA synthesis activities at the promoter.

    Directory of Open Access Journals (Sweden)

    Sarah L Noton

    Full Text Available Respiratory syncytial virus (RSV is an RNA virus in the Family Paramyxoviridae. Here, the activities performed by the RSV polymerase when it encounters the viral antigenomic promoter were examined. RSV RNA synthesis was reconstituted in vitro using recombinant, isolated polymerase and an RNA oligonucleotide template representing nucleotides 1-25 of the trailer complement (TrC promoter. The RSV polymerase was found to have two RNA synthesis activities, initiating RNA synthesis from the +3 site on the promoter, and adding a specific sequence of nucleotides to the 3' end of the TrC RNA using a back-priming mechanism. Examination of viral RNA isolated from RSV infected cells identified RNAs initiated at the +3 site on the TrC promoter, in addition to the expected +1 site, and showed that a significant proportion of antigenome RNAs contained specific nucleotide additions at the 3' end, demonstrating that the observations made in vitro reflected events that occur during RSV infection. Analysis of the impact of the 3' terminal extension on promoter activity indicated that it can inhibit RNA synthesis initiation. These findings indicate that RSV polymerase-promoter interactions are more complex than previously thought and suggest that there might be sophisticated mechanisms for regulating promoter activity during infection.

  7. Depletion of key protein components of the RISC pathway impairs pre-ribosomal RNA processing.

    Science.gov (United States)

    Liang, Xue-Hai; Crooke, Stanley T

    2011-06-01

    Little is known about whether components of the RNA-induced silencing complex (RISC) mediate the biogenesis of RNAs other than miRNA. Here, we show that depletion of key proteins of the RISC pathway by antisense oligonucleotides significantly impairs pre-rRNA processing in human cells. In cells depleted of Drosha or Dicer, different precursors to 5.8S rRNA strongly accumulated, without affecting normal endonucleolytic cleavages. Moderate yet distinct processing defects were also observed in Ago2-depleted cells. Physical links between pre-rRNA and these proteins were identified by co-immunoprecipitation analyses. Interestingly, simultaneous depletion of Dicer and Drosha led to a different processing defect, causing slower production of 28S rRNA and its precursor. Both Dicer and Ago2 were detected in the nuclear fraction, and reduction of Dicer altered the structure of the nucleolus, where pre-rRNA processing occurs. Together, these results suggest that Drosha and Dicer are implicated in rRNA biogenesis.

  8. An optimized lentiviral vector system for conditional RNAi and efficient cloning of microRNA embedded short hairpin RNA libraries.

    Science.gov (United States)

    Adams, Felix F; Heckl, Dirk; Hoffmann, Thomas; Talbot, Steven R; Kloos, Arnold; Thol, Felicitas; Heuser, Michael; Zuber, Johannes; Schambach, Axel; Schwarzer, Adrian

    2017-09-01

    RNA interference (RNAi) and CRISPR-Cas9-based screening systems have emerged as powerful and complementary tools to unravel genetic dependencies through systematic gain- and loss-of-function studies. In recent years, a series of technical advances helped to enhance the performance of virally delivered RNAi. For instance, the incorporation of short hairpin RNAs (shRNAs) into endogenous microRNA contexts (shRNAmiRs) allows the use of Tet-regulated promoters for synchronous onset of gene knockdown and precise interrogation of gene dosage effects. However, remaining challenges include lack of efficient cloning strategies, inconsistent knockdown potencies and leaky expression. Here, we present a simple, one-step cloning approach for rapid and efficient cloning of miR-30 shRNAmiR libraries. We combined a human miR-30 backbone retaining native flanking sequences with an optimized all-in-one lentiviral vector system for conditional RNAi to generate a versatile toolbox characterized by higher doxycycline sensitivity, reduced leakiness and enhanced titer. Furthermore, refinement of existing shRNA design rules resulted in substantially improved prediction of powerful shRNAs. Our approach was validated by accurate quantification of the knockdown potency of over 250 single shRNAmiRs. To facilitate access and use by the scientific community, an online tool was developed for the automated design of refined shRNA-coding oligonucleotides ready for cloning into our system. Copyright © 2017 Elsevier Ltd. All rights reserved.

  9. A comparison of alternative 60-mer probe designs in an in-situ synthesized oligonucleotide microarray

    Directory of Open Access Journals (Sweden)

    Fairbanks Benjamin D

    2006-04-01

    Full Text Available Abstract Background DNA microarrays have proven powerful for functional genomics studies. Several technologies exist for the generation of whole-genome arrays. It is well documented that 25mer probes directed against different regions of the same gene produce variable signal intensity values. However, the extent to which this is true for probes of greater length (60mers is not well characterized. Moreover, this information has not previously been reported for whole-genome arrays designed against bacteria, whose genomes may differ substantially in characteristics directly affecting microarray performance. Results We report here an analysis of alternative 60mer probe designs for an in-situ synthesized oligonucleotide array for the GC rich, β-proteobacterium Burkholderia cenocepacia. Probes were designed using the ArrayOligoSel3.5 software package and whole-genome microarrays synthesized by Agilent, Inc. using their in-situ, ink-jet technology platform. We first validated the quality of the microarrays as demonstrated by an average signal to noise ratio of >1000. Next, we determined that the variance of replicate probes (1178 total probes examined of identical sequence was 3.8% whereas the variance of alternative probes (558 total alternative probes examined designs was 9.5%. We determined that depending upon the definition, about 2.4% of replicate and 7.8% of alternative probes produced outlier conclusions. Finally, we determined none of the probe design subscores (GC content, internal repeat, binding energy and self annealment produced by ArrayOligoSel3.5 were predictive or probes that produced outlier signals. Conclusion Our analysis demonstrated that the use of multiple probes per target sequence is not essential for in-situ synthesized 60mer oligonucleotide arrays designed against bacteria. Although probes producing outlier signals were identified, the use of ratios results in less than 10% of such outlier conclusions. We also determined that

  10. Mechanism of inhibition of HIV-1 integrase by G-tetrad-forming oligonucleotides in Vitro.

    Science.gov (United States)

    Jing, N; Marchand, C; Liu, J; Mitra, R; Hogan, M E; Pommier, Y

    2000-07-14

    The G-tetrad-forming oligonucleotides and have been identified as potent inhibitors of human immunodeficiency virus type 1 integrase (HIV-1 IN) activity (Rando, R. F., Ojwang, J., Elbaggari, A., Reyes, G. R., Tinder, R., McGrath, M. S., and Hogan, M. E. (1995) J. Biol. Chem. 270, 1754-1760; Mazumder, A., Neamati, N., Ojwang, J. O., Sunder, S., Rando, R. F., and Pommier, Y. (1996) Biochemistry 35, 13762-13771; Jing, N., and Hogan, M. E. (1998) J. Biol. Chem. 273, 34992-34999). To understand the inhibition of HIV-1 IN activity by the G-quartet inhibitors, we have designed the oligonucleotides and, composed of three and four G-quartets with stem lengths of 19 and 24 A, respectively. The fact that increasing the G-quartet stem length from 15 to 24 A kept inhibition of HIV-1 IN activity unchanged suggests that the binding interaction occurs between a GTGT loop domain of the G-quartet inhibitors and a catalytic site of HIV-1 IN, referred to as a face-to-face interaction. Docking the NMR structure of (Jing and Hogan (1998)) into the x-ray structure of the core domain of HIV-1 IN, HIV-1 IN-(51-209) (Maignan, S., Guilloteau, J.-P. , Qing, Z.-L., Clement-Mella, C., and Mikol, V. (1998) J. Mol. Biol. 282, 359-368), was performed using the GRAMM program. The statistical distributions of hydrogen bonding between HIV-1 IN and were obtained from the analyses of 1000 random docking structures. The docking results show a high probability of interaction between the GTGT loop residues of the G-quartet inhibitors and the catalytic site of HIV-1 IN, in agreement with the experimental observation.

  11. Immobilization of oligonucleotide probes on silicon surfaces using biotin–streptavidin system examined with microscopic and spectroscopic techniques

    Energy Technology Data Exchange (ETDEWEB)

    Awsiuk, K., E-mail: kamil.awsiuk@uj.edu.pl [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Rysz, J. [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Petrou, P. [Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece); Budkowski, A. [M. Smoluchowski Institute of Physics, Jagiellonian University, Reymonta 4, Kraków 30-059 (Poland); Bernasik, A. [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, Kraków 30-059 (Poland); Kakabakos, S. [Institute of Nuclear and Radiological Sciences and Technology, Energy and Safety, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece); Marzec, M.M. [Faculty of Physics and Applied Computer Science, AGH-University of Science and Technology, Al. Mickiewicza 30, Kraków 30-059 (Poland); Raptis, I. [Institute for Advanced Materials, Physicochemical Processes, Nanotechnology and Microsystems, NCSR “Demokritos”, End Patriarchou Gregoriou Str., Aghia Paraskevi 15310 (Greece)

    2014-01-30

    To immobilize effectively oligonucleotide probes on SiO{sub 2} modified with (3-aminopropyl)triethoxysilane, four procedures based on streptavidin–biotin system are compared with Atomic Force Microscopy, Angle-Resolved X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry. The first approach involves: adsorption of biotinylated Bovine Serum Albumin, blocking free surface sites with BSA, binding of streptavidin and biotinylated oligonucleotide (b-oligo). Final steps are exchanged in the second procedure with immobilization of preformed streptavidin–b-oligo conjugate. The third approach consists of streptavidin adsorption, blocking with BSA and b-oligo binding. Finally, streptavidin–b-oligo conjugate is immobilized directly within the fourth method. Surface coverage with biomolecules, determined from ARXPS, accords with average AFM height, and is anti-correlated with the intensity of Si+ ions. Higher biomolecular coverage was achieved during the last steps of the first (2.45(±0.38) mg/m{sup 2}) and second (1.31(±0.22) mg/m{sup 2}) approach, as compared to lower surface density resulting from the third (0.58(±0.20) mg/m{sup 2}) and fourth (0.41(±0.11) mg/m{sup 2}) method. Phosphorus atomic concentration indicates effectiveness of oligonucleotide immobilization. Secondary ions intensities, characteristic for oligonucleotides, streptavidin, BSA, and proteins, allow additional insight into overlayer composition. These measurements verify the ARXPS results and show the superiority of the first two immobilization approaches in terms of streptavidin and oligonucleotide density achieved onto the surface.

  12. Immobilization of oligonucleotide probes on silicon surfaces using biotin–streptavidin system examined with microscopic and spectroscopic techniques

    International Nuclear Information System (INIS)

    Awsiuk, K.; Rysz, J.; Petrou, P.; Budkowski, A.; Bernasik, A.; Kakabakos, S.; Marzec, M.M.; Raptis, I.

    2014-01-01

    To immobilize effectively oligonucleotide probes on SiO 2 modified with (3-aminopropyl)triethoxysilane, four procedures based on streptavidin–biotin system are compared with Atomic Force Microscopy, Angle-Resolved X-ray Photoelectron Spectroscopy and Time-of-Flight Secondary Ion Mass Spectrometry. The first approach involves: adsorption of biotinylated Bovine Serum Albumin, blocking free surface sites with BSA, binding of streptavidin and biotinylated oligonucleotide (b-oligo). Final steps are exchanged in the second procedure with immobilization of preformed streptavidin–b-oligo conjugate. The third approach consists of streptavidin adsorption, blocking with BSA and b-oligo binding. Finally, streptavidin–b-oligo conjugate is immobilized directly within the fourth method. Surface coverage with biomolecules, determined from ARXPS, accords with average AFM height, and is anti-correlated with the intensity of Si+ ions. Higher biomolecular coverage was achieved during the last steps of the first (2.45(±0.38) mg/m 2 ) and second (1.31(±0.22) mg/m 2 ) approach, as compared to lower surface density resulting from the third (0.58(±0.20) mg/m 2 ) and fourth (0.41(±0.11) mg/m 2 ) method. Phosphorus atomic concentration indicates effectiveness of oligonucleotide immobilization. Secondary ions intensities, characteristic for oligonucleotides, streptavidin, BSA, and proteins, allow additional insight into overlayer composition. These measurements verify the ARXPS results and show the superiority of the first two immobilization approaches in terms of streptavidin and oligonucleotide density achieved onto the surface.

  13. The virion RNA species of the Kirsten murine sarcoma-leukemia virus complex released from a clonally related series of mouse cells

    International Nuclear Information System (INIS)

    Clewley, J.P.; Avery, R.J.

    1982-01-01

    We have characterized the virion RNA species of Kirsten sarcoma (KiSV) and Kirsten leukemia (KiLV) viruses released from a clonally related series of mouse cells (14). We have identified the KiLV and KiSV genome RNAs. In addition to the viral RNA species we find large amounts of a virus-like RNA (VL30 RNA), which is heterogeneous and shows variability in its expression. The amount of VL30 RNA in virions does not correlate with the state of transformation of the cells releasing the virus or the ability of the virus to transform other cells. Characterization of RNA rescued from non-producer cells has revealed a sarcoma virus (KiSVsub(SB3) with an oligonucleotide fingerprint different from that of a standard KiSV RNA, suggesting that it has lost some viral sequences. The oligonucleotide fingerprints of KiLV and VL30 RNAs are distinct from each other and from those reported for other murine leukemia virus RNAs. (Author)

  14. An RNA-binding compound that stabilizes the HIV-1 gRNA packaging signal structure and specifically blocks HIV-1 RNA encapsidation.

    Science.gov (United States)

    Ingemarsdotter, Carin K; Zeng, Jingwei; Long, Ziqi; Lever, Andrew M L; Kenyon, Julia C

    2018-03-14

    NSC260594, a quinolinium derivative from the NCI diversity set II compound library, was previously identified in a target-based assay as an inhibitor of the interaction between the HIV-1 (ψ) stem-loop 3 (SL3) RNA and Gag. This compound was shown to exhibit potent antiviral activity. Here, the effects of this compound on individual stages of the viral lifecycle were examined by qRT-PCR, ELISA and Western blot, to see if its actions were specific to the viral packaging stage. The structural effects of NSC260594 binding to the HIV-1 gRNA were also examined by SHAPE and dimerization assays. Treatment of cells with NSC260594 did not reduce the number of integration events of incoming virus, and treatment of virus producing cells did not affect the level of intracellular Gag protein or viral particle release as determined by immunoblot. However, NSC260594 reduced the incorporation of gRNA into virions by up to 82%, without affecting levels of gRNA inside the cell. This reduction in packaging correlated closely with the reduction in infectivity of the released viral particles. To establish the structural effects of NSC260594 on the HIV-1 gRNA, we performed SHAPE analyses to pinpoint RNA structural changes. NSC260594 had a stabilizing effect on the wild type RNA that was not confined to SL3, but that was propagated across the structure. A packaging mutant lacking SL3 did not show this effect. NSC260594 acts as a specific inhibitor of HIV-1 RNA packaging. No other viral functions are affected. Its action involves preventing the interaction of Gag with SL3 by stabilizing this small RNA stem-loop which then leads to stabilization of the global packaging signal region (psi or ψ). This confirms data, previously only shown in analyses of isolated SL3 oligonucleotides, that SL3 is structurally labile in the presence of Gag and that this is critical for the complete psi region to be able to adopt different conformations. Since replication is otherwise unaffected by NSC260594

  15. "Transcriptomics": molecular diagnosis of inborn errors of metabolism via RNA-sequencing.

    Science.gov (United States)

    Kremer, Laura S; Wortmann, Saskia B; Prokisch, Holger

    2018-01-25

    Exome wide sequencing techniques have revolutionized molecular diagnostics in patients with suspected inborn errors of metabolism or neuromuscular disorders. However, the diagnostic yield of 25-60% still leaves a large fraction of individuals without a diagnosis. This indicates a causative role for non-exonic regulatory variants not covered by whole exome sequencing. Here we review how systematic RNA-sequencing analysis (RNA-seq, "transcriptomics") lead to a molecular diagnosis in 10-35% of patients in whom whole exome sequencing failed to do so. Importantly, RNA-sequencing based discoveries cannot only guide molecular diagnosis but might also unravel therapeutic intervention points such as antisense oligonucleotide treatment for splicing defects as recently reported for spinal muscular atrophy.

  16. RNA modifications by oxidation

    DEFF Research Database (Denmark)

    Poulsen, Henrik E; Specht, Elisabeth; Broedbaek, Kasper

    2012-01-01

    to encompass various classes of novel regulatory RNAs, including, e.g., microRNAs. It is well known that DNA is constantly oxidized and repaired by complex genome maintenance mechanisms. Analogously, RNA also undergoes significant oxidation, and there are now convincing data suggesting that oxidation......The past decade has provided exciting insights into a novel class of central (small) RNA molecules intimately involved in gene regulation. Only a small percentage of our DNA is translated into proteins by mRNA, yet 80% or more of the DNA is transcribed into RNA, and this RNA has been found......, and the consequent loss of integrity of RNA, is a mechanism for disease development. Oxidized RNA is found in a large variety of diseases, and interest has been especially devoted to degenerative brain diseases such as Alzheimer disease, in which up to 50-70% of specific mRNA molecules are reported oxidized, whereas...

  17. Method for widespread microRNA-155 inhibition prolongs survival in ALS-model mice

    Science.gov (United States)

    Koval, Erica D.; Shaner, Carey; Zhang, Peter; du Maine, Xavier; Fischer, Kimberlee; Tay, Jia; Chau, B. Nelson; Wu, Gregory F.; Miller, Timothy M.

    2013-01-01

    microRNAs (miRNAs) are dysregulated in a variety of disease states, suggesting that this newly discovered class of gene expression repressors may be viable therapeutic targets. A microarray of miRNA changes in ALS-model superoxide dismutase 1 (SOD1)G93A rodents identified 12 miRNAs as significantly changed. Six miRNAs tested in human ALS tissues were confirmed increased. Specifically, miR-155 was increased 5-fold in mice and 2-fold in human spinal cords. To test miRNA inhibition in the central nervous system (CNS) as a potential novel therapeutic, we developed oligonucleotide-based miRNA inhibitors (anti-miRs) that could inhibit miRNAs throughout the CNS and in the periphery. Anti-miR-155 caused global derepression of targets in peritoneal macrophages and, following intraventricular delivery, demonstrated widespread functional distribution in the brain and spinal cord. After treating SOD1G93A mice with anti-miR-155, we significantly extended survival by 10 days and disease duration by 15 days (38%) while a scrambled control anti-miR did not significantly improve survival or disease duration. Therefore, antisense oligonucleotides may be used to successfully inhibit miRNAs throughout the brain and spinal cord, and miR-155 is a promising new therapeutic target for human ALS. PMID:23740943

  18. Magnetic bead/capture DNA/glucose-loaded nanoliposomes for amplifying the glucometer signal in the rapid screening of hepatitis C virus RNA.

    Science.gov (United States)

    Tu, Haijian; Lin, Kun; Lun, Yongzhi; Yu, Liuming

    2018-06-01

    A digital detection strategy based on a portable personal glucometer (PGM) was developed for the simple, rapid, and sensitive detection of hepatitis C virus (HCV) RNA, involving the release of glucose-loaded nanoliposomes due to coupling-site-specific cleavage by the endonuclease BamHI. The glucose-loaded nanoliposomes were synthesized using a reversed-phase evaporation method and provided an amplified signal at the PGM in the presence of HCV RNA. Initially, a 21-mer oligonucleotide complementary to HCV RNA was covalently conjugated to a magnetic bead through the amino group at the 5' end of the oligonucleotide, and then bound to a glucose-loaded liposome by typical carbodiimide coupling at its 3' end. In the presence of the target HCV RNA, the target hybridized with the oligonucleotide to form double-stranded DNA. The symmetrical duplex sequence 5'-GGATCC-3' between guanines was then catalytically cleaved by BamHI, which detached the glucose-loaded liposome from the magnetic bead. Following magnetic separation of the bead, the detached glucose-loaded liposome was lysed using Triton X-100 to release the glucose molecules within it, which were then detected as an amplified signal at the digital PGM. Under optimal conditions, the PGM signal increased with increasing HCV RNA, and displayed a strongly linear dependence on the level of HCV RNA for concentrations ranging from 10 pM to 1.0 μM. The detection limit (LOD) of the system was 1.9 pM. Good reproducibility and favorable specificity were achieved in the analysis of the target HCV RNA. Human serum samples containing HCV RNA were analyzed using this strategy, and the developed sensing platform was observed to yield satisfactory results based on a comparison with the corresponding results from a Cobas ® Amplicor HCV Test Analyzer. Graphical abstract A digital detection strategy utilizing a personal glucometer was developed for the detection of hepatitis C virus RNA. The strategy involved the use of the

  19. Assessing the Interplay between the Physicochemical Parameters of Ion-Pairing Reagents and the Analyte Sequence on the Electrospray Desorption Process for Oligonucleotides

    Science.gov (United States)

    Basiri, Babak; Murph, Mandi M.; Bartlett, Michael G.

    2017-08-01

    Alkylamines are widely used as ion-pairing agents during LC-MS of oligonucleotides. In addition to a better chromatographic separation, they also assist with the desorption of oligonucleotide ions into the gas phase, cause charge state reduction, and decrease cation adduction. However, the choice of such ion-pairing agents has considerable influence on the MS signal intensity of oligonucleotides as they can also cause significant ion suppression. Interestingly, optimal ion-pairing agents should be selected on a case by case basis as their choice is strongly influenced by the sequence of the oligonucleotide under investigation. Despite imposing major practical difficulties to analytical method development, such a highly variable system that responds very strongly to the nuances of the electrospray composition provides an excellent opportunity for a fundamental study of the electrospray ionization process. Our investigations using this system quantitatively revealed the major factors that influenced the ESI ionization efficiency of oligonucleotides. Parameters such as boiling point, proton affinity, partition coefficient, water solubility, and Henry's law constants for the ion-pairing reagents and the hydrophobic thymine content of the oligonucleotides were found to be the most significant contributors. Identification of these parameters also allowed for the development of a statistical predictive algorithm that can assist with the choice of an optimum IP agent for each particular oligonucleotide sequence. We believe that research in the field of oligonucleotide bioanalysis will significantly benefit from this algorithm (included in Supplementary Material) as it advocates for the use of lesser-known but more suitable ion-pair alternatives to TEA for many oligonucleotide sequences.

  20. Working with RNA

    DEFF Research Database (Denmark)

    Nielsen, Henrik

    2011-01-01

    Working with RNA is not a special discipline in molecular biology. However, RNA is chemically and structurally different from DNA and a few simple work rules have to be implemented to maintain the integrity of the RNA. Alkaline pH, high temperatures, and heavy metal ions should be avoided when po...

  1. Regulation of Peripheral Myelination through Transcriptional Buffering of Egr2 by an Antisense Long Non-coding RNA

    Directory of Open Access Journals (Sweden)

    Margot Martinez-Moreno

    2017-08-01

    Full Text Available Precise regulation of Egr2 transcription is fundamentally important to the control of peripheral myelination. Here, we describe a long non-coding RNA antisense to the promoter of Egr2 (Egr2-AS-RNA. During peripheral nerve injury, the expression of Egr2-AS-RNA is increased and correlates with decreased Egr2 transcript and protein levels. Ectopic expression of Egr2-AS-RNA in dorsal root ganglion (DRG cultures inhibits the expression of Egr2 mRNA and induces demyelination. In vivo inhibition of Egr2-AS-RNA using oligonucleotide GapMers released from a biodegradable hydrogel following sciatic nerve injury reverts the EGR2-mediated gene expression profile and significantly delays demyelination. Egr2-AS-RNA gradually recruits H3K27ME3, AGO1, AGO2, and EZH2 on the Egr2 promoter following sciatic nerve injury. Furthermore, expression of Egr2-AS-RNA is regulated through ERK1/2 signaling to YY1, while loss of Ser184 of YY1 regulates binding to Egr2-AS-RNA. In conclusion, we describe functional exploration of an antisense long non-coding RNA in peripheral nervous system (PNS biology.

  2. Disease Control in Animals Using Molecular Technology by Inactivation of ASO, RNAi and ss-siRNA Genes

    Directory of Open Access Journals (Sweden)

    Muhamad Ali

    2014-03-01

    Full Text Available Globalization causes high mobility of human and livestock, hence increase the transmission of infectious diseases, including avian influenza, severe acute respiratory syndrome (SARS, and swine influenza. Therefore, prevention of those diseases is required. Vaccines are effective to prevent infectious diseases; however, their development takes a long time and they cannot provide immediate protection in pandemic cases. This paper describes several gene silencing technologies including antisense oligonucleotide (ASO, RNA interference (RNAi and single strand-small interfering RNA (ss-siRNA for controlling diseases. The primary mechanism of these technologies is inhibition of gene expression, typically by causing the destruction of specific RNA molecule of the pathogen. The use of gene silencing technologies is expected to give new alternative that is more effective in eradication of infectious diseases in animals before threaten human being.

  3. Methods for RNA Analysis

    DEFF Research Database (Denmark)

    Olivarius, Signe

    of the transcriptome, 5’ end capture of RNA is combined with next-generation sequencing for high-throughput quantitative assessment of transcription start sites by two different methods. The methods presented here allow for functional investigation of coding as well as noncoding RNA and contribute to future...... RNAs rely on interactions with proteins, the establishment of protein-binding profiles is essential for the characterization of RNAs. Aiming to facilitate RNA analysis, this thesis introduces proteomics- as well as transcriptomics-based methods for the functional characterization of RNA. First, RNA...

  4. Synthesis of modified oligonucleotides for repair and replication studies of single and double radio-induced DNA lesions

    International Nuclear Information System (INIS)

    Muller, E.

    2002-01-01

    Several oxidative processes induce the formation of DNA lesions. In order to evaluate the biological and structural significance of such damage, several DNA lesions were inserted into synthetic oligonucleotides at defined sites. The research work aimed at describing the preparation of oligonucleotides t hat contained DNA damage and the evaluation of the biological properties of the lesions. A first part described the incorporation of radiation-induced lesions, namely (5'S,6S)-5',6-cyclo-5,6-dihydro-2'-deoxyuridine and (5'S,5S,6S)-5',6-cyclo-5-hydroxy-5,6-dihydro-2'-desoxyuridine into oligonucleotides. The modified DNA fragments were characterised by several spectroscopic and biochemical analyses including ESI MS, MALDI-TOF MS, CLHP and enzymatic digestions. During in vitro DNA synthesis by Taq DNA polymerase and Klenow exo fragment, the pyrimidine cyclo-nucleosides were found to block the progression of the enzymes. Then, repair studies by ADN N glycosylases, operating in the base excision repair pathway, have shown that the anhydro-nucleoside lesions were not recognised nor excised by Fpg, endo III, endo VIII, yNtg1 yNtg2 and yOgg1. Interestingly, the Latococcus lactis Fpg protein recognises (formation of a non covalent complex) but do not excise the damage. The incorporation into oligonucleotides of the (5R*) and (5S*) diastereoisomers of 1-[2-deoxy-β-D-erythro-pentofuranosyl]-5-hydroxy-hydantoin, generated by several oxidative processes was then described. In vitro DNA replication assays using modified oligonucleotides matrix showed a lethal potential of the latter base damage. Repair studies by ADN N-glycosylases showed that the damage was substrate for Fpg, endo III, endo VIII, Ntg1, Ntg2 and Fpg-L1. The rates of excision as inferred from the determination of the Michaelis kinetics constants were found to be affected by the presence of the damage. MALDI-TOF MS was used in order to gain insights into mechanistic aspects of oligonucleotides cleavage by the

  5. Solving RNA's structural secrets: interaction with antibodies and crystal structure of a nuclease resistant RNA

    International Nuclear Information System (INIS)

    Wallace, S.T.

    1998-10-01

    This Ph.D. thesis concerns the structural characterization of RNA. The work is split into two sections: 1) in vitro selection and characterization of RNAs which bind antibiotics and 2) crystal structure of a nuclease resistant RNA molecule used in antisense applications. Understanding antibiotic-RNA interactions is crucial in aiding rational drug design. We were interested in studying antibiotic interactions with RNAs small enough to characterize at the molecular and possibly at the atomic level. In order to do so, we previously performed in vitro selection to find small RNAs which bind to the peptide antibiotic viomycin and the aminoglycoside antibiotic streptomycin. The characterization of the viomycin-binding RNAs revealed the necessity of a pseudoknot-structure in order to interact with the antibiotic. The RNAs which were selected to interact with streptomycin require the presence of magnesium to bind the antibiotic. One of the RNAs, upon interacting with streptomycin undergoes a significant conformational change spanning the entire RNA sequence needed to bind the antibiotic. In a quest to design oligodeoxynucleotides (ODNs) which are able to specifically bid and inactivate the mRNA of a gene, it is necessary to fulfill two criteria: 1) increase binding affinity between the ODN and the target RNA and 2) increase the ODN's resistance to nuclease degradation. An ODN with an aminopropyl modification at the 2' position of its ribose has emerged as the most successful candidate at fulfilling both criteria. It is the most nuclease resistant modification known to date. We were interested in explaining how this modification is able to circumvent degradation by nucleases. A dodecamer containing a single 2'-O-aminopropyl modified nucleotide was crystallized and the structure was solved to a resolution of 1.6 A. In an attempt to explain the nuclease resistance, the crystal coordinates were modeled into the active exonuclease site of DNA polymerase I. We propose the

  6. Cytoplasmic Z-RNA

    International Nuclear Information System (INIS)

    Zarling, D.A.; Calhoun, C.J.; Hardin, C.C.; Zarling, A.H.

    1987-01-01

    Specific immunochemical probes for Z-RNA were generated and characterized to search for possible Z-RNA-like double helices in cells. Z-RNA was detected in the cytoplasm of fixed protozoan cells by immunofluorescence microscopy using these anti-Z-RNA IgCs. In contrast, autoimmune or experimentally elicited anti-DNA antibodies, specifically reactive with B-DNA or Z-DNA, stained the nuclei. Pre-or nonimmune IgGs did not bind to the cells. RNase A or T1 digestion eliminated anti-Z-RNA IgG binding to cytoplasmic determinants; however, DNase I or mung bean nuclease had no effect. Doxorubicin and ethidium bromide prevented anti-Z-RNA antibody binding; however, actinomycin D, which does not bind double-stranded RNA, did not. Anti-Z-RNA immunofluorescence was specifically blocked in competition assays by synthetic Z-RNA but not Z-DNA, A-RNA, or single-stranded RNAs. Thus, some cytoplasmic sequences in fixed cells exist in the left-handed Z-RNA conformation

  7. microRNA-10b Is Overexpressed and Critical for Cell Survival and Proliferation in Medulloblastoma.

    Directory of Open Access Journals (Sweden)

    Rekha Pal

    Full Text Available This study demonstrates the effects of miRNA-10b on medulloblastoma proliferation through transcriptional induction of the anti-apoptotic protein BCL2. Using a cancer specific miRNA-array, high expression of miRNA-10b in medulloblastoma cell lines compared to a normal cerebellar control was shown, and this was confirmed with real time PCR (RT-PCR. Two medulloblastoma cell lines (DAOY and UW228 were transiently transfected with control miRNA, miRNA-10b inhibitor or miRNA-10b mimic and subjected to RT-PCR, MTT, apoptosis, clonogenic assay and western blot analysis. Transfection of miRNA-10b inhibitor induced a significant down-regulation of miRNA-10b expression, inhibited proliferation, and induced apoptosis, while miRNA-10b mimic exerted an opposite effect. Inhibition of miRNA-10b abrogated the colony-forming capability of medulloblastoma cells, and markedly down-regulated the expression of BCL2. Down-regulation of BCL2 by antisense oligonucleotides or siRNA also significantly down-regulated miRNA-10b, suggesting that BCL2 is a major mediator of the effects of miRNA-10b. ABT-737 and ABT-199, potent inhibitors of BCL2, downregulated the expression of miRNA-10b and increased apoptosis. Analysis of miRNA-10b levels in 13 primary medulloblastoma samples revealed that the 2 patients with the highest levels of miRNA-10b had multiple recurrences (4.5 and died within 8 years of diagnosis, compared with the 11 patients with low levels of miRNA-10b who had a mean of 1.2 recurrences and nearly 40% long-term survival. The data presented here indicate that miRNA-10b may act as an oncomir in medulloblastoma tumorigenesis, and reveal a previously unreported mechanism with Bcl-2 as a mediator of the effects of miRNA-10b upon medulloblastoma cell survival.

  8. Expedited quantification of mutant ribosomal RNA by binary deoxyribozyme (BiDz) sensors.

    Science.gov (United States)

    Gerasimova, Yulia V; Yakovchuk, Petro; Dedkova, Larisa M; Hecht, Sidney M; Kolpashchikov, Dmitry M

    2015-10-01

    Mutations in ribosomal RNA (rRNA) have traditionally been detected by the primer extension assay, which is a tedious and multistage procedure. Here, we describe a simple and straightforward fluorescence assay based on binary deoxyribozyme (BiDz) sensors. The assay uses two short DNA oligonucleotides that hybridize specifically to adjacent fragments of rRNA, one of which contains a mutation site. This hybridization results in the formation of a deoxyribozyme catalytic core that produces the fluorescent signal and amplifies it due to multiple rounds of catalytic action. This assay enables us to expedite semi-quantification of mutant rRNA content in cell cultures starting from whole cells, which provides information useful for optimization of culture preparation prior to ribosome isolation. The method requires less than a microliter of a standard Escherichia coli cell culture and decreases analysis time from several days (for primer extension assay) to 1.5 h with hands-on time of ∼10 min. It is sensitive to single-nucleotide mutations. The new assay simplifies the preliminary analysis of RNA samples and cells in molecular biology and cloning experiments and is promising in other applications where fast detection/quantification of specific RNA is required. © 2015 Gerasimova et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society.

  9. Advances in the delivery of RNA therapeutics: from concept to clinical reality.

    Science.gov (United States)

    Kaczmarek, James C; Kowalski, Piotr S; Anderson, Daniel G

    2017-06-27

    The rapid expansion of the available genomic data continues to greatly impact biomedical science and medicine. Fulfilling the clinical potential of genetic discoveries requires the development of therapeutics that can specifically modulate the expression of disease-relevant genes. RNA-based drugs, including short interfering RNAs and antisense oligonucleotides, are particularly promising examples of this newer class of biologics. For over two decades, researchers have been trying to overcome major challenges for utilizing such RNAs in a therapeutic context, including intracellular delivery, stability, and immune response activation. This research is finally beginning to bear fruit as the first RNA drugs gain FDA approval and more advance to the final phases of clinical trials. Furthermore, the recent advent of CRISPR, an RNA-guided gene-editing technology, as well as new strides in the delivery of messenger RNA transcribed in vitro, have triggered a major expansion of the RNA-therapeutics field. In this review, we discuss the challenges for clinical translation of RNA-based therapeutics, with an emphasis on recent advances in delivery technologies, and present an overview of the applications of RNA-based drugs for modulation of gene/protein expression and genome editing that are currently being investigated both in the laboratory as well as in the clinic.

  10. MicroRNA-221 and -222 Regulate Radiation Sensitivity by Targeting the PTEN Pathway

    International Nuclear Information System (INIS)

    Zhang Chunzhi; Kang Chunsheng; Wang Ping; Cao Yongzhen; Lv Zhonghong; Yu Shizhu; Wang Guangxiu; Zhang Anling; Jia Zhifan; Han Lei; Yang Chunying; Ishiyama, Hiromichi; Teh, Bin S.; Xu Bo; Pu Peiyu

    2011-01-01

    Purpose: MicroRNAs (miRNAs) are noncoding RNAs inhibiting expression of numerous target genes by posttranscriptional regulation. miRNA-221 and miRNA-222 (miRNA-221/-222) expression is elevated in radioresistant tumor cell lines; however, it is not known whether and how miRNAs control cellular responses to ionizing irradiation. Methods and Materials: We used bioinformatic analyses, luciferase reporter assay, and genetic knockdown and biochemical assays to characterize the regulation pathways of miRNA-221/-222 in response to radiation treatment. Results: We identified the PTEN gene as a target of miRNA-221/-222. Furthermore, we found that knocking down miRNA-221/-222 by antisense oligonucleotides upregulated PTEN expression. Upregulated PTEN expression suppressed AKT activity and increased radiation-induced apoptosis, resulting in enhancement of radiosensitivity in tumor cells. Conclusions: miRNA-221/-222 control radiation sensitivity by regulating the PTEN/AKT pathway and can be explored as novel targets for radiosensitization.

  11. Distributed biotin-streptavidin transcription roadblocks for mapping cotranscriptional RNA folding.

    Science.gov (United States)

    Strobel, Eric J; Watters, Kyle E; Nedialkov, Yuri; Artsimovitch, Irina; Lucks, Julius B

    2017-07-07

    RNA folding during transcription directs an order of folding that can determine RNA structure and function. However, the experimental study of cotranscriptional RNA folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structure at nucleotide resolution. To address this, we previously developed cotranscriptional selective 2΄-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-Seq) to simultaneously probe all intermediate RNA transcripts during transcription by stalling elongation complexes at catalytically dead EcoRIE111Q roadblocks. While effective, the distribution of elongation complexes using EcoRIE111Q requires laborious PCR using many different oligonucleotides for each sequence analyzed. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent biotin-streptavidin (SAv) roadblocking strategy that simplifies the preparation of roadblocking DNA templates. We first determine the properties of biotin-SAv roadblocks. We then show that randomly distributed biotin-SAv roadblocks can be used in cotranscriptional SHAPE-Seq experiments to identify the same RNA structural transitions related to a riboswitch decision-making process that we previously identified using EcoRIE111Q. Lastly, we find that EcoRIE111Q maps nascent RNA structure to specific transcript lengths more precisely than biotin-SAv and propose guidelines to leverage the complementary strengths of each transcription roadblock in cotranscriptional SHAPE-Seq. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  12. Reversing multidrug resistance in Caco-2 by silencing MDR1, MRP1, MRP2, and BCL-2/BCL-xL using liposomal antisense oligonucleotides.

    Directory of Open Access Journals (Sweden)

    Yu-Li Lo

    Full Text Available Multidrug resistance (MDR is a major impediment to chemotherapy. In the present study, we designed antisense oligonucleotides (ASOs against MDR1, MDR-associated protein (MRP1, MRP2, and/or BCL-2/BCL-xL to reverse MDR transporters and induce apoptosis, respectively. The cationic liposomes (100 nm composed of N-[1-(2,3-dioleyloxypropyl]-n,n,n-trimethylammonium chloride and dioleoyl phosphotidylethanolamine core surrounded by a polyethylene glycol (PEG shell were prepared to carry ASOs and/or epirubicin, an antineoplastic agent. We aimed to simultaneously suppress efflux pumps, provoke apoptosis, and enhance the chemosensitivity of human colon adenocarcinoma Caco-2 cells to epirubicin. We evaluated encapsulation efficiency, particle size, cytotoxicity, intracellular accumulation, mRNA levels, cell cycle distribution, and caspase activity of these formulations. We found that PEGylated liposomal ASOs significantly reduced Caco-2 cell viability and thus intensified epirubicin-mediated apoptosis. These formulations also decreased the MDR1 promoter activity levels and enhanced the intracellular retention of epirubicin in Caco-2 cells. Epirubicin and ASOs in PEGylated liposomes remarkably decreased mRNA expression levels of human MDR1, MRP1, MRP2, and BCL-2. The combined treatments all significantly increased the mRNA expressions of p53 and BAX, and activity levels of caspase-3, -8, and -9. The formulation of epirubicin and ASOs targeting both pump resistance of MDR1, MRP1, and MRP2 and nonpump resistance of BCL-2/BCL-xL demonstrated more superior effect to all the other formulations used in this study. Our results provide a novel insight into the mechanisms by which PEGylated liposomal ASOs against both resistance types act as activators to epirubicin-induced apoptosis through suppressing MDR1, MRP1, and MRP2, as well as triggering intrinsic mitochondrial and extrinsic death receptor pathways. The complicated regulation of MDR highlights the necessity

  13. Beneficial metabolic effects of CB1R anti-sense oligonucleotide treatment in diet-induced obese AKR/J mice.

    Directory of Open Access Journals (Sweden)

    Yuting Tang

    Full Text Available An increasing amount of evidence supports pleiotropic metabolic roles of the cannibinoid-1 receptor (CB1R in peripheral tissues such as adipose, liver, skeletal muscle and pancreas. To further understand the metabolic consequences of specific blockade of CB1R function in peripheral tissues, we performed a 10-week-study with an anti-sense oligonucleotide directed against the CB1R in diet-induced obese (DIO AKR/J mice. DIO AKR/J mice were treated with CB1R ASO Isis-414930 (6.25, 12.5 and 25 mg/kg/week or control ASO Isis-141923 (25 mg/kg/week via intraperitoneal injection for 10 weeks. At the end of the treatment, CB1R mRNA from the 25 mg/kg/week CB1R ASO group in the epididymal fat and kidney was decreased by 81% and 63%, respectively. Body weight gain was decreased in a dose-dependent fashion, significantly different in the 25 mg/kg/week CB1R ASO group (46.1±1.0 g vs veh, 51.2±0.9 g, p<0.05. Body fat mass was reduced in parallel with attenuated body weight gain. CB1R ASO treatment led to decreased fed glucose level (at week 8, 25 mg/kg/week group, 145±4 mg/dL vs veh, 195±10 mg/dL, p<0.05. Moreover, CB1R ASO treatment dose-dependently improved glucose excursion during an oral glucose tolerance test, whereas control ASO exerted no effect. Liver steatosis was also decreased upon CB1R ASO treatment. At the end of the study, plasma insulin and leptin levels were significantly reduced by 25 mg/kg/week CB1R ASO treatment. SREBP1 mRNA expression was decreased in both epididymal fat and liver. G6PC and fatty acid translocase/CD36 mRNA levels were also reduced in the liver. In summary, CB1R ASO treatment in DIO AKR/J mice led to improved insulin sensitivity and glucose homeostasis. The beneficial effects of CB1R ASO treatment strongly support the notion that selective inhibition of the peripheral CB1R, without blockade of central CB1R, may serve as an effective approach for treating type II diabetes, obesity and the metabolic syndrome.

  14. Study on biodistribution and imaging of radioiodinated antisense oligonucleotides in nude mice bearing human lymphoma

    International Nuclear Information System (INIS)

    Wang, R.F.; Shen, J.; Zhang, C.L.; Liu, M.; Guo, F.Q.

    2005-01-01

    The incidence of sporadic lymphoma has risen due to an increase in immunosuppressed patients, particularly those with human immunodeficiency virus (HIV) infection. Sometimes suspect lymphoma has an undetectable location and we can not get the pathological specimen. Management of lymphoma is also difficult because the persistence of a significant number of residual tumor cells after intensive treatment. These relative failures can be attributed to make us choose this study for opening a new diagnostic and therapeutic field of lymphoma from molecular level. Immunoglobulin (Ig) heavy chain framework region (FR) of V1 family have been verified to be a major determinant of malignant phenotype of V1 family B-cell lymphoma. Most of targets for tumor antisense therapy study are protooncogenes, such as c-myc, bc1-2, which are broad -spectrum tumor imaging agents. The aim of this study was to investigate the possibility of using radioiodine labeled FR antisense oligonucleotides (ASONs) as an imaging agent or antisense therapeutic radiopharmaceutical in lymphoma. A 18-mer partial phosphorothioate oligonucleotide sequence was synthesized and grafted in 5 ' with a tyramine group which was further labeled with 125 I or 131 I using the chloramine T method. Normal CD-1 mice were injected via a tail vein with 148 kBq of 125 I-FR-ASON (2∼3 μ g). Animals were sacrificed at 1, 2, 4 and 24 h and tissue samples were studied. Liposome-mediated 3.33 MBq of 131 I-FR-ASON (7 ∼ 9μ g) was injected intratumorally into tumor-bearing BALB/c mice (6 weeks after inoculation of 10 7 Namalwa cells) meanwhile liposome-mediated 131 I labeled sense oligonucleotides served as controls. Biodistribution was monitored by sequential scintigraphy and organ radioactivity measurement 24 h after injection. The percentage of the injected dose per gram (%ID/g) of tumor and tumor/ non-tumor tissue ratios (T/NT) were calculated for each group of mice and the difference between two groups was assessed. The 5

  15. DNA radio-induced tandem lesions: formation, introduction in oligonucleotides and repair

    International Nuclear Information System (INIS)

    Bourdat, Anne-Gaelle

    2000-01-01

    Cell killing induced by excited photosensitizers, ionizing radiation or radiomimetic drugs can not be only explained by the formation of single DNA lesions. Thus, multiply damaged sites, are likely to have harmful biological consequences. One example of tandem base damage induced by ".OH radical in X-irradiated aqueous solution of DNA oligomers is N-(2-deoxy-β-D-erythro-pentofuranosyl)-formyl-amine (dβF)/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo). In order to investigate the biological significance of such a tandem lesion, both 8-oxodGuo and dβF were introduced in synthetic oligonucleotides at vicinal positions using the solid phase phosphoramidite method with the 'Pac phosphoramidite' chemistry. The purity of the synthetic DNA fragments and the integrity of modified nucleosides was confirmed using different complementary techniques: HPLC, PAGE, ESI MS, MALDI-TOF MS and capillary electrophoresis. Using the above synthetic substrates, investigations were carried out in order to determine the substrate specificity and the excision mechanism of three glycosylases involved in the base excision repair pathway: endonuclease III, Fpg and yOggl. Both tandem lesions were substrates for the BER enzymes. However, the tandem lesion are not completely excised by the repair enzymes. The rates of excision as inferred from the determination of the ratios of Vm/Km Michaelis kinetics constants were not found to be significantly affected by the presence of the tandem lesions. MALDI-TOF mass spectrometry was used in order to gain insights into mechanistic aspects of oligonucleotide cleavage by the BER enzymes. During in vitro DNA synthesis by Taq DNA polymerase, Klenow fragment exo- and DNA polymerase β, tandem base damage were found to block the progression of the enzymes. Finally, the level of tandem base damage in the DNA exposed to γ-ray using the liquid chromatography coupled to electro-spray ionization tandem mass spectrometry was determined. Both dβF-8-oxodGuo and 8

  16. Whole genome DNA copy number changes identified by high density oligonucleotide arrays

    Directory of Open Access Journals (Sweden)

    Huang Jing

    2004-05-01

    Full Text Available Abstract Changes in DNA copy number are one of the hallmarks of the genetic instability common to most human cancers. Previous micro-array-based methods have been used to identify chromosomal gains and losses; however, they are unable to genotype alleles at the level of single nucleotide polymorphisms (SNPs. Here we describe a novel algorithm that uses a recently developed high-density oligonucleotide array-based SNP genotyping method, whole genome sampling analysis (WGSA, to identify genome-wide chromosomal gains and losses at high resolution. WGSA simultaneously genotypes over 10,000 SNPs by allele-specific hybridisation to perfect match (PM and mismatch (MM probes synthesised on a single array. The copy number algorithm jointly uses PM intensity and discrimination ratios between paired PM and MM intensity values to identify and estimate genetic copy number changes. Values from an experimental sample are compared with SNP-specific distributions derived from a reference set containing over 100 normal individuals to gain statistical power. Genomic regions with statistically significant copy number changes can be identified using both single point analysis and contiguous point analysis of SNP intensities. We identified multiple regions of amplification and deletion using a panel of human breast cancer cell lines. We verified these results using an independent method based on quantitative polymerase chain reaction and found that our approach is both sensitive and specific and can tolerate samples which contain a mixture of both tumour and normal DNA. In addition, by using known allele frequencies from the reference set, statistically significant genomic intervals can be identified containing contiguous stretches of homozygous markers, potentially allowing the detection of regions undergoing loss of heterozygosity (LOH without the need for a matched normal control sample. The coupling of LOH analysis, via SNP genotyping, with copy number

  17. Crystal structures of two eukaryotic nucleases involved in RNA metabolism

    DEFF Research Database (Denmark)

    Jonstrup, Anette Thyssen; Midtgaard, Søren Fuglsang; Van, Lan Bich

    RNA serves a number of functions in the cell: mRNAs are the carriers of information between gene and protein, tRNAs and rRNAs are involved in the synthesis of proteins, whereas a number of additional RNA species are responsible for other functions in the cell. The quality of the different RNAs...... RNAs. We have solved the structures of two nucleases involved in 3'-5' degradation of RNA; the S. pombe Pop2p and the S. cerevisiae Rrp6p. Pop2p is part of the main cytoplasmatic deadenylation complex in yeast, which also contains the nuclease Ccr4p. Deadenylation, where the poly(A)-tail is removed...... specific transcripts. Here, we present the crystal structure of the S. pombe Pop2p protein to 1.4 Å resolution. The high resolution structure provides a clear picture of the active site architecture. Structural alignment of single nucleotides and poly(A)-oligonucleotides from earlier co-crystal structures...

  18. Primary structure of the α-subunit of Na+, K+-ATPase. II. Isolation, reverse transcription, and cloning of messenger RNA

    International Nuclear Information System (INIS)

    Petrukhin, K.E.; Broude, N.E.; Arsenyan, S.G.; Grishin, A.V.; Dzhandzhugazyan, K.N.; Modyanov, N.N.

    1986-01-01

    The messenger RNA coding the α-subunit of Na + ,K + -ATPase has been isolated from the outer medullary layer of porcine kidneys. The mRNA gives a specific hybridization band in the 25S-26S region with three oligonucleotide probes synthesized on the basis of information on the structure of three peptides isolated from a tryptic hydrolyzate of the α-subunit of Na + ,K + -ATPase. The translation of the mRNA in Xenopus laevis oocytes followed by immunochemical identification of the products of synthesis confirmed the presence of the mRNA of the α-subunit of Na + ,K + -ATPase in an enriched fraction of poly(A + )-RNA. This preparation has been used for the synthesis of cloning of double-stranded cDNA

  19. Primary structure of the. cap alpha. -subunit of Na/sup +/, K/sup +/-ATPase. II. Isolation, reverse transcription, and cloning of messenger RNA

    Energy Technology Data Exchange (ETDEWEB)

    Petrukhin, K.E.; Broude, N.E.; Arsenyan, S.G.; Grishin, A.V.; Dzhandzhugazyan, K.N.; Modyanov, N.N.

    1986-10-01

    The messenger RNA coding the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase has been isolated from the outer medullary layer of porcine kidneys. The mRNA gives a specific hybridization band in the 25S-26S region with three oligonucleotide probes synthesized on the basis of information on the structure of three peptides isolated from a tryptic hydrolyzate of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase. The translation of the mRNA in Xenopus laevis oocytes followed by immunochemical identification of the products of synthesis confirmed the presence of the mRNA of the ..cap alpha..-subunit of Na/sup +/,K/sup +/-ATPase in an enriched fraction of poly(A/sup +/)-RNA. This preparation has been used for the synthesis of cloning of double-stranded cDNA.

  20. Label-Free Electrochemical Detection of the Specific Oligonucleotide Sequence of Dengue Virus Type 1 on Pencil Graphite Electrodes

    Science.gov (United States)

    Souza, Elaine; Nascimento, Gustavo; Santana, Nataly; Ferreira, Danielly; Lima, Manoel; Natividade, Edna; Martins, Danyelly; Lima-Filho, José

    2011-01-01

    A biosensor that relies on the adsorption immobilization of the 18-mer single-stranded nucleic acid related to dengue virus gene 1 on activated pencil graphite was developed. Hybridization between the probe and its complementary oligonucleotides (the target) was investigated by monitoring guanine oxidation by differential pulse voltammetry (DPV). The pencil graphite electrode was made of ordinary pencil lead (type 4B). The polished surface of the working electrode was activated by applying a potential of 1.8 V for 5 min. Afterward, the dengue oligonucleotides probe was immobilized on the activated electrode by applying 0.5 V to the electrode in 0.5 M acetate buffer (pH 5.0) for 5 min. The hybridization process was carried out by incubating at the annealing temperature of the oligonucleotides. A time of five minutes and concentration of 1 μM were found to be the optimal conditions for probe immobilization. The electrochemical detection of annealing between the DNA probe (TS-1P) immobilized on the modified electrode, and the target (TS-1T) was achieved. The target could be quantified in a range from 1 to 40 nM with good linearity and a detection limit of 0.92 nM. The specificity of the electrochemical biosensor was tested using non-complementary sequences of dengue virus 2 and 3. PMID:22163916

  1. New approach to the determination phosphorothioate oligonucleotides by ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry.

    Science.gov (United States)

    Studzińska, Sylwia; Mounicou, Sandra; Szpunar, Joanna; Łobiński, Ryszard; Buszewski, Bogusław

    2015-01-15

    This text presents a novel method for the separation and detection of phosphorothioate oligonucleotides with the use of ion pair ultra high performance liquid chromatography coupled with inductively coupled plasma mass spectrometry The research showed that hexafluoroisopropanol/triethylamine based mobile phases may be successfully used when liquid chromatography is coupled with such elemental detection. However, the concentration of both HFIP and TEA influences the final result. The lower concentration of HFIP, the lower the background in ICP-MS and the greater the sensitivity. The method applied for the analysis of serum samples was based on high resolution inductively coupled plasma mass spectrometry. Utilization of this method allows determination of fifty times lower quantity of phosphorothioate oligonucleotides than in the case of quadrupole mass analyzer. Monitoring of (31)P may be used to quantify these compounds at the level of 80 μg L(-1), while simultaneous determination of sulfur is very useful for qualitative analysis. Moreover, the results presented in this paper demonstrate the practical applicability of coupling LC with ICP-MS in determining phosphorothioate oligonucleotides and their metabolites in serum within 7 min with a very good sensitivity. The method was linear in the concentration range between 0.2 and 3 mg L(-1). The limit of detection was in the range of 0.07 and 0.13 mg L(-1). Accuracy varied with concentration, but was in the range of 3%. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Development of oligonucleotide microarrays for simultaneous multi-species identification of Phellinus tree-pathogenic fungi.

    Science.gov (United States)

    Tzean, Yuh; Shu, Po-Yao; Liou, Ruey-Fen; Tzean, Shean-Shong

    2016-03-01

    Polyporoid Phellinus fungi are ubiquitously present in the environment and play an important role in shaping forest ecology. Several species of Phellinus are notorious pathogens that can affect a broad variety of tree species in forest, plantation, orchard and urban habitats; however, current detection methods are overly complex and lack the sensitivity required to identify these pathogens at the species level in a timely fashion for effective infestation control. Here, we describe eight oligonucleotide microarray platforms for the simultaneous and specific detection of 17 important Phellinus species, using probes generated from the internal transcribed spacer regions unique to each species. The sensitivity, robustness and efficiency of this Phellinus microarray system was subsequently confirmed against template DNA from two key Phellinus species, as well as field samples collected from tree roots, trunks and surrounding soil. This system can provide early, specific and convenient detection of Phellinus species for forestry, arboriculture and quarantine inspection, and could potentially help to mitigate the environmental and economic impact of Phellinus-related diseases. © 2015 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  3. Experimental design, modeling and optimization of polyplex formation between DNA oligonucleotides and branched polyethylenimine.

    Science.gov (United States)

    Clima, Lilia; Ursu, Elena L; Cojocaru, Corneliu; Rotaru, Alexandru; Barboiu, Mihail; Pinteala, Mariana

    2015-09-28

    The complexes formed by DNA and polycations have received great attention owing to their potential application in gene therapy. In this study, the binding efficiency between double-stranded oligonucleotides (dsDNA) and branched polyethylenimine (B-PEI) has been quantified by processing of the images captured from the gel electrophoresis assays. The central composite experimental design has been employed to investigate the effects of controllable factors on the binding efficiency. On the basis of experimental data and the response surface methodology, a multivariate regression model has been constructed and statistically validated. The model has enabled us to predict the binding efficiency depending on experimental factors, such as concentrations of dsDNA and B-PEI as well as the initial pH of solution. The optimization of the binding process has been performed using simplex and gradient methods. The optimal conditions determined for polyplex formation have yielded a maximal binding efficiency close to 100%. In order to reveal the mechanism of complex formation at the atomic-scale, a molecular dynamic simulation has been carried out. According to the computation results, B-PEI amine hydrogen atoms have interacted with oxygen atoms from dsDNA phosphate groups. These interactions have led to the formation of hydrogen bonds between macromolecules, stabilizing the polyplex structure.

  4. A novel analysis strategy for HLA typing using a sequence-specific oligonucleotide probe method.

    Science.gov (United States)

    Won, D I

    2017-11-01

    The technique of reverse sequence-specific oligonucleotide probes (SSOPs) is commonly used in human leukocyte antigen (HLA) typing. In the conventional method for data analysis (exact pattern matching, EPM), the larger is the number of mismatched probes, the longer the time for final typing assignment. A novel strategy, filtering and scoring (FnS), has been developed to easily assign the best-fit allele pair. In the FnS method, candidate alleles and allele pairs were filtered based on (1) subject's ethnicity, and (2) the measured partial reaction pattern with only definitely negative or positive probes. Then, the complete reaction pattern for all probes (CRPoAPs) were compared between the raw sample and expected residual allele pairs to obtain mismatch scores. To compare the FnS and EPM methods, each analysis time (minutes:seconds) for reverse SSOP HLA typing with intermediate resolution (n = 507) was measured. The analysis time with FnS method was shorter than that of the EPM method [00:21 (00:08-01:47) and 01:04 (00:15-23:45), respectively, P typing in a comprehensive and quantitative comparison between measured and expected CRPoAPs of candidate allele pairs. Therefore, this analysis strategy might be useful in a clinical setting. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  5. DNA polymorphisms in the Sahiwal breed of Zebu cattle revealed by synthetic oligonucleotide probes

    International Nuclear Information System (INIS)

    Shashikanth; Yadav, B.R.

    2005-01-01

    Genomic DNA of 15 randomly selected unrelated animals and from two sire families (11 animals) of the Sahiwal breed of Zebu cattle were investigated. Four oligonucleotide probes - (GTG) 5 , (TCC) 5 , (GT) 8 and (GT) 12 - were used on genomic DNA digested with restriction enzymes AluI, HinfI, MboI, EcoRI and HaeIII in different combinations. All four probes produced multiloci fingerprints with differing levels of polymorphisms. Total bands and shared bands in the fingerprints of each individual were in the range of 2.5 to 23.0 KB. Band number ranged from 9 to 17, with 0.48 average band sharing. Probes (GT) 8 , (GT) 12 and (TCC) 5 produced fingerprinting patterns of medium to low polymorphism, whereas probe (GTG) 5 produced highly polymorphic patterns. Probe (GTG) 5 in combination with the HaeIII enzyme was highly polymorphic with a heterozygosity level of 0.85, followed by (GT) 8 , (TCC) 5 and (GT) 12 with heterozygosity levels of 0.70, 0.65 and 0.30, respectively. Probe GTG 5 or its complementary sequence CAC 5 produced highly polymorphic fingerprints, indicating that the probe can be used for analysing population structure, parentage verification and identifying loci controlling quantitative traits and fertility status. (author)

  6. Spotting and validation of a genome wide oligonucleotide chip with duplicate measurement of each gene

    International Nuclear Information System (INIS)

    Thomassen, Mads; Skov, Vibe; Eiriksdottir, Freyja; Tan, Qihua; Jochumsen, Kirsten; Fritzner, Niels; Brusgaard, Klaus; Dahlgaard, Jesper; Kruse, Torben A.

    2006-01-01

    The quality of DNA microarray based gene expression data relies on the reproducibility of several steps in a microarray experiment. We have developed a spotted genome wide microarray chip with oligonucleotides printed in duplicate in order to minimise undesirable biases, thereby optimising detection of true differential expression. The validation study design consisted of an assessment of the microarray chip performance using the MessageAmp and FairPlay labelling kits. Intraclass correlation coefficient (ICC) was used to demonstrate that MessageAmp was significantly more reproducible than FairPlay. Further examinations with MessageAmp revealed the applicability of the system. The linear range of the chips was three orders of magnitude, the precision was high, as 95% of measurements deviated less than 1.24-fold from the expected value, and the coefficient of variation for relative expression was 13.6%. Relative quantitation was more reproducible than absolute quantitation and substantial reduction of variance was attained with duplicate spotting. An analysis of variance (ANOVA) demonstrated no significant day-to-day variation

  7. Kinetic analysis of Yersinia pestis DNA adenine methyltransferase activity using a hemimethylated molecular break light oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Robert J Wood

    Full Text Available BACKGROUND: DNA adenine methylation plays an important role in several critical bacterial processes including mismatch repair, the timing of DNA replication and the transcriptional control of gene expression. The dependence of bacterial virulence on DNA adenine methyltransferase (Dam has led to the proposal that selective Dam inhibitors might function as broad spectrum antibiotics. METHODOLOGY/PRINCIPAL FINDINGS: Herein we report the expression and purification of Yersinia pestis Dam and the development of a continuous fluorescence based assay for DNA adenine methyltransferase activity that is suitable for determining the kinetic parameters of the enzyme and for high throughput screening against potential Dam inhibitors. The assay utilised a hemimethylated break light oligonucleotide substrate containing a GATC methylation site. When this substrate was fully methylated by Dam, it became a substrate for the restriction enzyme DpnI, resulting in separation of fluorophore (fluorescein and quencher (dabcyl and therefore an increase in fluorescence. The assays were monitored in real time using a fluorescence microplate reader in 96 well format and were used for the kinetic characterisation of Yersinia pestis Dam, its substrates and the known Dam inhibitor, S-adenosylhomocysteine. The assay has been validated for high throughput screening, giving a Z-factor of 0.71+/-0.07 indicating that it is a sensitive assay for the identification of inhibitors. CONCLUSIONS/SIGNIFICANCE: The assay is therefore suitable for high throughput screening for inhibitors of DNA adenine methyltransferases and the kinetic characterisation of the inhibition.

  8. Pyrene–nucleobase conjugates: synthesis, oligonucleotide binding and confocal bioimaging studies

    Directory of Open Access Journals (Sweden)

    Artur Jabłoński

    2017-11-01

    Full Text Available Fluorescent pyrene–linker–nucleobase (nucleobase = thymine, adenine conjugates with carbonyl and hydroxy functionalities in the linker were synthesized and characterized. X-ray single-crystal structure analysis performed for the pyrene–C(OCH2CH2–thymine (2 conjugate reveals dimers of molecules 2 stabilized by hydrogen bonds between the thymine moieties. The photochemical characterization showed structure-dependent fluorescence properties of the investigated compounds. The conjugates bearing a carbonyl function represent weak emitters as compared to compounds with a hydroxy function in the linker. The self-assembly properties of pyrene nucleobases were investigated in respect to their binding to single and double strand oligonucleotides in water and in buffer solution. In respect to the complementary oligothymidine T10 template in water, compounds 3 and 5 both show a self-assembling behavior according to canonical base–base pairing. However, in buffer solution, derivative 5 was much more effective than 3 in binding to the T10 template. Furthermore the adenine derivative 5 binds to the double-stranded (dA10–T10 template with a self-assembly ratio of 112%. Such a high value of a self-assembly ratio can be rationalized by a triple-helix-like binding, intercalation, or a mixture of both. Remarkably, compound 5 also shows dual staining pattern in living HeLa cells. Confocal microscopy confirmed that 5 predominantly stains mitochondria but it also accumulates in the nucleoli of the cells.

  9. Ammonium and arsenic trioxide are potent facilitators of oligonucleotide function when delivered by gymnosis

    Science.gov (United States)

    Zhang, Xiaowei; Castanotto, Daniela; Liu, Xueli; Shemi, Amotz; Stein, Cy A

    2018-01-01

    Abstract Oligonucleotide (ON) concentrations employed for therapeutic applications vary widely, but in general are high enough to raise significant concerns for off target effects and cellular toxicity. However, lowering ON concentrations reduces the chances of a therapeutic response, since typically relatively small amounts of ON are taken up by targeted cells in tissue culture. It is therefore imperative to identify new strategies to improve the concentration dependence of ON function. In this work, we have identified ammonium ion (NH4+) as a non-toxic potent enhancer of ON activity in the nucleus and cytoplasm following delivery by gymnosis. NH4+ is a metabolite that has been extensively employed as diuretic, expectorant, for the treatment of renal calculi and in a variety of other diseases. Enhancement of function can be found in attached and suspension cells, including in difficult-to-transfect Jurkat T and CEM T cells. We have also demonstrated that NH4+ can synergistically interact with arsenic trioxide (arsenite) to further promote ON function without producing any apparent increased cellular toxicity. These small, inexpensive, widely distributed molecules could be useful not only in laboratory experiments but potentially in therapeutic ON-based combinatorial strategy for clinical applications. PMID:29522198

  10. [Antiarrhythmic effect of oligonucleotides accompanied by activation of HSP70 protein in the heart of rats].

    Science.gov (United States)

    Kruglov, S V; Terekhina, O L; Smirnova, E A; Kashaeva, O V; Belkina, L M

    2015-01-01

    The mechanisms of the protective effect of oligonucleotides (OGN) during pathological processes are poorlyunderstood. The goal of this work was to study the effect of OGN on arrhythmias induced by myocardial ischemia and reperfusion, and the HSP70 level in the heart. As a source of OGN was used the drug "Derinat" ("Technomedservis", Russia). In male Wistar rats were pre-treated the drug for 7 days (i/m, 7.5 mg/kg).The intensity of the arrhythmias was assessed by ECG during 10 min occlusion of the left coronary artery and subsequent 5 min of reperfusion. Protein HSP70 determined in the left ventricle of the heart by Western-blot analysis. During ischemia, this drug reduced duration of extrasystolia by 13 times and the incidence of ventricular tachycardia by 1.5 times. During reperfusion the drug reduced the incidence of ventricular fibrillation, a more than 2-fold, as compared with the control (respectively 23% vs 56%) and by 5 times its duration (8,4 ± 2,3 48,1 ± sec vs 18 7 sec). "Derinat" increased the HSP70 level in the heart by 65% compared with control. These data support the fact that the activation of HSP70 synthesis, induced by OGN is one of the mechanisms that increases the heart resistance to the ischemic and reperfusion damages.

  11. Oligonucleotide-stabilized fluorescent silver nanoclusters for the specific and sensitive detection of biotin.

    Science.gov (United States)

    Xiong, Xiaoli; Tang, Yan; Zhao, Jingjin; Zhao, Shulin

    2016-02-21

    A novel biotin fluorescent probe based on oligonucleotide-stabilized silver nanoclusters (DNA-AgNCs) was synthesized by employing a biotinylated cytosine-rich sequence as a synthesized template. The fluorescence properties of the DNA-AgNCs are related to the modified position of the DNA. When biotin is linked to the middle thymine base of the DNA sequence, the DNA-AgNCs emit the strongest fluorescence. Moreover, the stability of the DNA-AgNCs was affected by avidin through biotin-avidin binding, quenching the fluorescence of the DNA-AgNCs. In contrast, if free biotin is further introduced into this system, the quenching is apparently weakened by competition, leading to the restoration of fluorescence. This phenomenon can be utilized for the detection of biotin. Under the optimal conditions, the fluorescence recovery is linearly proportional to the concentration of biotin in the range of 10 nM-1.0 μM with a detection limit of 6.0 nM. This DNA-AgNCs probe with excellent fluorescent properties is sensitive and selective for the detection of biotin and has been applied for the determination of biotin in wheat flour.

  12. Suppression of human immunodeficiency virus type 1 activity in vitro by oligonucleotides which form intramolecular tetrads.

    Science.gov (United States)

    Rando, R F; Ojwang, J; Elbaggari, A; Reyes, G R; Tinder, R; McGrath, M S; Hogan, M E

    1995-01-27

    An oligonucleotide (I100-15) composed of only deoxyguanosine and thymidine was able to inhibit human immunodeficiency virus type-1 (HIV-1) in culture assay systems. I100-15 did not block virus entry into cells but did reduce viral-specific transcripts. As assessed by NMR and polyacrylamide gel methods, I100-15 appears to form a structure in which two stacked guanosine tetrads are connected by three two-base long loops. Structure/activity experiments indicated that formation of intramolecular guanosine tetrads was necessary to achieve maximum antiviral activity. The single deoxyguanosine nucleotide present in each loop was found to be extremely important for the overall antiviral activity. The toxicity of I100-15 was determined to be well above the 50% effective dose (ED50) in culture which yielded a high therapeutic index (> 100). The addition of a cholesterol moiety to the 3' terminus of I100-15 (I100-23) reduced the ED50 value to less than 50 nM (from 0.12 microM for I100-15) and increased the duration of viral suppression to greater than 21 days (versus 7-10 days for I100-15) after removal of the drug from infected cell cultures. The favorable therapeutic index of such molecules coupled with the prolonged suppression of HIV-1, suggest that such compounds further warrant investigation as potential therapeutic agents.

  13. Analysis of Structural Flexibility of Damaged DNA Using Thiol-Tethered Oligonucleotide Duplexes.

    Directory of Open Access Journals (Sweden)

    Masashi Fujita

    Full Text Available Bent structures are formed in DNA by the binding of small molecules or proteins. We developed a chemical method to detect bent DNA structures. Oligonucleotide duplexes in which two mercaptoalkyl groups were attached to the positions facing each other across the major groove were prepared. When the duplex contained the cisplatin adduct, which was proved to induce static helix bending, interstrand disulfide bond formation under an oxygen atmosphere was detected by HPLC analyses, but not in the non-adducted duplex, when the two thiol-tethered nucleosides were separated by six base pairs. When the insert was five and seven base pairs, the disulfide bond was formed and was not formed, respectively, regardless of the cisplatin adduct formation. The same reaction was observed in the duplexes containing an abasic site analog and the (6–4 photoproduct. Compared with the cisplatin case, the disulfide bond formation was slower in these duplexes, but the reaction rate was nearly independent of the linker length. These results indicate that dynamic structural changes of the abasic site- and (6–4 photoproduct-containing duplexes could be detected by our method. It is strongly suggested that the UV-damaged DNA-binding protein, which specifically binds these duplexes and functions at the first step of global-genome nucleotide excision repair, recognizes the easily bendable nature of damaged DNA.

  14. Correlation test to assess low-level processing of high-density oligonucleotide microarray data

    Directory of Open Access Journals (Sweden)

    Bergh Jonas

    2005-03-01

    Full Text Available Abstract Background There are currently a number of competing techniques for low-level processing of oligonucleotide array data. The choice of technique has a profound effect on subsequent statistical analyses, but there is no method to assess whether a particular technique is appropriate for a specific data set, without reference to external data. Results We analyzed coregulation between genes in order to detect insufficient normalization between arrays, where coregulation is measured in terms of statistical correlation. In a large collection of genes, a random pair of genes should have on average zero correlation, hence allowing a correlation test. For all data sets that we evaluated, and the three most commonly used low-level processing procedures including MAS5, RMA and MBEI, the housekeeping-gene normalization failed the test. For a real clinical data set, RMA and MBEI showed significant correlation for absent genes. We also found that a second round of normalization on the probe set level improved normalization significantly throughout. Conclusion Previous evaluation of low-level processing in the literature has been limited to artificial spike-in and mixture data sets. In the absence of a known gold-standard, the correlation criterion allows us to assess the appropriateness of low-level processing of a specific data set and the success of normalization for subsets of genes.

  15. SWPhylo - A Novel Tool for Phylogenomic Inferences by Comparison of Oligonucleotide Patterns and Integration of Genome-Based and Gene-Based Phylogenetic Trees.

    Science.gov (United States)

    Yu, Xiaoyu; Reva, Oleg N

    2018-01-01

    Modern phylogenetic studies may benefit from the analysis of complete genome sequences of various microorganisms. Evolutionary inferences based on genome-scale analysis are believed to be more accurate than the gene-based alternative. However, the computational complexity of current phylogenomic procedures, inappropriateness of standard phylogenetic tools to process genome-wide data, and lack of reliable substitution models which correlates with alignment-free phylogenomic approaches deter microbiologists from using these opportunities. For example, the super-matrix and super-tree approaches of phylogenomics use multiple integrated genomic loci or individual gene-based trees to infer an overall consensus tree. However, these approaches potentially multiply errors of gene annotation and sequence alignment not mentioning the computational complexity and laboriousness of the methods. In this article, we demonstrate that the annotation- and alignment-free comparison of genome-wide tetranucleotide frequencies, termed oligonucleotide usage patterns (OUPs), allowed a fast and reliable inference of phylogenetic trees. These were congruent to the corresponding whole genome super-matrix trees in terms of tree topology when compared with other known approaches including 16S ribosomal RNA and GyrA protein sequence comparison, complete genome-based MAUVE, and CVTree methods. A Web-based program to perform the alignment-free OUP-based phylogenomic inferences was implemented at http://swphylo.bi.up.ac.za/. Applicability of the tool was tested on different taxa from subspecies to intergeneric levels. Distinguishing between closely related taxonomic units may be enforced by providing the program with alignments of marker protein sequences, eg, GyrA.

  16. SWPhylo – A Novel Tool for Phylogenomic Inferences by Comparison of Oligonucleotide Patterns and Integration of Genome-Based and Gene-Based Phylogenetic Trees

    Science.gov (United States)

    Yu, Xiaoyu; Reva, Oleg N

    2018-01-01

    Modern phylogenetic studies may benefit from the analysis of complete genome sequences of various microorganisms. Evolutionary inferences based on genome-scale analysis are believed to be more accurate than the gene-based alternative. However, the computational complexity of current phylogenomic procedures, inappropriateness of standard phylogenetic tools to process genome-wide data, and lack of reliable substitution models which correlates with alignment-free phylogenomic approaches deter microbiologists from using these opportunities. For example, the super-matrix and super-tree approaches of phylogenomics use multiple integrated genomic loci or individual gene-based trees to infer an overall consensus tree. However, these approaches potentially multiply errors of gene annotation and sequence alignment not mentioning the computational complexity and laboriousness of the methods. In this article, we demonstrate that the annotation- and alignment-free comparison of genome-wide tetranucleotide frequencies, termed oligonucleotide usage patterns (OUPs), allowed a fast and reliable inference of phylogenetic trees. These were congruent to the corresponding whole genome super-matrix trees in terms of tree topology when compared with other known approaches including 16S ribosomal RNA and GyrA protein sequence comparison, complete genome-based MAUVE, and CVTree methods. A Web-based program to perform the alignment-free OUP-based phylogenomic inferences was implemented at http://swphylo.bi.up.ac.za/. Applicability of the tool was tested on different taxa from subspecies to intergeneric levels. Distinguishing between closely related taxonomic units may be enforced by providing the program with alignments of marker protein sequences, eg, GyrA. PMID:29511354

  17. Accurate and rapid modeling of iron-bleomycin-induced DNA damage using tethered duplex oligonucleotides and electrospray ionization ion trap mass spectrometric analysis.

    Science.gov (United States)

    Harsch, A; Marzilli, L A; Bunt, R C; Stubbe, J; Vouros, P

    2000-05-01

    Bleomycin B(2)(BLM) in the presence of iron [Fe(II)] and O(2)catalyzes single-stranded (ss) and double-stranded (ds) cleavage of DNA. Electrospray ionization ion trap mass spectrometry was used to monitor these cleavage processes. Two duplex oligonucleotides containing an ethylene oxide tether between both strands were used in this investigation, allowing facile monitoring of all ss and ds cleavage events. A sequence for site-specific binding and cleavage by Fe-BLM was incorporated into each analyte. One of these core sequences, GTAC, is a known hot-spot for ds cleavage, while the other sequence, GGCC, is a hot-spot for ss cleavage. Incubation of each oligo-nucleotide under anaerobic conditions with Fe(II)-BLM allowed detection of the non-covalent ternary Fe-BLM/oligonucleotide complex in the gas phase. Cleavage studies were then performed utilizing O(2)-activated Fe(II)-BLM. No work-up or separation steps were required and direct MS and MS/MS analyses of the crude reaction mixtures confirmed sequence-specific Fe-BLM-induced cleavage. Comparison of the cleavage patterns for both oligonucleotides revealed sequence-dependent preferences for ss and ds cleavages in accordance with previously established gel electrophoresis analysis of hairpin oligonucleotides. This novel methodology allowed direct, rapid and accurate determination of cleavage profiles of model duplex oligonucleotides after exposure to activated Fe-BLM.

  18. RNA decay by messenger RNA interferases

    DEFF Research Database (Denmark)

    Christensen-Dalsgaard, Mikkel; Overgaard, Martin; Winther, Kristoffer Skovbo

    2008-01-01

    Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mR...... cleaving enzymes such as RelE of Escherichia coli and the corresponding antitoxin RelB. In particular, we describe a set of plasmid vectors useful for the detailed analysis of cleavage sites in model mRNAs.......Two abundant toxin-antitoxin (TA) gene families, relBE and mazEF, encode mRNA cleaving enzymes whose ectopic overexpression abruptly inhibits translation and thereby induces a bacteriostatic condition. Here we describe and discuss protocols for the overproduction, purification, and analysis of mRNA...

  19. Marburg virus VP35 can both fully coat the backbone and cap the ends of dsRNA for interferon antagonism.

    Directory of Open Access Journals (Sweden)

    Shridhar Bale

    2012-09-01

    Full Text Available Filoviruses, including Marburg virus (MARV and Ebola virus (EBOV, cause fatal hemorrhagic fever in humans and non-human primates. All filoviruses encode a unique multi-functional protein termed VP35. The C-terminal double-stranded (dsRNA-binding domain (RBD of VP35 has been implicated in interferon antagonism and immune evasion. Crystal structures of the VP35 RBD from two ebolaviruses have previously demonstrated that the viral protein caps the ends of dsRNA. However, it is not yet understood how the expanses of dsRNA backbone, between the ends, are masked from immune surveillance during filovirus infection. Here, we report the crystal structure of MARV VP35 RBD bound to dsRNA. In the crystal structure, molecules of dsRNA stack end-to-end to form a pseudo-continuous oligonucleotide. This oligonucleotide is continuously and completely coated along its sugar-phosphate backbone by the MARV VP35 RBD. Analysis of dsRNA binding by dot-blot and isothermal titration calorimetry reveals that multiple copies of MARV VP35 RBD can indeed bind the dsRNA sugar-phosphate backbone in a cooperative manner in solution. Further, MARV VP35 RBD can also cap the ends of the dsRNA in solution, although this arrangement was not captured in crystals. Together, these studies suggest that MARV VP35 can both coat the backbone and cap the ends, and that for MARV, coating of the dsRNA backbone may be an essential mechanism by which dsRNA is masked from backbone-sensing immune surveillance molecules.

  20. mRNA fragments in in vitro culture media are associated with bovine preimplantation embryonic development.

    Science.gov (United States)

    Kropp, Jenna; Khatib, Hasan

    2015-01-01

    In vitro production (IVP) systems have been used to bypass problems of fertilization and early embryonic development. However, embryos produced by IVP are commonly selected for implantation based on morphological assessment, which is not a strong indicator of establishment and maintenance of pregnancy. Thus, there is a need to identify additional indicators of embryonic developmental potential. Previous studies have identified microRNA expression in in vitro culture media to be indicative of embryo quality in both bovine and human embryos. Like microRNAs, mRNAs have been shown to be secreted from cells into the extracellular environment, but it is unknown whether or not these RNAs are secreted by embryos. Thus, the objective of the present study was to determine whether mRNAs are secreted into in vitro culture media and if their expression in the media is indicative of embryo quality. In vitro culture medium was generated and collected from both blastocyst and degenerate (those which fail to develop from the morula to blastocyst stage) embryos. Small-RNA sequencing revealed that many mRNA fragments were present in the culture media. A total of 17 mRNA fragments were differentially expressed between blastocyst and degenerate conditioned media. Differential expression was confirmed by quantitative real-time PCR for fragments of mRNA POSTN and VSNL-1, in four additional biological replicates of media. To better understand the mechanisms of mRNA secretion into the media, the expression of a predicted RNA binding protein of POSTN, PUM2, was knocked down using an antisense oligonucleotide gapmer. Supplementation of a PUM2 gapmer significantly reduced blastocyst development and decreased secretion of POSTN mRNA into the media. Overall, differential mRNA expression in the media was repeatable and sets the framework for future study of mRNA biomarkers in in vitro culture media to improve predictability of reproductive performance.

  1. Identification of special fragments containing the 5' end of polivirus RNA after ribonuclease III digestion

    Energy Technology Data Exchange (ETDEWEB)

    Harris, T.J.R.; Dunn, J.J.; Wimmer, E.

    1978-11-01

    The small protein (VPg) covalently linked to the 5' end of the poliovirus Type 1 (PV-1) RNA has been labeled in vitro with /sup 125/I usingthe Bolton and Hunter reagent. The RNA is not degraded under the conditions used and nearly all the label enters VPg and not the polynucleotide chain. When this /sup 125/I-labeled RNA is cleaved with RNase III at low monovalent salt concentrations, one major /sup 125/I-labeled fragment, approximately 100 nucleotides long, is produced. The corresponding fragment from similar digests of /sup 32/P-labeled RNA has also been identified. The /sup 32/P-labeled fragment changes electrophoretic mobility after protease treatment indicating that it contains VPg. Furthermore, the RNase T1 oligonucleotide known to be at the 5' terminus of poliovirus RNA is found in T1 digests of the purified fragment. These results confirm that the fragment is derived from the 5' end of the RNA. This fragment will be useful in studies concerning the initiation of protein synthesis during poliovirus infection.

  2. The True Story and Advantages of RNA Phage Capsids as Nanotools.

    Science.gov (United States)

    Pumpens, Paul; Renhofa, Regina; Dishlers, Andris; Kozlovska, Tatjana; Ose, Velta; Pushko, Peter; Tars, Kaspars; Grens, Elmars; Bachmann, Martin F

    2016-01-01

    RNA phages are often used as prototypes for modern recombinant virus-like particle (VLP) technologies. Icosahedral RNA phage VLPs can be formed from coat proteins (CPs) and are efficiently produced in bacteria and yeast. Both genetic fusion and chemical coupling have been successfully used for the production of numerous chimeras based on RNA phage VLPs. In this review, we describe advances in RNA phage VLP technology along with the history of the Leviviridae family, including its taxonomical organization, genomic structure, and important role in the development of molecular biology. Comparative 3D structures of different RNA phage VLPs are used to explain the level of VLP tolerance to foreign elements displayed on VLP surfaces. We also summarize data that demonstrate the ability of CPs to tolerate different organic (peptides, oligonucleotides, and carbohydrates) and inorganic (metal ions) compounds either chemically coupled or noncovalently added to the outer and/or inner surfaces of VLPs. Finally, we present lists of nanotechnological RNA phage VLP applications, such as experimental vaccines constructed by genetic fusion and chemical coupling methodologies, nanocontainers for targeted drug delivery, and bioimaging tools. © 2016 S. Karger AG, Basel.

  3. Detection of HIV-RNA-positive monocytes in peripheral blood of HIV-positive patients by simultaneous flow cytometric analysis of intracellular HIV RNA and cellular immunophenotype.

    Science.gov (United States)

    Patterson, B K; Mosiman, V L; Cantarero, L; Furtado, M; Bhattacharya, M; Goolsby, C

    1998-04-01

    Determinations of plasma HIV viral RNA copy numbers help to define the kinetics of HIV-1 infection in vivo and to monitor antiretroviral therapy. However, questions remain regarding the identity of various infected cell types contributing to this free virus pool and to the in vivo lifecycle of HIV during disease progression. Characterization of a novel fluorescence in situ hybridization (FISH) assay employing a pool of labeled oligonucleotide probes directed against HIV RNA was done followed by coupling of the FISH assay with simultaneous surface immunophenotyping to address these questions. In vitro characterizations of this assay using tumor necrosis factor-alpha stimulated and unstimulated ACH-2 cells demonstrated the ability to detect < 5% HIV RNA positive cells with a sensitivity of < 30 RNA copies per cell. Peripheral blood mononuclear cells from 39 HIV-seropositive patients on no, single, combination, or triple drug therapy and 8 HIV-seronegative patients were examined. The majority of HIV-positive patients (24/39) harbored monocytes positive for HIV RNA and a significantly higher fraction of patients with high plasma viral load carried positive monocytes (13/16) than did patients in the low plasma viral load group (11/23). These results demonstrate the effectiveness of a novel FISH assay for identifying and monitoring HIV-infected cell populations in the peripheral blood of HIV-positive patients. In addition, monocytes are a major source of cellular HIV virus in the peripheral blood of HIV patients, even with progression of disease.

  4. STAT3 Gene Silencing by Aptamer-siRNA Chimera as Selective Therapeutic for Glioblastoma

    Directory of Open Access Journals (Sweden)

    Carla Lucia Esposito

    2018-03-01

    Full Text Available Glioblastoma (GBM is the most frequent and aggressive primary brain tumor in adults, and despite advances in neuro-oncology, the prognosis for patients remains dismal. The signal transducer and activator of transcription-3 (STAT3 has been reported as a key regulator of the highly aggressive mesenchymal GBM subtype, and its direct silencing (by RNAi oligonucleotides has revealed a great potential as an anti-cancer therapy. However, clinical use of oligonucleotide-based therapies is dependent on safer ways for tissue-specific targeting and increased membrane penetration. The objective of this study is to explore the use of nucleic acid aptamers as carriers to specifically drive a STAT3 siRNA to GBM cells in a receptor-dependent manner. Using an aptamer that binds to and antagonizes the oncogenic receptor tyrosine kinase PDGFRβ (Gint4.T, here we describe the design of a novel aptamer-siRNA chimera (Gint4.T-STAT3 to target STAT3. We demonstrate the efficient delivery and silencing of STAT3 in PDGFRβ+ GBM cells. Importantly, the conjugate reduces cell viability and migration in vitro and inhibits tumor growth and angiogenesis in vivo in a subcutaneous xenograft mouse model. Our data reveals Gint4.T-STAT3 conjugate as a novel molecule with great translational potential for GBM therapy.

  5. Development of DNA affinity techniques for the functional characterization of purified RNA polymerase II transcription factors

    International Nuclear Information System (INIS)

    Garfinkel, S.; Thompson, J.A.; Cohen, R.B.; Brendler, T.; Safer, B.

    1987-01-01

    Affinity adsorption, precipitation, and partitioning techniques have been developed to purify and characterize RNA Pol II transcription components from whole cell extracts (WCE) (HeLa) and nuclear extracts (K562). The titration of these extracts with multicopy constructs of the Ad2 MLP but not pUC8, inhibits transcriptional activity. DNA-binding factors precipitated by this technique are greatly enriched by centrifugation. Using this approach, factors binding to the upstream promoter sequence (UPS) of the Ad2 MLP have been rapidly isolated by Mono Q, Mono S, and DNA affinity chromatography. By U.V. crosslinking to nucleotides containing specific 32 P-phosphodiester bonds within the recognition sequence, this factor is identified as a M/sub r/ = 45,000 polypeptide. To generate an assay system for the functional evaluation of single transcription components, a similar approach using synthetic oligonucleotide sequences spanning single promoter binding sites has been developed. The addition of a synthetic 63-mer containing the UPS element of the Ad2 MLP to HeLa WCE inhibited transcription by 60%. The addition of partially purified UPS binding protein, but not RNA Pol II, restored transcriptional activity. The addition of synthetic oligonucleotides containing other regulatory sequences not present in the Ad2 MLP was without effect

  6. Topology of RNA-RNA interaction structures

    DEFF Research Database (Denmark)

    Andersen, Jørgen Ellegaard; Huang, Fenix Wenda; Penner, Robert

    2012-01-01

    Abstract The topological filtration of interacting RNA complexes is studied, and the role is analyzed of certain diagrams called irreducible shadows, which form suitable building blocks for more general structures. We prove that, for two interacting RNAs, called interaction structures, there exist...

  7. RNA Localization in Astrocytes

    DEFF Research Database (Denmark)

    Thomsen, Rune

    2012-01-01

    , regulation of the blood brain barrier and glial scar tissue formation. Despite the involvement in various CNS functions only a limited number of studies have addressed mRNA localization in astrocytes. This PhD project was initially focused on developing and implementing methods that could be used to asses mRNA......Messenger RNA (mRNA) localization is a mechanism by which polarized cells can regulate protein synthesis to specific subcellular compartments in a spatial and temporal manner, and plays a pivotal role in multiple physiological processes from embryonic development to cell differentiation...... localization in astrocyte protrusions, and following look into the subcellular localization pattern of specific mRNA species of both primary astrocytes isolated from cortical hemispheres of newborn mice, and the mouse astrocyte cell line, C8S. The Boyden chamber cell fractionation assay was optimized, in a way...

  8. Electrocatalytic miRNA Detection Using Cobalt Porphyrin-Modified Reduced Graphene Oxide

    Directory of Open Access Journals (Sweden)

    Camille De Souza

    2014-06-01

    Full Text Available Metalated porphyrins have been described to bind nucleic acids. Additionally, cobalt porphyrins present catalytic properties towards oxygen reduction. In this work, a carboxylic acid-functionalized cobalt porphyrin was physisorbed on reduced graphene oxide, then immobilized on glassy carbon electrodes. The carboxylic groups were used to covalently graft amino-terminated oligonucleotide probes which are complementary to a short microRNA target. It was shown that the catalytic oxygen electroreduction on cobalt porphyrin increases upon hybridization of miRNA strand (“signal-on” response. Current changes are amplified compared to non-catalytic amperometric system. Apart from oxygen, no added reagent is necessary. A limit of detection in the sub-nanomolar range was reached. This approach has never been described in the literature.

  9. Basic science for the clinician 49: expanding the description of the RNA universe.

    Science.gov (United States)

    Sigal, Leonard H

    2009-03-01

    We have come a long way in paying RNA its due respect. Originally thought to be nothing more than a shuttle of information from DNA to protein, a bearer of amino acids to the ribosome, and a splicer of messenger RNA, we now know that other RNA species are pivotal in controlling cellular functions that assure normal development and differentiation of immune cells, modulation of inflammatory mechanisms, control of proliferation of a number of hematologic lineages, and spermatogenesis (clearly, vital for the maintenance of the species!). In the future, ribozymes, antisense RNA and oligonucleotides, decoy RNA, peptide-nucleic acid chimeras, and other RNAs will probably be part of the routine armamentarium in a variety of medical practices. Targeting these to the appropriate cell may allow for highly directed therapies, maximizing efficacy and minimizing toxicity. It is a new world, an RNA world, and we will all benefit from the insights broadly outlined in this article. When I was in college and medical school, RNA was known to come in only a few varieties. There was messenger RNA, ribosomal RNA, transfer RNA, and double-stranded RNA in some viruses. And that was that! My, how times have changed!! The truth, as always, is much more complicated than we had thought. We now know that RNA is involved in splicing of mRNA and in cleaving RNA. And, recent studies have revealed even more: DNA transcription, mRNA stability, and levels of protein synthesis are all, to some degree, controlled by an entirely different set of RNAs, such as small RNAs, which come in at least 3 different broad varieties. Thus, there are now at least 10 varieties of RNAs of which I am aware at the time I write these words, and who is to say that there are not more out there? Just as the entire repertoire of the known classes of small RNAs has not yet been described, there may be different RNAs out there yet to be identified. If, in fact, the bio-universe was initially determined by RNA, not DNA, there

  10. Inhibition effects of 125I-triplex forming oligonucleotide to hepatoma cells

    International Nuclear Information System (INIS)

    Lv Zhongwei; Hou Min; Cai Haidong; Yuan Xueyu; Yang Yuehua; Yuan Shidong; He Junmin

    2007-01-01

    Objective: Triplex forming oligonucleotide (TFO) has been reported as a new antigene strategy. The purpose of this study was to observe the inhibition effects of 125 I-TFO on hepatoma cells and to investigate the possibility of using 125 I-TFO as an antigene radiotherapy technique for hepatocellular carcinoma (HCC) related to HBV. Methods: TFO complementary to the initiator of S gene of HBV was synthesized and labeled with 125 I. HepG2.2.15 cells, in which HBV genome was integrated, were incubated with 125 I-TFO, TFO and 125 I respectively. After incubation, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) of each group were assayed with ELISA and the survival rate of cells in each group was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) reduction assay. Results: 125 I-TFO showed a high stability with a radiolabeling rate of >93%. The radiochemical purity of labeled compound was 90.8%, 81.1% and 73.2% respectively after 12, 48 and 72 h at 37 degree C. The peak inhibition effect of 125 I-TFO on synthesizing HBsAg and HBeAg by HepG2.2.15 cells were found at 48 h after transfection, with significantly the highest inhibition rate of 45.2% for HBsAg and 74.5% for HBeAg expression among the three groups(P 125 I-TFO may inhibit the antigen expression of HBV and the growth of hepatocarcinoma cells, thus it may provide a new approach to develop gene-based radiotherapeutic pharmaceuticals for anti-HBV and HCC. (authors)

  11. Elucidation of the Biotransformation Pathways of a Galnac3-conjugated Antisense Oligonucleotide in Rats and Monkeys

    Directory of Open Access Journals (Sweden)

    Colby S Shemesh

    2016-01-01

    Full Text Available Triantennary N-acetyl galactosamine (GalNAc3 is a high-affinity ligand for hepatocyte-specific asialoglycoprotein receptors. Conjugation with GalNAc3 via a trishexylamino (THA-C6 cluster significantly enhances antisense oligonucleotide (ASO potency. Herein, the biotransformation, disposition, and elimination of the THA cluster of ION-681257, a GalNAc3-conjugated ASO currently in clinical development, are investigated in rats and monkey. Rats were administered a single subcutaneous dose of 3H-radiolabeled (3H placed in THA or nonradiolabeled ION-681257. Mass balance included radiometric profiling and metabolite fractionation with characterization by mass spectrometry. GalNAc3-conjugated ASOs were extensively distributed into liver. The THA-C6 triantenerrary GalNAc3 conjugate at the 5′-end of the ASO was rapidly metabolized and excreted with 25.67 ± 1.635% and 71.66 ± 4.17% of radioactivity recovered in urine and feces within 48 hours postdose. Unchanged drug, short-mer ASOs, and linker metabolites were detected in urine. Collectively, 14 novel linker associated metabolites were discovered including oxidation at each branching arm, initially by monooxidation at the β-position followed by dioxidation at the α-arm, and lastly, tri and tetra oxidations on the two remaining β-arms. Metabolites in bile and feces were identical to urine except for oxidized linear and cyclic linker metabolites. Enzymatic reaction phenotyping confirmed involvement of N-acetyl-β-glucosaminidase, deoxyribonuclease II, alkaline phosphatase, and alcohol + aldehyde dehydrogenases on the complex metabolism pathway for THA supplementing in vivo findings. Lastly, excreta from monkeys treated with ION-681257 revealed the identical series as observed in rat. In summary, our findings provide an improved understanding of GalNAc3-conjugated-ASO metabolism pathways which facilitate similar development programs.

  12. Customized oligonucleotide microarray gene expression-based classification of neuroblastoma patients outperforms current clinical risk stratification.

    Science.gov (United States)

    Oberthuer, André; Berthold, Frank; Warnat, Patrick; Hero, Barbara; Kahlert, Yvonne; Spitz, Rüdiger; Ernestus, Karen; König, Rainer; Haas, Stefan; Eils, Roland; Schwab, Manfred; Brors, Benedikt; Westermann, Frank; Fischer, Matthias

    2006-11-01

    To develop a gene expression-based classifier for neuroblastoma patients that reliably predicts courses of the disease. Two hundred fifty-one neuroblastoma specimens were analyzed using a customized oligonucleotide microarray comprising 10,163 probes for transcripts with differential expression in clinical subgroups of the disease. Subsequently, the prediction analysis for microarrays (PAM) was applied to a first set of patients with maximally divergent clinical courses (n = 77). The classification accuracy was estimated by a complete 10-times-repeated 10-fold cross validation, and a 144-gene predictor was constructed from this set. This classifier's predictive power was evaluated in an independent second set (n = 174) by comparing results of the gene expression-based classification with those of risk stratification systems of current trials from Germany, Japan, and the United States. The first set of patients was accurately predicted by PAM (cross-validated accuracy, 99%). Within the second set, the PAM classifier significantly separated cohorts with distinct courses (3-year event-free survival [EFS] 0.86 +/- 0.03 [favorable; n = 115] v 0.52 +/- 0.07 [unfavorable; n = 59] and 3-year overall survival 0.99 +/- 0.01 v 0.84 +/- 0.05; both P model, the PAM predictor classified patients of the second set more accurately than risk stratification of current trials from Germany, Japan, and the United States (P < .001; hazard ratio, 4.756 [95% CI, 2.544 to 8.893]). Integration of gene expression-based class prediction of neuroblastoma patients may improve risk estimation of current neuroblastoma trials.

  13. Rescue of peripheral vestibular function in Usher syndrome mice using a splice-switching antisense oligonucleotide.

    Science.gov (United States)

    Vijayakumar, Sarath; Depreux, Frederic F; Jodelka, Francine M; Lentz, Jennifer J; Rigo, Frank; Jones, Timothy A; Hastings, Michelle L

    2017-09-15

    Usher syndrome type 1C (USH1C/harmonin) is associated with profound retinal, auditory and vestibular dysfunction. We have previously reported on an antisense oligonucleotide (ASO-29) that dramatically improves auditory function and balance behavior in mice homozygous for the harmonin mutation Ush1c c.216G > A following a single systemic administration. The findings were suggestive of improved vestibular function; however, no direct vestibular assessment was made. Here, we measured vestibular sensory evoked potentials (VsEPs) to directly assess vestibular function in Usher mice. We report that VsEPs are absent or abnormal in Usher mice, indicating profound loss of vestibular function. Strikingly, Usher mice receiving ASO-29 treatment have normal or elevated vestibular response thresholds when treated during a critical period between postnatal day 1 and 5, respectively. In contrast, treatment of mice with ASO-29 treatment at P15 was minimally effective at rescuing vestibular function. Interestingly, ASO-29 treatment at P1, P5 or P15 resulted in sufficient vestibular recovery to support normal balance behaviors, suggesting a therapeutic benefit to balance with ASO-29 treatment at P15 despite the profound vestibular functional deficits that persist with treatment at this later time. These findings provide the first direct evidence of an effective treatment of peripheral vestibular function in a mouse model of USH1C and reveal the potential for using antisense technology to treat vestibular dysfunction. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  14. Inhibition effects of {sup 125}I-triplex forming oligonucleotide to hepatoma cells

    Energy Technology Data Exchange (ETDEWEB)

    Zhongwei, Lv; Min, Hou; Haidong, Cai; Xueyu, Yuan; Yuehua, Yang; Shidong, Yuan [Department of Nuclear Medicine, 10th People' s Hospital, Tongji Univ., Shanghai (China); Junmin, He

    2007-08-15

    Objective: Triplex forming oligonucleotide (TFO) has been reported as a new antigene strategy. The purpose of this study was to observe the inhibition effects of {sup 125}I-TFO on hepatoma cells and to investigate the possibility of using {sup 125}I-TFO as an antigene radiotherapy technique for hepatocellular carcinoma (HCC) related to HBV. Methods: TFO complementary to the initiator of S gene of HBV was synthesized and labeled with {sup 125}I. HepG2.2.15 cells, in which HBV genome was integrated, were incubated with {sup 125}I-TFO, TFO and {sup 125}I respectively. After incubation, hepatitis B e antigen (HBeAg) and hepatitis B surface antigen (HBsAg) of each group were assayed with ELISA and the survival rate of cells in each group was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-trazolium bromide (MTT) reduction assay. Results: {sup 125}I-TFO showed a high stability with a radiolabeling rate of >93%. The radiochemical purity of labeled compound was 90.8%, 81.1% and 73.2% respectively after 12, 48 and 72 h at 37 degree C. The peak inhibition effect of {sup 125}I-TFO on synthesizing HBsAg and HBeAg by HepG2.2.15 cells were found at 48 h after transfection, with significantly the highest inhibition rate of 45.2% for HBsAg and 74.5% for HBeAg expression among the three groups(P<0.01 ). As the transfection time prolonged its inhibition effects were stronger. Conclusion: {sup 125}I-TFO may inhibit the antigen expression of HBV and the growth of hepatocarcinoma cells, thus it may provide a new approach to develop gene-based radiotherapeutic pharmaceuticals for anti-HBV and HCC. (authors)

  15. Charge transfer through DNA/DNA duplexes and DNA/RNA hybrids: complex theoretical and experimental studies.

    Science.gov (United States)

    Kratochvílová, Irena; Vala, Martin; Weiter, Martin; Špérová, Miroslava; Schneider, Bohdan; Páv, Ondřej; Šebera, Jakub; Rosenberg, Ivan; Sychrovský, Vladimír

    2013-01-01

    Oligonucleotides conduct electric charge via various mechanisms and their characterization and understanding is a very important and complicated task. In this work, experimental (temperature dependent steady state fluorescence spectroscopy, time-resolved fluorescence spectroscopy) and theoretical (Density Functional Theory) approaches were combined to study charge transfer processes in short DNA/DNA and RNA/DNA duplexes with virtually equivalent sequences. The experimental results were consistent with the theoretical model - the delocalized nature of HOMO orbitals and holes, base stacking, electronic coupling and conformational flexibility formed the conditions for more effective short distance charge transfer processes in RNA/DNA hybrids. RNA/DNA and DNA/DNA charge transfer properties were strongly connected with temperature affected structural changes of molecular systems - charge transfer could be used as a probe of even tiny changes of molecular structures and settings. © 2013. Published by Elsevier B.V. All rights reserved.

  16. The Deinococcus-Thermus phylum and the effect of rRNA composition on phylogenetic tree construction

    Science.gov (United States)

    Weisburg, W. G.; Giovannoni, S. J.; Woese, C. R.

    1989-01-01

    Through comparative analysis of 16S ribosomal RNA sequences, it can be shown that two seemingly dissimilar types of eubacteria Deinococcus and the ubiquitous hot spring organism Thermus are distantly but specifically related to one another. This confirms an earlier report based upon 16S rRNA oligonucleotide cataloging studies (Hensel et al., 1986). Their two lineages form a distinctive grouping within the eubacteria that deserved the taxonomic status of a phylum. The (partial) sequence of T. aquaticus rRNA appears relatively close to those of other thermophilic eubacteria. e.g. Thermotoga maritima and Thermomicrobium roseum. However, this closeness does not reflect a true evolutionary closeness; rather it is due to a "thermophilic convergence", the result of unusually high G+C composition in the rRNAs of thermophilic bacteria. Unless such compositional biases are taken into account, the branching order and root of phylogenetic trees can be inco