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Sample records for antisense peptide nucleic

  1. Peptide nucleic acid (PNA) antisense effects in Escherichia coli

    DEFF Research Database (Denmark)

    Good, L; Nielsen, P E

    1999-01-01

    Antisense peptide nucleic acid (PNA) can be used to control cell growth, gene expression and growth phenotypes in the bacteria Escherichia coli. PNAs targeted to the RNA components of the ribosome can inhibit translation and cell growth, and PNAs targeted to mRNA can limit gene expression with gene...... and sequence specificity. In an E. coli cell extract, efficient inhibition is observed when using PNA concentrations in the nanomolar range, whereas micromolar concentrations are required for inhibition in growing cells. A mutant strain of E. coli that is more permeable to antibiotics also is more susceptible...... to antisense PNAs than the wild type. This chapter details methods for testing the antisense activities of PNA in E. coli. As an example of the specific antisense inhibition possible, we show the effects of an anti-beta-galactosidase PNA in comparison to control PNAs. With improvements in cell uptake...

  2. Cell number and transfection volume dependent peptide nucleic acid antisense activity by cationic delivery methods

    DEFF Research Database (Denmark)

    Llovera Nadal, Laia; Berthold, Peter; Nielsen, Peter E;

    2012-01-01

    Efficient intracellular delivery is essential for high activity of nucleic acids based therapeutics, including antisense agents. Several strategies have been developed and practically all rely on auxiliary transfection reagents such as cationic lipids, cationic polymers and cell penetrating...... peptides as complexing agents and carriers of the nucleic acids. However, uptake mechanisms remain rather poorly understood, and protocols always require optimization of transfection parameters. Considering that cationic transfection complexes bind to and thus may up-concentrate on the cell surface, we...... have now quantitatively compared the cellular activity (in the pLuc705 HeLa cell splice correction system) of PNA antisense oligomers using lipoplex delivery of cholesterol- and bisphosphonate-PNA conjugates, polyplex delivery via a PNA-polyethyleneimine conjugate and CPP delivery via a PNA...

  3. Cellular delivery and antisense effects of peptide nucleic acid conjugated to polyethyleneimine via disulfide linkers

    DEFF Research Database (Denmark)

    Berthold, Peter R; Shiraishi, Takehiko; Nielsen, Peter E

    2010-01-01

    Peptide nucleic acid (PNA) is potentially an attractive antisense and antigene agent for which more efficient cellular delivery systems are still warranted. The cationic polymer polyethylenimine (PEI) is commonly used for cellular transfection of DNA and RNA complexes, but is not readily applicable...... for PNA due to the (inherent) charge neutrality of PNA. However, PEI could function as an efficient scaffold for PNA via chemical conjugation. Accordingly, we modified PEI with the amine-reactive heterobifunctional linker agent N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP) (with and without a PEG...... moiety) and further reacted this with a cysteine PNA. The level of modification was determined spectrophotometrically with high accuracy, and the PNA transfection efficiency of the conjugates was evaluated in an antisense luciferase splice-correction assay using HeLa pLuc705 cells. We find that PEI...

  4. Electroporation-based delivery of cell-penetrating peptide conjugates of peptide nucleic acids for antisense inhibition of intracellular bacteria.

    Science.gov (United States)

    Ma, Sai; Schroeder, Betsy; Sun, Chen; Loufakis, Despina Nelie; Cao, Zhenning; Sriranganathan, Nammalwar; Lu, Chang

    2014-10-01

    Cell penetrating peptides (CPPs) have been used for a myriad of cellular delivery applications and were recently explored for delivery of antisense agents such as peptide nucleic acids (PNAs) for bacterial inhibition. Although these molecular systems (i.e. CPP-PNAs) have shown ability to inhibit growth of bacterial cultures in vitro, they show limited effectiveness in killing encapsulated intracellular bacteria in mammalian cells such as macrophages, presumably due to difficulty involved in the endosomal escape of the reagents. In this report, we show that electroporation delivery dramatically increases the bioavailability of CPP-PNAs to kill Salmonella enterica serovar Typhimurium LT2 inside macrophages. Electroporation delivers the molecules without involving endocytosis and greatly increases the antisense effect. The decrease in the average number of Salmonella per macrophage under a 1200 V cm(-1) and 5 ms pulse was a factor of 9 higher than that without electroporation (in an experiment with a multiplicity of infection of 2 : 1). Our results suggest that electroporation is an effective approach for a wide range of applications involving CPP-based delivery. The microfluidic format will allow convenient functional screening and testing of PNA-based reagents for antisense applications.

  5. Potent Antibacterial Antisense Peptide-Peptide Nucleic Acid Conjugates Against Pseudomonas aeruginosa

    DEFF Research Database (Denmark)

    Ghosal, Anubrata; Nielsen, Peter E

    2012-01-01

    Pseudomonas aeruginosa is an opportunistic pathogen causing severe infections in hospital settings, especially with immune compromised patients, and the increasing prevalence of multidrug resistant strains urges search for new drugs with novel mechanisms of action. In this study we introduce...... significantly reduced bacterial survival. These results open the possibility of development of antisense antibacterials for treatment of Pseudomonas infections....

  6. Improved cellular activity of antisense peptide nucleic acids by conjugation to a cationic peptide-lipid (CatLip) domain

    DEFF Research Database (Denmark)

    Koppelhus, Uffe; Shiraishi, Takehiko; Zachar, Vladimir;

    2008-01-01

    for future in vivo applications. We find that simply conjugating a lipid domain (fatty acid) to the cationic peptide (a CatLip conjugate) increases the biological effect of the corresponding PNA (CatLip) conjugates in a luciferase cellular antisense assay up to 2 orders of magnitude. The effect increases...

  7. Peptide nucleic acid (PNA) cell penetrating peptide (CPP) conjugates as carriers for cellular delivery of antisense oligomers

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2012-01-01

    splicing correction of the mutated luciferase gene in the HeLa pLuc705 cell line, reporting cellular (nuclear) uptake of the antisense PNA via luciferase activity measurement. Carrier CPP-PNA constructs were studied in terms of construct modification (with octaarginine and/or decanoic acid) and carrier PNA...... that the carrier might facilitate endosomal escape. Furthermore, 50% downregulation of luciferase expression at 60 nM siRNA was obtained using this carrier CPP-PNA delivery strategy (with CQ co-treatment) for a single stranded antisense RNA targeting normal luciferase mRNA. These results indicated that CPP...

  8. Improved cellular uptake of antisense Peptide nucleic acids by conjugation to a cell-penetrating Peptide and a lipid domain

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2011-01-01

    Unaided cellular uptake of RNA interference agents such as antisense oligonucleotides and siRNA is extremely poor, and in vivo bioavailability is also limited. Thus, effective delivery strategies for such potential drugs are in high demand. Recently, a novel approach using a class of short cationic...

  9. Comparative study on imaging of 99Tcm-survivin mRNA antisense peptide nucleic acid in tumor and inflammation animal models

    International Nuclear Information System (INIS)

    Objective: To investigate the value of 99Tcm labeled survivin mRNA antisense peptide nucleic acid (PNA) as an imaging agent in the specific diagnosis for carcinoma. Methods: Survivin mRNA antisense PNA was labeled directly with 99Tcm by the ligand-exchange method. Twenty nude mice with lung carcinoma A549 xenografts were randomly divided into 4 groups. Three groups were used for biodistribution study and one group was used for imaging study. Other twenty mice infected by staphylococcus aureus underwent the same procedure. The biodistribution and imaging of 99Tcm-survivin mRNA antisense PNA was studied at 1, 2 and 4 h respectively after the intravenous injection in nude mice bearing lung carcinoma A549 xenografts or inflammation models. SPSS 13.0 was used in the study and all data were analyzed by t test. Results: Biodistribution results showed that the highest radioactivity was found in the liver,and then in the kidney. Four hours after the administration of the imaging agent, the radioactivity ratios of target-to-non target (T/NT, tumor or inflammatory lesions to the contralateral regions) in tumor model group were significantly higher than those in inflammation model group (3.69 ± 1.13 vs 2.03 ± 0.47, t=3.01, P=0.02). Tumors were clearly visible in the tumor model groups at 0.5 h and still clearly seen at 4 h after the injection of antisense PNA. On the contrary,inflammatory lesions could not be seen clearly. Conclusion: 99Tcm labeled survivin mRNA antisense PNA can be used to distinguish tumor from inflammation and it may provide a new feasible method for specific tumor diagnosis. (authors)

  10. Peptide Nucleic Acid Synthons

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  11. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2003-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  12. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  13. Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    2002-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  14. Subnanomolar antisense activity of phosphonate-peptide nucleic acid (PNA) conjugates delivered by cationic lipids to HeLa cells

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Hamzavi, Ramin; Nielsen, Peter E

    2008-01-01

    as inferred from induced luciferase activity as a consequence of pre-mRNA splicing correction by the antisense-PNA. Antisense activity depended on the number of phosphonate moieties and the most potent hexa-bis-phosphonate-PNA showed at least 20-fold higher activity than that of an optimized PNA/DNA hetero...

  15. Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds known as peptide nucleic acids, bind complementary DNA and RNA strands, and generally do so more strongly than the corresponding DNA or RNA strands while exhibiting increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from...

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

    OpenAIRE

    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 ...

  17. Nucleic Acid Backbone Structure Variations: Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    Nielsen, Peter E.

    2010-01-01

    Synthetic analogues and mimics of the natural genetic material deoxyribonucleic acid (DNA) are potential gene therapeutic (antisense or antigene) drugs. One of these mimics, peptide nucleic acids (PNAs), are chemically closer to peptides and proteins than to DNA, but nonetheless have retained many...... of the structural properties of DNA. These molecules have found applications as probes in genetic diagnostics and are also being developed into antisense (RNA (ribonucleic acid) interference) gene therapeutic drugs, targeting selected genes through sequence-specific recognition of (messenger or micro......)RNA and in the future also antigene applications targeting the double-stranded DNA of the genes themselves leading to gene silencing or guiding specific gene repair. Finally, the special chemical and structural properties of PNA suggest that these or similar molecules might have played a role in the prebiotic origin...

  18. Inhibiting the growth of methicillin-resistant Staphylococcus aureus in vitro with antisense peptide nucleic acid conjugates targeting the ftsZ gene

    Directory of Open Access Journals (Sweden)

    Shumei Liang

    2015-01-01

    Conclusion: Our results demonstrate that the potent effects of PNAs on bacterial growth and cell viability were mediated by the down-regulation or even knock-out of ftsZ gene expression. This highlights the utility of ftsZ as a promising target for the development of new antisense antibacterial agents to treat MRSA infections.

  19. Peptide Nucleic Acids Complexes of Two Peptide Nucleic Acid Strands and One

    DEFF Research Database (Denmark)

    1999-01-01

    Peptide nucleic acids and analogues of peptide nucleic acids are used to form duplex, triplex, and other structures with nucleic acids and to modify nucleic acids. The peptide nucleic acids and analogues thereof also are used to modulate protein activity through, for example, transcription arrest...

  20. Histidine-Containing Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2000-01-01

    Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics.......Peptide nucleic acids containing histidine moieties are provided. These compounds have applications including diagnostics, research and potential therapeutics....

  1. Synthetic Procedures for Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2004-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  2. Double-Stranded Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    2001-01-01

    A novel class of compounds, known as peptide nucleic acids, form double-stranded structures with one another and with ssDNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker....

  3. Cleaving Double-Stranded DNA with Peptide Nucleic Acids

    DEFF Research Database (Denmark)

    1997-01-01

    Peptide nucleic acids and analogues of peptide nucleic acids are used to form duplex, triplex, and other structures with nucleic acids and to modify nucleic acids. The peptide nucleic acids and analogues thereof also are used to modulate protein activity through, for example, transcription arrest...

  4. Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA)

    DEFF Research Database (Denmark)

    Robaczewska, Magdalena; Narayan, Ramamurthy; Seigneres, Beatrice;

    2005-01-01

    BACKGROUND/AIMS: Peptide nucleic acids (PNAs) appear as promising new antisense agents, that have not yet been examined as hepatitis B virus (HBV) inhibitors. Our aim was to study the ability of PNAs targeting the duck HBV (DHBV) encapsidation signal epsilon to inhibit reverse transcription (RT...

  5. An introduction to peptide nucleic acid

    DEFF Research Database (Denmark)

    Nielsen, P E; Egholm, M

    1999-01-01

    Peptide Nucleic Acid (PNA) is a powerful new biomolecular tool with a wide range of important applications. PNA mimics the behaviour of DNA and binds complementary nucleic acid strands. The unique chemical, physical and biological properties of PNA have been exploited to produce powerful...

  6. Antibacterial Peptide Nucleic Acid-Antimicrobial Peptide (PNA-AMP) Conjugates

    DEFF Research Database (Denmark)

    Hansen, Anna Mette; Bonke, Gitte; Larsen, Camilla Josephine;

    2016-01-01

    Antisense peptide nucleic acid (PNA) oligomers constitute a novel class of potential antibiotics that inhibit bacterial growth via specific knockdown of essential gene expression. However, discovery of efficient, nontoxic delivery vehicles for such PNA oligomers has remained a challenge....... In the present study we show that antimicrobial peptides (AMPs) with an intracellular mode of action can be efficient vehicles for bacterial delivery of an antibacterial PNA targeting the essential acpP gene. The results demonstrate that buforin 2-A (BF2-A), drosocin, oncocin 10, Pep-1-K, KLW-9,13-a, (P59→W59...

  7. Screening of effective antisense peptide nucleic acids targeting gyrA from multidrug-resistant Acinetobacter baumannii and their antimicrobial effects in vitro%多重耐药鲍曼不动杆菌gyrA基因高效反义肽核酸序列筛选及其体外抗菌活性观察

    Institute of Scientific and Technical Information of China (English)

    王慧娟; 何云燕; 夏云; 王立朋; 梁树梅

    2013-01-01

    Objective To screen the effective antisense peptide nucleic acids targeting gyrA gene from multidrug-resistant Acinetobacter baumannii,and to evaluate their antimicrobial effects in vitro.Methods Two RNA folding computer programs,Mfold and RNA structure 4.6,were used to predict the secondary structure of gyrA mRNA,and then 10 antisense oligonucleotides were designed based on free energy theory.The full length of gyrA mRNA was transcribed in vitro and labeled by digoxigenin-ll-uridine-5'-triphosphate.Dot blothybridization was used to screen the gyrA mRNA accessible sites which showed strong binding affinity to the antisense oligonucleotides.Peptide nucleic acid (PNA) was synthesized based on the sequence of antisense oligonucleotide showing high affinity.Another PNA oligomer containing 6 mismatched nucleotides was used as a negative control.Both the 2 PNAs were conjugated to cell penetrating peptide (CPPs) (KFF)3 K to form peptide-PNA (PPNA).After the bacterial culture was treated with different concentrations of PPNA,OD600 and viable cell counts were measured to evaluate the growth inhibitory effect of the antisense oligonucleotide.Reverse transcript (RT)-PCR was applied to evaluate the level of gyrA expression.Results Of the 10 antisense oligonucleotides,5 showed binding affinity to gyrA mRNA and one of them showed strong binding affinity.PPNA designed based on the oligonucleotide significantly inhibited the growth of the bacterium and gyrA gene expression at a dose of 5 μmol/L,and exhibited anti-bactericidal effect at a dose of l0 μmol/L.Mismatched PPNA had no effect on the bacterial growth.Conclusion Combination of computer-aided prediction with dot blot hybridization is a high-flux and rapid way to screen effective antisense oligonucleotides in vitro.The screened anti-gyrA PPNA exerts significant inhibitory effect on the growth and gene expression in the bacterium in vitro.%目的 筛选出能与多重耐药鲍曼不动杆菌gyrA基因的mRNA紧密结合的

  8. Peptide Nucleic Acids Having Enhanced Binding Affinity and Sequence Specificity

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. Methods of increasing binding affinity and sequence specificity of peptide nucleic aci...

  9. Peptide Nucleic Acids Having Amino Acid Side Chains

    DEFF Research Database (Denmark)

    1998-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than the corresponding DNA or RNA strands, and exhibit increased sequence specificity and solubility. The peptide nucleic acids comprise ligands selected from a group consisting of nat...

  10. Peptide Nucleic Acids Having 2,6-Diaminopurine Nucleobases

    DEFF Research Database (Denmark)

    2003-01-01

    A novel class of compounds, known as peptide nucleic acids, bind complementary DNA and RNA strands more strongly than a corresponding DNA strand, and exhibit increased sequence specificity and binding affinity. The peptide nucleic acids of the invention comprise ligands selected from a group cons...

  11. Gene Targeting and Expression Modulation by Peptide Nucleic Acids (PNA)

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    Peptide nucleic acids (PNA) are artificial structural mimics of nucleic acids capable of sequence specific hybridization to both RNA and DNA. Thus they have obvious potential as gene targeting agents for drug discovery approaches. An overview with emphasis on recent progress on RNA "interference"...

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

    International Nuclear Information System (INIS)

    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)

  13. Ribonuclease H1-dependent hepatotoxicity caused by locked nucleic acid-modified gapmer antisense oligonucleotides.

    Science.gov (United States)

    Kasuya, Takeshi; Hori, Shin-Ichiro; Watanabe, Ayahisa; Nakajima, Mado; Gahara, Yoshinari; Rokushima, Masatomo; Yanagimoto, Toru; Kugimiya, Akira

    2016-01-01

    Gapmer antisense oligonucleotides cleave target RNA effectively in vivo, and is considered as promising therapeutics. Especially, gapmers modified with locked nucleic acid (LNA) shows potent knockdown activity; however, they also cause hepatotoxic side effects. For developing safe and effective gapmer drugs, a deeper understanding of the mechanisms of hepatotoxicity is required. Here, we investigated the cause of hepatotoxicity derived from LNA-modified gapmers. Chemical modification of gapmer's gap region completely suppressed both knockdown activity and hepatotoxicity, indicating that the root cause of hepatotoxicity is related to intracellular gapmer activity. Gene silencing of hepatic ribonuclease H1 (RNaseH1), which catalyses gapmer-mediated RNA knockdown, strongly supressed hepatotoxic effects. Small interfering RNA (siRNA)-mediated knockdown of a target mRNA did not result in any hepatotoxic effects, while the gapmer targeting the same position on mRNA as does the siRNA showed acute toxicity. Microarray analysis revealed that several pre-mRNAs containing a sequence similar to the gapmer target were also knocked down. These results suggest that hepatotoxicity of LNA gapmer is caused by RNAseH1 activity, presumably because of off-target cleavage of RNAs inside nuclei. PMID:27461380

  14. A Simple Three-Step Method for Design and Affinity Testing of New Antisense Peptides: An Example of Erythropoietin

    Directory of Open Access Journals (Sweden)

    Nikola Štambuk

    2014-05-01

    Full Text Available Antisense peptide technology is a valuable tool for deriving new biologically active molecules and performing peptide–receptor modulation. It is based on the fact that peptides specified by the complementary (antisense nucleotide sequences often bind to each other with a higher specificity and efficacy. We tested the validity of this concept on the example of human erythropoietin, a well-characterized and pharmacologically relevant hematopoietic growth factor. The purpose of the work was to present and test simple and efficient three-step procedure for the design of an antisense peptide targeting receptor-binding site of human erythropoietin. Firstly, we selected the carboxyl-terminal receptor binding region of the molecule (epitope as a template for the antisense peptide modeling; Secondly, we designed an antisense peptide using mRNA transcription of the epitope sequence in the 3'→5' direction and computational screening of potential paratope structures with BLAST; Thirdly, we evaluated sense–antisense (epitope–paratope peptide binding and affinity by means of fluorescence spectroscopy and microscale thermophoresis. Both methods showed similar Kd values of 850 and 816 µM, respectively. The advantages of the methods were: fast screening with a small quantity of the sample needed, and measurements done within the range of physicochemical parameters resembling physiological conditions. Antisense peptides targeting specific erythropoietin region(s could be used for the development of new immunochemical methods. Selected antisense peptides with optimal affinity are potential lead compounds for the development of novel diagnostic substances, biopharmaceuticals and vaccines.

  15. Peptide nucleic acids and their potential applications in biotechnology

    DEFF Research Database (Denmark)

    Buchardt, O.; Egholm, M.; Berg, R.H.;

    1993-01-01

    Peptide nucleic acids (PNAs) are novel DNA mimics in which the sugar-phosphate backbone has been replaced with a backbone based on amino acids1-3. PNAs exhibit sequence-specific binding to DNA and RNA with higher affinities and specificities than unmodified DNA. They,are resistant to nuclease...

  16. Biological activity and biotechnological aspects of peptide nucleic acid.

    Science.gov (United States)

    Lundin, Karin E; Good, Liam; Strömberg, Roger; Gräslund, Astrid; Smith, C I Edvard

    2006-01-01

    During the latest decades a number of different nucleic acid analogs containing natural nucleobases on a modified backbone have been synthesized. An example of this is peptide nucleic acid (PNA), a DNA mimic with a noncyclic peptide-like backbone, which was first synthesized in 1991. Owing to its flexible and neutral backbone PNA displays very good hybridization properties also at low-ion concentrations and has subsequently attracted large interest both in biotechnology and biomedicine. Numerous modifications have been made, which could be of value for particular settings. However, the original PNA does so far perform well in many diverse applications. The high biostability makes it interesting for in vivo use, although the very limited diffusion over lipid membranes requires further modifications in order to make it suitable for treatment in eukaryotic cells. The possibility to use this nucleic acid analog for gene regulation and gene editing is discussed. Peptide nucleic acid is now also used for specific genetic detection in a number of diagnostic techniques, as well as for site-specific labeling and hybridization of functional molecules to both DNA and RNA, areas that are also discussed in this chapter.

  17. Recent Advances in Chemical Modification of Peptide Nucleic Acids

    Directory of Open Access Journals (Sweden)

    Eriks Rozners

    2012-01-01

    Full Text Available Peptide nucleic acid (PNA has become an extremely powerful tool in chemistry and biology. Although PNA recognizes single-stranded nucleic acids with exceptionally high affinity and sequence selectivity, there is considerable ongoing effort to further improve properties of PNA for both fundamental science and practical applications. The present paper discusses selected recent studies that improve on cellular uptake and binding of PNA to double-stranded DNA and RNA. The focus is on chemical modifications of PNA's backbone and heterocyclic nucleobases. The paper selects representative recent studies and does not attempt to provide comprehensive coverage of the broad and vibrant field of PNA modification.

  18. Peptide nucleic acids arrest the growth of gastric cancer cells SGC7901

    Institute of Scientific and Technical Information of China (English)

    王宽; 张岂凡; 王锡山; 薛英威; 庞达; 傅松滨

    2004-01-01

    Background Peptide nucleic acid (PNA) has many characteristics useful in molecular biology. This paper described an effective way to raise the cell ingestion rate of PNA so as to kill gastric cancer cells.Methods Heteroduplexes of PNAs and oligonucleotides, wrapped by Lipofectamine 2000, were used to infect SGC7901 cells. The inhibitive effect of heteroduplexes was evaluated by analyzing cell clone forming and cell growth rate. Telomerase activity of SGC7901 cells was detected by polymerase chain reaction enzyme-linked immunosorbent assay (PCR-ELISA) and silver staining assay.Results PNAs showed a dose-dependent inhibition of cell proliferation. The percentage of proliferation inhibition was 99.4% after 7 days; the rate of cloning inhibition was 98.2% after 8 days;whereas for oligonucleotide groups, at the same concentration, the percentages were 50. 1% and 67. 5% respectively. Antisense PNA-DNA-Lipofectamine 2000 group (AP-D-L group) exhibited significantly different percentages from the control groups (P<0.05). The test result indicated that telomerase activity of the AP-D-L group was inhibited (P<0.05). At the same time, the impact on cell morphology was observed.Conclusions The results showed that PNAs are potent antisense reagents. The telomeraseassociated therapies are very promising for the treatment of malignant tumours.

  19. Synthesis and photoactivity of phenylazonaphthalene peptide nucleic acid monomers

    Institute of Scientific and Technical Information of China (English)

    Jin Du Li; Miao Chen; Sheng Liu; Hao Bo Zhang; Zhi Lu Liu

    2008-01-01

    Phenylazonaphthalene peptide nucleic acid (PNA) monomers were successfully synthesized,and their photoisomerization was examined.The new PNA monomers showed reversible trans-cis isomerization with UV and visible light irradiation,which might be the foundation of photo-regulating the hybridization between PNA containing phenylazonaphthalene unit and DNA.Simultaneously,the fluorescence of the new PNA monomers might make them especially useful as structural probes.

  20. Effects of Peptide Nucleic Acids against Ki-67 Gene on the Proliferation and Apoptosis of Human Renal Carcinoma Cell Line

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    To investigate the effects of anti-sense peptide nucleic acids (PNAs) targeting Ki-67gene on modulation of the proliferation and apoptosis of human renal carcinoma cell lines, human renal carcinoma cell line 786-0 cells were treated with anti-sense PNAs at different concentrations (1.0 μmol/L, 2.0 μmol/L, 10.0 μmol/L). The Ki-67 expression of 786-0 cells was detected by immunohistochemical technique and Western blot method respectively. The proliferation of 786-0 cells was studied by cell growth curves and 3H-thymidine incorporation. The apoptosis of 786-0 cells was detected by TUNEL assay. The control groups were treated with anti-sense oligonucleotide (ASODNs)targeting Ki-67 gene. Our results showed that the Ki-67 expression of 786-0 cells treated with anti-sense PNAs (16.9±0.7) was significantly inhibited as compared with that of the control groups (28.6±0.4) (P<0.01). The Ki-67 protein rate of 786-0 cells treated with anti-sense PNAs (42.1±2.2)was significantly reduced when compared with that of the control groups (83.6±1.4) (P<0.01). Proliferation of 786-0 cells treated with anti-sense PNAs (20.7±1.5) was significantly inhibited as compared with that of the control groups (58.6±1.4) (P<0.01). The apoptosis rate of 786-0 cells treated with anti-sense PNAs (28.7±2.3) was significantly increased higher compared with that of the control groups (13.8±1.0) (P<0.01). From these finds we are led to conclude that anti-sense PNAs targeting Ki-67 gene have stronger effects on the inhibition of the proliferation and induction of apoptosis of human renal carcinoma cells than ASODNs targeting Ki-67 gene. The strategies using anti-sense PNAs targeting Ki-67 gene may be a promising approach for the treatment of renal cell carcinoma.

  1. Purification of noncoding RNA and bound proteins using FLAG peptide-conjugated antisense-oligonucleotides.

    Science.gov (United States)

    Adachi, Shungo; Natsume, Tohru

    2015-01-01

    To understand the function of certain RNAs, including noncoding RNAs, it is important to identify the proteins that interact with the RNAs. Here we describe the method for purification of ribonucleoprotein (RNP) complexes composed of specific cellular RNAs by pull-down with FLAG peptide-conjugated antisense oligonucleotide (ASO). Using this method, we identified a novel protein component of U7 snRNP complex.

  2. Logic gates and antisense DNA devices operating on a translator nucleic Acid scaffold.

    Science.gov (United States)

    Shlyahovsky, Bella; Li, Yang; Lioubashevski, Oleg; Elbaz, Johann; Willner, Itamar

    2009-07-28

    A series of logic gates, "AND", "OR", and "XOR", are designed using a DNA scaffold that includes four "footholds" on which the logic operations are activated. Two of the footholds represent input-recognition strands, and these are blocked by complementary nucleic acids, whereas the other two footholds are blocked by nucleic acids that include the horseradish peroxidase (HRP)-mimicking DNAzyme sequence. The logic gates are activated by either nucleic acid inputs that hybridize to the respective "footholds", or by low-molecular-weight inputs (adenosine monophosphate or cocaine) that yield the respective aptamer-substrate complexes. This results in the respective translocation of the blocking nucleic acids to the footholds carrying the HRP-mimicking DNAzyme sequence, and the concomitant release of the respective DNAzyme. The released product-strands then self-assemble into the hemin/G-quadruplex-HRP-mimicking DNAzyme that biocatalyzes the formation of a colored product and provides an output signal for the different logic gates. The principle of the logic operation is, then, implemented as a possible paradigm for future nanomedicine. The nucleic acid inputs that bind to the blocked footholds result in the translocation of the blocking nucleic acids to the respective footholds carrying the antithrombin aptamer. The released aptamer inhibits, then, the hydrolytic activity of thrombin. The system demonstrates the regulation of a biocatalytic reaction by a translator system activated on a DNA scaffold. PMID:19507821

  3. DNA-like double helix formed by peptide nucleic acid

    DEFF Research Database (Denmark)

    Wittung, P; Nielsen, Peter E.; Buchardt, O;

    1994-01-01

    Although the importance of the nucleobases in the DNA double helix is well understood, the evolutionary significance of the deoxyribose phosphate backbone and the contribution of this chemical entity to the overall helical structure and stability of the double helix is not so clear. Peptide nucleic...... acid (PNA) is a DNA analogue with a backbone consisting of N-(2-aminoethyl)glycine units (Fig. 1) which has been shown to mimic DNA in forming Watson-Crick complementary duplexes with normal DNA. Using circular dichroism spectroscopy we show here that two complementary PNA strands can hybridize to one...

  4. Formulation of nucleic acid with pH-responsive amphipathic peptides for pulmonary delivery

    OpenAIRE

    Liang, Wanling; 梁婉玲

    2014-01-01

    Nucleic acids could be used as therapeutic agents for the treatment of many different diseases, but poor delivery limits their clinical application. A series of pH-responsive amphipathic peptides containing histidine or 2,3-diaminopropionic acid (Dap) derivatives, LAH and LADap peptides, were investigated in this study as nucleic acid carriers for the treatment of respiratory infectious disease. LAH and LADap peptides are cationic, amphipathic pH-responsive peptides. The major attractive ...

  5. Role of SbmA in the Uptake of Peptide Nucleic Acid (PNA)-Peptide Conjugates in E. coli

    DEFF Research Database (Denmark)

    Ghosal, Anubrata; Vitali, Ally; Stach, James E M;

    2013-01-01

    Antisense PNA oligomers targeting essential genes (acpP or ftsZ) and conjugated to the delivery peptide L((KFF)(3)K) show complete growth inhibition of wild type E. coli strain (MG1655) with submicromolar MIC. In this study we show that resistant mutants generated against such PNA-peptide conjuga...

  6. Synthesis of New Chrial Building Blocks for Novel Peptide Nucleic Acids

    Institute of Scientific and Technical Information of China (English)

    WU,Jie; XU,Xiao-Yu; LIU,Ke-Liang

    2003-01-01

    N-Boc protected amino acids of analogues of peptide nucleic acid (PNA),which are a class of conformationally constrained building blocks based on 4-aminoproline backbone with chirality at 2-c and 4-c,have been synthesized.Those monomers can be used for the construction of novel peptide nucleic acid analogues.

  7. Detection of target DNA using fluorescent cationic polymer and peptide nucleic acid probes on solid support

    Directory of Open Access Journals (Sweden)

    Leclerc Mario

    2005-04-01

    Full Text Available Abstract Background Nucleic acids detection using microarrays requires labelling of target nucleic acids with fluorophores or other reporter molecules prior to hybridization. Results Using surface-bound peptide nucleic acids (PNA probes and soluble fluorescent cationic polythiophenes, we show a simple and sensitive electrostatic approach to detect and identify unlabelled target nucleic acid on microarray. Conclusion This simple methodology opens exciting possibilities for applied genetic analysis for the diagnosis of infections, identification of genetic mutations, and forensic inquiries. This electrostatic strategy could also be used with other nucleic acid detection methods such as electrochemistry, silver staining, metallization, quantum dots, or electrochemical dyes.

  8. Affinity capture of (Arg sup 8 )vasopressin-receptor complex using immobilized antisense peptide

    Energy Technology Data Exchange (ETDEWEB)

    Feng Xian Lu; Aiyar, N.; Chaiken, I. (SmithKline Beecham, King of Prussia, PA (United States))

    1991-05-01

    Solubilized noncovalent complexes of (Arg{sup 8})-vasopressin (AVP) with receptor proteins from rat liver membranes were isolated by selective binding to silica-immobilized antisense (AS) peptide. The affinity chromatographic support was prepared with a chemically synthesized AS peptide whose sequence is encoded by the AS DNA corresponding to the 20 amino-terminal residues of the AVP bovine neurophysin II biosynthetic precursor (pro-AVP/BNPII-(20-1)), region that includes the AVP sequence at residues 1-9. The AS peptide-AVP interaction mechanism hypothesized, contact by hydropathic complementarity at multiple sites along the peptide chains, led to the prediction that AVP bound to its receptor would still have enough free surface to interact with immobilized AS peptide. To test this prediction of a three-way interaction, ({sup 3}H)AVP-receptor was obtained as a solubilized, partially purified fraction from rat liver membrane. Covalently crosslinked ({sup 3}H)AVP complex also was bound to the AS peptide column; binding was blocked by competition with unlabeled AVP in the elution buffer. Since the AVP-linked 31- and 38-kDa proteins have the same apparent molecular mass on SDS/PAGE as found previously by photoaffinity labeling, the authors conclude that the AS peptide column has affinity-captured AVP-receptor complexes. The 15-kDa protein appears to be an active AVP-receptor fragment of one or both of the larger proteins. It is generally concluded that immobilized AS peptides may be useful to isolate peptide and protein receptor complexes in other systems as well.

  9. Efficient gene silencing by delivery of locked nucleic acid antisense oligonucleotides, unassisted by transfection reagents.

    Science.gov (United States)

    Stein, C A; Hansen, J Bo; Lai, Johnathan; Wu, SiJian; Voskresenskiy, Anatoliy; Høg, Anja; Worm, Jesper; Hedtjärn, Maj; Souleimanian, Naira; Miller, Paul; Soifer, Harris S; Castanotto, Daniella; Benimetskaya, Luba; Ørum, Henrik; Koch, Troels

    2010-01-01

    For the past 15-20 years, the intracellular delivery and silencing activity of oligodeoxynucleotides have been essentially completely dependent on the use of a delivery technology (e.g. lipofection). We have developed a method (called 'gymnosis') that does not require the use of any transfection reagent or any additives to serum whatsoever, but rather takes advantage of the normal growth properties of cells in tissue culture in order to promote productive oligonucleotide uptake. This robust method permits the sequence-specific silencing of multiple targets in a large number of cell types in tissue culture, both at the protein and mRNA level, at concentrations in the low micromolar range. Optimum results were obtained with locked nucleic acid (LNA) phosphorothioate gap-mers. By appropriate manipulation of oligonucleotide dosing, this silencing can be continuously maintained with little or no toxicity for >240 days. High levels of oligonucleotide in the cell nucleus are not a requirement for gene silencing, contrary to long accepted dogma. In addition, gymnotic delivery can efficiently deliver oligonucleotides to suspension cells that are known to be very difficult to transfect. Finally, the pattern of gene silencing of in vitro gymnotically delivered oligonucleotides correlates particularly well with in vivo silencing. The establishment of this link is of particular significance to those in the academic research and drug discovery and development communities.

  10. Peptide nucleic acid: a new artificial biomacromolecular with great potential applications in molecular biology and biomedicine

    Institute of Scientific and Technical Information of China (English)

    WANG Jin-ke; LU Zu-hong; HE Nong-yue

    2001-01-01

    @@ Peptide nucleic acid (PNA) is a DNA mimic that was originally developed by Peter E Nielsen in 1991 as a reagent for sequence-specific recognition of double stranded (ds) DNA by a conventional triple helix type principle.

  11. Peptide nucleic acid (PNA) binding-mediated gene regulation

    Institute of Scientific and Technical Information of China (English)

    2004-01-01

    Peptide nucleic acids (PNAs) are synthetic oligonucleotides with chemically modified backbones. PNAs can bind to both DNA and RNA targets in a sequence-specific manner to form PNA/DNA and PNA/RNA duplex structures. When bound to double-stranded DNA (dsDNA) targets, the PNA molecule replaces one DNA strand in the duplex by strand invasion to form a PNA/DNA/PNA [or (PNA)2/DNA] triplex structure and the displaced DNA strand exists as a singlestranded D-loop. PNA has been used in many studies as research tools for gene regulation and gene targeting. The Dloops generated from the PNA binding have also been demonstrated for its potential in initiating transcription and inducing gene expression. PNA provides a powerful tool to study the mechanism of transcription and an innovative strategy to regulate target gene expression. An understanding of the PNA-mediated gene regulation will have important clinical implications in treatment of many human diseases including genetic, cancerous, and age-related diseases.

  12. 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-high-density......The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled...... the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half...

  13. Dual Toxic-Peptide-Coding Staphylococcus aureus RNA under Antisense Regulation Targets Host Cells and Bacterial Rivals Unequally

    Directory of Open Access Journals (Sweden)

    Marie-Laure Pinel-Marie

    2014-04-01

    Full Text Available Produced from the pathogenicity islands of Staphylococcus aureus clinical isolates, stable SprG1 RNA encodes two peptides from a single internal reading frame. These two peptides accumulate at the membrane, and inducing their expression triggers S. aureus death. Replacement of the two initiation codons by termination signals reverses this toxicity. During growth, cis-antisense RNA SprF1 is expressed, preventing mortality by reducing SprG1 RNA and peptide levels. The peptides are secreted extracellularly, where they lyse human host erythrocytes, a process performed more efficiently by the longer peptide. The two peptides also inactivate Gram-negative and -positive bacteria, with the shorter peptide more effective against S. aureus rivals. Two peptides are secreted from an individual RNA containing two functional initiation codons. Thus, we present an unconventional type I toxin-antitoxin system expressed from a human pathogen producing two hemolytic and antibacterial peptides from a dual-coding RNA, negatively regulated by a dual-acting antisense RNA.

  14. 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

    Full Text Available BACKGROUND: 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. METHODOLOGY/PRINCIPAL FINDINGS: 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. CONCLUSION/SIGNIFICANCE: 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

  15. Nucleic Acid-Peptide Complex Phase Controlled by DNA Hybridization

    Science.gov (United States)

    Vieregg, Jeffrey; Lueckheide, Michael; Leon, Lorraine; Marciel, Amanda; Tirrell, Matthew

    When polyanions and polycations are mixed, counterion release drives formation of polymer-rich complexes that can either be solid (precipitates) or liquid (coacervates) depending on the properties of the polyelectrolytes. These complexes are important in many fields, from encapsulation of industrial polymers to membrane-free segregation of biomolecules such as nucleic acids and proteins. Condensation of long double-stranded DNA has been studied for several decades, but comparatively little attention has been paid to the polyelectrolyte behavior of oligonucleotides. We report here studies of DNA oligonucleotides (10 - 88 nt) complexed with polylysine (10 - 100 aa). Unexpectedly, we find that the phase of the resulting complexes is controlled by the hybridization state of the nucleic acid, with double-stranded DNA forming precipitates and single-stranded DNA forming coacervates. Stability increases with polyelectrolyte length and decreases with solution salt concentration, with complexes of the longer double-stranded polymers undergoing precipitate/coacervate/soluble transitions as ionic strength is increased. Mixing coacervates formed by complementary single-stranded oligonucleotides results in precipitate formation, raising the possibility of stimulus-responsive material design.

  16. Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD).

    Science.gov (United States)

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

    Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most promising methods for restoration of dystrophin expression. This approach has been tested extensively targeting different exons in numerous models both in vitro and in vivo. During the past 10 years, there has been a considerable progress by using DMD animal models involving three types of antisense oligonucleotides (2'-O-methyl phosphorothioate (2OME-PS), phosphorodiamidate morpholino oligomer (PMO)) and peptide nucleic acid (PNA). PMID:21686247

  17. Structural diversity of target-specific homopyrimidine peptide nucleic acid–dsDNA complexes

    OpenAIRE

    Bentin, Thomas; Hansen, Georg I.; Nielsen, Peter E.

    2006-01-01

    Sequence-selective recognition of double-stranded (ds) DNA by homopyrimidine peptide nucleic acid (PNA) oligomers can occur by major groove triplex binding or by helix invasion via triplex P-loop formation. We have compared the binding of a decamer, a dodecamer and a pentadecamer thymine–cytosine homopyrimidine PNA oligomer to a sequence complementary homopurine target in duplex DNA using gel-shift and chemical probing analyses. We find that all three PNAs form stable triplex invasion complex...

  18. Recognition of Double Stranded RNA by Guanidine-Modified Peptide Nucleic Acids (GPNA)

    OpenAIRE

    Gupta, Pankaj; Muse, Oluwatoyosi; Rozners, Eriks

    2011-01-01

    Double helical RNA has become an attractive target for molecular recognition because many non-coding RNAs play important roles in control of gene expression. Recently, we discovered that short peptide nucleic acids (PNA) bind strongly and sequence selectively to a homopurine tract of double helical RNA via triple helix formation. Herein we tested if the molecular recognition of RNA can be enhanced by α-guanidine modification of PNA. Our study was motivated by the discovery of Ly and co-worker...

  19. Question 1: Peptide Nucleic Acids and the Origin and Homochirality of Life

    Science.gov (United States)

    Nielsen, Peter E.

    2007-10-01

    The possibilities of pseudo peptide DNA mimics like PNA (peptide nucleic acid) having a role for the prebiotic origin of life prior to an RNA world is discussed. In particular a scenario is proposed in which protocells with an achiral genetic material through several generations stepwise is converted into a chiral genetic material, e.g., by incorporation of RNA units. Provided that a sufficiently large sequence space is occupied, a selection process based on catalytic function in which a single cell (first common ancestor) has a definite evolutionary advantage, selection of this cell would by contingency also lock it into homochirality.

  20. Question 1: Peptide nucleic acids and the origin and homochirality of life.

    Science.gov (United States)

    Nielsen, Peter E

    2007-10-01

    The possibilities of pseudo peptide DNA mimics like PNA (peptide nucleic acid) having a role for the prebiotic origin of life prior to an RNA world is discussed. In particular a scenario is proposed in which protocells with an achiral genetic material through several generations stepwise is converted into a chiral genetic material, e.g., by incorporation of RNA units. Provided that a sufficiently large sequence space is occupied, a selection process based on catalytic function in which a single cell (first common ancestor) has a definite evolutionary advantage, selection of this cell would by contingency also lock it into homochirality.

  1. Construction of Prokaryotic Expressing Vector of Antisense Nucleic Acid of LasR and Its Effect on the Virulence of Pseudomonas Aeruginosus

    Institute of Scientific and Technical Information of China (English)

    ZHANG Ling; ZHOU Junli; LI Jingming; LIAO Fang

    2007-01-01

    To construct a pUCP18/lasRantisense plasmid carrying the reversed gene and analyze its effect on the virulence of Pseudomonas aeruginosus, LasR gene was amplified from the genome of Pseudomonas aeruginosus by PCR and reversely recombined with plasmid pUCP18. The recombinant pUCP18/lasRantisense was verified by enzyme digestion, PCR and sequencing. The biological effects of pUCP18/lasRantisense were examined by using RT-PCR, NAD method and the assay of pyocyanin. Our results showed that the expected full length lasR fragment (721 bp) was extended from Pseudomonas aeruginosus gene with PCR. And it is consistent with LasR gene of Pseudomonas aeruginosa in GenBank (No. NC_002516). The recombinant plasmid was successfully constructed and transferred into Pseudomonas aeruginosus. The antisense nucleic acid of LasR gene could reduce the virulence of Pseudomonas aeruginosus and might serve as a new target site for treatment purpose.

  2. Enhanced cellular delivery of cell-penetrating peptide-peptide nucleic acid conjugates by photochemical internalization

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Nielsen, Peter E

    2011-01-01

    )) or tetraphenylporphyrin tetrasulfonic acid (TPPS). Cellular uptake of the PNA conjugates were evaluated by using a sensitive cellular method with HeLa pLuc705 cells based on the splicing correction of luciferase gene by targeting antisense oligonucleotides to a cryptic splice site of the mutated luciferase gene....... The cellular efficacy of CPP conjugates were evaluated by measuring luciferase activity as a result of splicing correction and was also confirmed by RT-PCR analysis of luciferase pre-mRNA....

  3. Gene-Specific Effects of Antisense Phosphorodiamidate Morpholino Oligomer-Peptide Conjugates on Escherichia coli and Salmonella enterica Serovar Typhimurium in Pure Culture and in Tissue Culture

    OpenAIRE

    Lucas D Tilley; Hine, Orion S.; Kellogg, Jill A.; Hassinger, Jed N.; Weller, Dwight D.; Iversen, Patrick L.; Geller, Bruce L.

    2006-01-01

    The objective was to improve efficacy of antisense phosphorodiamidate morpholino oligomers (PMOs) by improving their uptake into bacterial cells. Four different bacterium-permeating peptides, RFFRFFRFFXB, RTRTRFLRRTXB, RXXRXXRXXB, and KFFKFFKFFKXB (X is 6-aminohexanoic acid and B is β-alanine), were separately coupled to two different PMOs that are complementary to regions near the start codons of a luciferase reporter gene (luc) and a gene required for viability (acpP). Luc peptide-PMOs targ...

  4. Preparation of surfactant-stabilized gold nanoparticle-peptide nucleic acid conjugates

    Energy Technology Data Exchange (ETDEWEB)

    Duy, Janice, E-mail: janice.duy@umit.maine.ed [University of Maine, Graduate School of Biomedical Sciences (United States); Connell, Laurie B. [University of Maine, School of Marine Sciences (United States); Eck, Wolfgang [University of Heidelberg, Applied Physical Chemistry (Germany); Collins, Scott D. [University of Maine, Department of Chemistry (United States); Smith, Rosemary L. [University of Maine, Electrical and Computer Engineering Department (United States)

    2010-09-15

    A simple, two-step method of producing stable and functional peptide nucleic acid (PNA)-conjugated gold nanoparticles using a surfactant stabilization step is presented. PNA are DNA analogs with superior chemical stability and target discrimination, but their use in metallic nanoparticle systems has been limited by the difficulty of producing stable colloids of nanoparticle-PNA conjugates. In this work, the nonionic surfactant Tween 20 (polyoxyethylene (20) sorbitan monolaurate) was used to sterically shield gold surfaces prior to the addition of thiolated PNA, producing conjugates which remain dispersed in solution and retain the ability to hybridize to complementary nucleic acid sequences. The conjugates were characterized using transmission electron microscopy, dynamic light scattering, and UV-visible absorbance spectroscopy. PNA attachment to gold nanoparticles was confirmed with an enzyme-linked immunoassay, while the ability of nanoparticle-bound PNA to hybridize to its complement was demonstrated using labeled DNA.

  5. A Peptide & Peptide Nucleic Acid Synthesis Technology for Transporter Molecules and Theranostics - The SPPS

    OpenAIRE

    Pipkorn, Ruediger; Braun, Klaus; Wiessler, Manfred; Waldeck, Waldemar; Schrenk, Hans-Hermann; Koch, Mario; Semmler, Wolfhard; Komljenovic, Dorde

    2014-01-01

    Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and functi...

  6. A peptide & peptide nucleic acid synthesis technology for transporter molecules and theranostics--the SPPS.

    Science.gov (United States)

    Pipkorn, Ruediger; Braun, Klaus; Wiessler, Manfred; Waldeck, Waldemar; Schrenk, Hans-Hermann; Koch, Mario; Semmler, Wolfhard; Komljenovic, Dorde

    2014-01-01

    Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and function of proteins is widely investigated, especially by using computer-assisted methods. Nowadays the solid phase synthesis (SPPS) chemistry emerges as a key technology and is considered as a promising methodology to design peptides for the investigation of molecular pharmacological processes at the transcriptional level. SPPS syntheses could be carried out in core facilities producing peptides for large-scale scientific implementations as presented here. PMID:24843319

  7. A Peptide & Peptide Nucleic Acid Synthesis Technology for Transporter Molecules and Theranostics - The SPPS

    Science.gov (United States)

    Pipkorn, Ruediger; Braun, Klaus; Wiessler, Manfred; Waldeck, Waldemar; Schrenk, Hans-Hermann; Koch, Mario; Semmler, Wolfhard; Komljenovic, Dorde

    2014-01-01

    Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and function of proteins is widely investigated, especially by using computer-assisted methods. Nowadays the solid phase synthesis (SPPS) chemistry emerges as a key technology and is considered as a promising methodology to design peptides for the investigation of molecular pharmacological processes at the transcriptional level. SPPS syntheses could be carried out in core facilities producing peptides for large-scale scientific implementations as presented here. PMID:24843319

  8. Electrostatic Binding and Hydrophobic Collapse of Peptide-Nucleic Acid Aggregates Quantified Using Force Spectroscopy

    CERN Document Server

    Camunas-Soler, Joan; Bizarro, Cristiano V; de Loreno, Sara; Fuentes-Perez, Maria Eugenia; Ramsch, Roland; Vilchez, Susana; Solans, Conxita; Moreno-Herrero, Fernando; Albericio, Fernando; Eritja, Ramon; Giralt, Ernest; Dev, Sukhendu B; Ritort, Felix

    2014-01-01

    Knowledge of the mechanisms of interaction between self-aggregating peptides and nucleic acids or other polyanions is key to the understanding of many aggregation processes underlying several human diseases (e.g. Alzheimer's and Parkinson's diseases). Determining the affinity and kinetic steps of such interactions is challenging due to the competition between hydrophobic self-aggregating forces and electrostatic binding forces. Kahalalide F (KF) is an anticancer hydrophobic peptide which contains a single positive charge that confers strong aggregative properties with polyanions. This makes KF an ideal model to elucidate the mechanisms by which self-aggregation competes with binding to a strongly charged polyelectrolyte such as DNA. We use optical tweezers to apply mechanical forces to single DNA molecules and show that KF and DNA interact in a two-step kinetic process promoted by the electrostatic binding of DNA to the aggregate surface followed by the stabilization of the complex due to hydrophobic interact...

  9. In situ synthesis of peptide nucleic acids in porous silicon for drug delivery and biosensing.

    Science.gov (United States)

    Beavers, Kelsey R; Mares, Jeremy W; Swartz, Caleb M; Zhao, Yiliang; Weiss, Sharon M; Duvall, Craig L

    2014-07-16

    Peptide nucleic acids (PNA) are a unique class of synthetic molecules that have a peptide backbone and can hybridize with nucleic acids. Here, a versatile method has been developed for the automated, in situ synthesis of PNA from a porous silicon (PSi) substrate for applications in gene therapy and biosensing. Nondestructive optical measurements were performed to monitor single base additions of PNA initiated from (3-aminopropyl)triethoxysilane attached to the surface of PSi films, and mass spectrometry was conducted to verify synthesis of the desired sequence. Comparison of in situ synthesis to postsynthesis surface conjugation of the full PNA molecules showed that surface mediated, in situ PNA synthesis increased loading 8-fold. For therapeutic proof-of-concept, controlled PNA release from PSi films was characterized in phosphate buffered saline, and PSi nanoparticles fabricated from PSi films containing in situ grown PNA complementary to micro-RNA (miR) 122 generated significant anti-miR activity in a Huh7 psiCHECK-miR122 cell line. The applicability of this platform for biosensing was also demonstrated using optical measurements that indicated selective hybridization of complementary DNA target molecules to PNA synthesized in situ on PSi films. These collective data confirm that we have established a novel PNA-PSi platform with broad utility in drug delivery and biosensing.

  10. Cellular uptake of antisense oligonucleotides after complexing or conjugation with cell-penetrating model peptides.

    Science.gov (United States)

    Oehlke, J; Birth, P; Klauschenz, E; Wiesner, B; Beyermann, M; Oksche, A; Bienert, M

    2002-08-01

    The uptake by mammalian cells of phosphorothioate oligonucleotides was compared with that of their respective complexes or conjugates with cationic, cell-penetrating model peptides of varying helix-forming propensity and amphipathicity. An HPLC-based protocol for the synthesis and purification of disulfide bridged conjugates in the 10-100 nmol range was developed. Confocal laser scanning microscopy (CLSM) in combination with gel-capillary electrophoresis and laser induced fluorescence detection (GCE-LIF) revealed cytoplasmic and nuclear accumulationin all cases. The uptake differences between naked oligonucleotides and their respective peptide complexes or conjugates were generally confined to one order of magnitude. No significant influence of the structural properties of the peptide components upon cellular uptake was found. Our results question the common belief that the increased biological activity of oligonucleotides after derivatization with membrane permeable peptides may be primarily due to improved membrane translocation.

  11. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Eysturskard, Jonhard; Nielsen, Peter E

    2010-01-01

    ABSTRACT: BACKGROUND: Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human...... cancer gene in JAR cells. METHODS: We screened 10 different 15 mer PNAs targeting intron2 at both the 5;- and the 3;-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3;-splice site of intron3 with a complementarity of 4 bases to intron......3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT). RESULTS: We show that several of these PNAs effectively...

  12. 肽核酸在分子生物学技术中的应用%Application of Peptide Nucleic Acid in Molecular Biotechnology

    Institute of Scientific and Technical Information of China (English)

    王建华; 郭泽琴

    2013-01-01

    Peptide nucleic acid ( PNA) is a nucleic acid mimic in which the deoxyribose phosphate backbone has been replaced by a pseudo-peptide polymer to which the nucleobases are linked. PNA forms complexes with DNA following the Watson-Crick base-pairing rules. The PNA-DNA complexes exhibited high thermal stability and mismatch sensitivity. The high stability of these hybrids has been explained by the absence of negative charges along the PNA backbone. Furthermore, PNA is resistant to biological degradation and can bind complementary RNA or DNA sequences with extraordinary high affinity and specificity. PNA possesses many of the properties desired for a good antisense agent or antigene drugs, such as the detection of a single base mutate in nucleic acids, the diagnosis and detection by PCR molecular beacon, quantitative analysis of fluorescence in situ hybridization, genechip and biosensor technology and so on. Thus, the above-mentioned applications of PNA in molecular biotechnology in recent years and the prospect of its application were summarized.%肽核酸(PNA)作为一种人工合成的核酸类似物,以中性的肽链酰胺2-氨基乙基甘氨酸键取代了DNA中的戊糖磷酸二酯键骨架,其余部分与DNA相同.PNA可通过Watson-Crick碱基配对的形式识别并结合DNA或RNA序列,形成稳定的双螺旋结构.与传统的DNA或RNA相比,PNA具有生物学稳定性高、杂交特异性强、杂合体的稳定性高和杂交速度快等明显优点,使PNA具有良好的物理化学性质和生物学特性,在检测目的核酸序列中单碱基突变、PCR基因分子诊断与检测、荧光原位杂交定量分析、基因芯片和生物传感器技术等调控水平和临床应用上有自己的特点.简要综述了近年来肽核酸在上述分子生物学技术中的运用以及应用前景的展望.

  13. Synthesis and optical properties of pyrrolidinyl peptide nucleic acid carrying a clicked Nile red label

    Directory of Open Access Journals (Sweden)

    Nattawut Yotapan

    2014-09-01

    Full Text Available DNA or its analogues with an environment-sensitive fluorescent label are potentially useful as a probe for studying the structure and dynamics of nucleic acids. In this work, pyrrolidinyl peptide nucleic acid (acpcPNA was labeled at its backbone with Nile red, a solvatochromic benzophenoxazine dye, by means of click chemistry. The optical properties of the Nile red-labeled acpcPNA were investigated by UV–vis and fluorescence spectroscopy in the absence and in the presence of DNA. In contrast to the usual quenching observed in Nile red-labeled DNA, the hybridization with DNA resulted in blue shifting and an enhanced fluorescence regardless of the neighboring bases. More pronounced blue shifts and fluorescence enhancements were observed when the DNA target carried a base insertion in close proximity to the Nile red label. The results indicate that the Nile red label is located in a more hydrophobic environment in acpcPNA–DNA duplexes than in the single-stranded acpcPNA. The different fluorescence properties of the acpcPNA hybrids of complementary DNA and DNA carrying a base insertion are suggestive of different interactions between the Nile red label and the duplexes.

  14. Label-free potentiometry for detecting DNA hybridization using peptide nucleic acid and DNA probes.

    Science.gov (United States)

    Goda, Tatsuro; Singi, Ankit Balram; Maeda, Yasuhiro; Matsumoto, Akira; Torimura, Masaki; Aoki, Hiroshi; Miyahara, Yuji

    2013-02-07

    Peptide nucleic acid (PNA) has outstanding affinity over DNA for complementary nucleic acid sequences by forming a PNA-DNA heterodimer upon hybridization via Watson-Crick base-pairing. To verify whether PNA probes on an electrode surface enhance sensitivity for potentiometric DNA detection or not, we conducted a comparative study on the hybridization of PNA and DNA probes on the surface of a 10-channel gold electrodes microarray. Changes in the charge density as a result of hybridization at the solution/electrode interface on the self-assembled monolayer (SAM)-formed microelectrodes were directly transformed into potentiometric signals using a high input impedance electrometer. The charge readout allows label-free, reagent-less, and multi-parallel detection of target oligonucleotides without any optical assistance. The differences in the probe lengths between 15- to 22-mer dramatically influenced on the sensitivity of the PNA and DNA sensors. Molecular type of the capturing probe did not affect the degree of potential shift. Theoretical model for charged rod-like duplex using the Gouy-Chapman equation indicates the dominant effect of electrostatic attractive forces between anionic DNA and underlying electrode at the electrolyte/electrode interface in the potentiometry.

  15. Disrupting protein expression with Peptide Nucleic Acids reduces infection by obligate intracellular Rickettsia.

    Science.gov (United States)

    Pelc, Rebecca S; McClure, Jennifer C; Kaur, Simran J; Sears, Khandra T; Rahman, M Sayeedur; Ceraul, Shane M

    2015-01-01

    Peptide Nucleic Acids (PNAs) are single-stranded synthetic nucleic acids with a pseudopeptide backbone in lieu of the phosphodiester linked sugar and phosphate found in traditional oligos. PNA designed complementary to the bacterial Shine-Dalgarno or start codon regions of mRNA disrupts translation resulting in the transient reduction in protein expression. This study examines the use of PNA technology to interrupt protein expression in obligate intracellular Rickettsia sp. Their historically intractable genetic system limits characterization of protein function. We designed PNA targeting mRNA for rOmpB from Rickettsia typhi and rickA from Rickettsia montanensis, ubiquitous factors important for infection. Using an in vitro translation system and competitive binding assays, we determined that our PNAs bind target regions. Electroporation of R. typhi and R. montanensis with PNA specific to rOmpB and rickA, respectively, reduced the bacteria's ability to infect host cells. These studies open the possibility of using PNA to suppress protein synthesis in obligate intracellular bacteria.

  16. Evidence for extensive non-endocytotic translocation of peptide nucleic acids across mammalian plasma membranes.

    Science.gov (United States)

    Oehlke, Johannes; Turner, Yvonne; Pritz, Stephan; Bienert, Michael

    2011-09-01

    The ability of peptide nucleic acids (PNA) to enter and to cross filter-grown MDCK, HEK and CHO cells was studied by means of a protocol based on capillary electrophoresis combined with laser-induced fluorescence detection. The used approach avoided possible errors encountered in protocols based on confocal laserscanning microscopy and FACS analysis. In contradiction to the commonly anticipated unability of PNA to cross biomembranes, extensive translocation of unmodified PNA into and across the investigated cell types was found. The transport mode comprised a variety of energy dependent and -independent as well as temperature sensitive mechanisms being probably destined to natural substrates and hijacked by PNA. The presented results suggest active as well as passive export mechanisms rather than poor penetration into cells to be responsible for the only weak biological activity of unmodified PNA.

  17. DNA detection using water-soluble conjugated polymers and peptide nucleic acid probes

    Science.gov (United States)

    Gaylord, Brent S.; Heeger, Alan J.; Bazan, Guillermo C.

    2002-08-01

    The light-harvesting properties of cationic conjugated polymers are used to sensitize the emission of a dye on a specific peptide nucleic acid (PNA) sequence for the purpose of homogeneous, "real-time" DNA detection. Signal transduction is controlled by hybridization of the neutral PNA probe and the negative DNA target. Electrostatic interactions bring the hybrid complex and cationic polymer within distances required for Förster energy transfer. Conjugated polymer excitation provides fluorescein emission >25 times higher than that obtained by exciting the dye, allowing detection of target DNA at concentrations of 10 pM with a standard fluorometer. A simple and highly sensitive assay with optical amplification that uses the improved hybridization behavior of PNA/DNA complexes is thus demonstrated.

  18. Targeted gene correction using psoralen, chlorambucil and camptothecin conjugates of triplex forming peptide nucleic acid (PNA)

    DEFF Research Database (Denmark)

    Birkedal, Henrik; Nielsen, Peter E

    2011-01-01

    Gene correction activation effects of a small series of triplex forming peptide nucleic acid (PNA) covalently conjugated to the DNA interacting ligands psoralen, chlorambucil and camptothecin targeted proximal to a stop codon mutation in an EGFP reporter gene were studied. A 15-mer homopyrimidine...... interstrand crosslinked adducts with dsDNA dramatically decreased the frequency of targeted repair/correction. The PNA conjugates were also studied in mammalian cell lines upon transfection of PNA bound EGFP reporter vector and scoring repair of the EGFP gene by FACS analysis of functional EGFP expression...... suggest that simple triplex forming PNAs have little effect on proximal gene correctional events whereas PNA conjugates capable of forming DNA adducts and interstrand crosslinks are strong inhibitors. Most interestingly the PNA conjugated to the topoisomerase inhibitor, camptothecin enhanced repair...

  19. Sequence-selective targeting of duplex DNA by peptide nucleic acids

    DEFF Research Database (Denmark)

    Nielsen, Peter E

    2010-01-01

    Sequence-selective gene targeting constitutes an attractive drug-discovery approach for genetic therapy, with the aim of reducing or enhancing the activity of specific genes at the transcriptional level, or as part of a methodology for targeted gene repair. The pseudopeptide DNA mimic peptide...... nucleic acid (PNA) can recognize duplex DNA with high sequence specificity and affinity in triplex, duplex and double-duplex invasive modes or non-invasive triplex modes. Novel PNA modification has improved the affinity for DNA recognition via duplex invasion, double-duplex invasion and triplex...... recognition considerably. Such modifications have also resulted in new approaches to targeted gene repair and sequence-selective double-strand cleavage of genomic DNA....

  20. Locked nucleic acid

    DEFF Research Database (Denmark)

    Jepsen, Jan Stenvang; Sørensen, Mads D; Wengel, Jesper;

    2004-01-01

    Locked nucleic acid (LNA) is a class of nucleic acid analogs possessing very high affinity and excellent specificity toward complementary DNA and RNA, and LNA oligonucleotides have been applied as antisense molecules both in vitro and in vivo. In this review, we briefly describe the basic...

  1. The Prebiotic Synthesis of Ethylenediamine Monoacetic Acid, The Repeating Unit of Peptide Nucleic Acids

    Science.gov (United States)

    Nelson, Kevin E.; Miller, Stanley L.

    1992-01-01

    The polymerization of ribonucleic acids or their precursors constitutes an important event in prebiotic chemistry. The various problems using ribonucleotides to make RNA suggest that there may have been a precursor. An attractive possibility are the peptide nucleic acids (PNA). PNAs are nucleotide analogs that make use of a polymer of ethylenediamine monoacetic acid (EDMA or 2-amninoethyl glycine) with the bases attached by an acetic acid. EDMA is an especially attractive alternative to the ribose phosphate or deoxyribose phosphate backbone because it contains no chiral centers and is potentially prebiotic, but there is no reported prebiotic synthesis. We have synthesized both EDMA and ethylenediamine diacetic acid (EDDA) from the prebiotic compounds ethylenediamine, formaldehyde, and hydrogen cyanide. The yields of EDMA range from 11 to 79% along with some sEDDA and uEDDA. These reactions work with concentrations of 10(exp -1)M and as low as 10(exp -4)M, and the reaction is likely to be effective at even lower concentrations. Ethylenediamine is a likely prebiotic compound, but it has not yet been demonstrated, although compounds such as ethanolamine and cysteamine have been proven to be prebiotic. Under neutral pH and heating at l00 C, EDMA is converted to the lactam, monoketopiperazine (MKP). The cyclization occurs and has an approximate ratio of MKP/EDMA = 3 at equilibrium. We have measured the solubilities of EDMA center dot H20 as 6.4 m, EDMA center dot HCl center dot H20 as 13.7 m, and EDMA center dot 2HCl center dot H20 as 3.4 m. These syntheses together with the high solubility of EDMA suggest that EDMA would concentrate in drying lagoons and might efficiently form polymers. Given the instability of ribose and the poor polymerizability of nucleotides, the prebiotic presence of EDMA and the possibility of its polymerization raises the possibility that PNAs are the progenitors of present day nucleic acids. A pre-RNA world may have existed in which PNAs or

  2. Development of an antisense RNA delivery system using conjugates of the MS2 bacteriophage capsids and HIV-1 TAT cell-penetrating peptide.

    Science.gov (United States)

    Wei, Baojun; Wei, Yuxiang; Zhang, Kuo; Wang, Jing; Xu, Ruihuan; Zhan, Sien; Lin, Guigao; Wang, Wei; Liu, Min; Wang, Lunan; Zhang, Rui; Li, Jinming

    2009-05-01

    RNA-based therapeutic strategies are used widely due to their highly specific mode of action. However, the major obstacle in any RNA-based therapy is cellular delivery and stability in the cells. The self-assembly of the MS2 bacteriophage capsids has been used to develop virus-like particles (VLPs) for drug delivery. In this study, we utilized the heterobifunctional crosslinker, sulfosuccinimidyl-4-(p-maleimidophenyl)-butyrate (sulfo-SMPB), to conjugate the human immunodeficiency virus-1 (HIV-1) Tat peptide and MS2 VLPs; the antisense RNA against the 5'-untranslated region (UTR) and the internal ribosome entry site (IRES) of the hepatitis C virus (HCV) was packaged into these particles by using a two-plasmid coexpression system. The MS2 VLPs conjugated with the Tat peptide were then transferred into Huh-7 cells containing an HCV reporter system. The packaged antisense RNA showed an inhibitory effect on the translation of HCV. This paper describes our initial results with this system using the Tat peptide. PMID:18823738

  3. THE APPLICATION OF PEPTIDE NUCLEIC ACID PROBES FOR RAPID DETECTION AND ENUMERATION OF EUBACTERIA, STAPHYLOCOCCUS AUREUS AND PSEUDOMONAS AERUGINOSA IN RECREATIONAL BEACHES OF S. FLORIDA. (R828830)

    Science.gov (United States)

    A novel chemiluminescent in situ hybridization technique using peptide nucleic acids (PNA) was adapted for the detection of bacteria in beach sand and recreational waters in South Florida. The simultaneous detection and enumeration of eubacteria and the novel indicators, S...

  4. Visualization of the mycelia of wood-rotting fungi by fluorescence in situ hybridization using a peptide nucleic acid probe.

    Science.gov (United States)

    Nakada, Yuji; Nakaba, Satoshi; Matsunaga, Hiroshi; Funada, Ryo; Yoshida, Makoto

    2013-01-01

    White rot fungus, Phanerochaete chrysosporium, and brown rot fungus, Postia placenta, grown on agar plates, were visualized by fluorescence in situ hybridization (FISH) using a peptide nucleic acid (PNA) probe. Mycelia grown on wood chips were also clearly detected by PNA-FISH following blocking treatment. To the best of our knowledge, this is the first report on the visualization of fungi in wood by FISH. PMID:23391931

  5. Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids

    OpenAIRE

    Kim, Ki-Hyun; Nielsen, Peter E.; Glazer, Peter M.

    2007-01-01

    Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable ...

  6. Using Triple Helix Forming Peptide Nucleic Acids for Sequence-selective Recognition of Double-stranded RNA

    OpenAIRE

    Hnedzko, Dziyana; Cheruiyot, Samwel K.; Rozners, Eriks

    2014-01-01

    Non-coding RNAs play important roles in regulation of gene expression. Specific recognition and inhibition of these biologically important RNAs that form complex double-helical structures will be highly useful for fundamental studies in biology and practical applications in medicine. This protocol describes a strategy developed in our laboratory for sequence-selective recognition of double-stranded RNA (dsRNA) using triple helix forming peptide nucleic acids (PNAs) that bind in the major grov...

  7. Fluorescence in situ hybridization method using a peptide nucleic acid probe for identification of Lactobacillus spp. in milk samples

    OpenAIRE

    Machado, António; Almeida, Carina; Carvalho, Ana; Boyen, Filip; Haesebrouck, Freddy; L. R. Rodrigues; Cerca, Nuno; Azevedo, N. F.

    2013-01-01

    Lactobacillus species constitute one of the dominant and beneficial bacteria in our body and are used in developed countries as a microbial adjuvant. Identification of these probiotic bacteria is traditionally performed by culture-based techniques. However, such methods are very time-consuming and can give inaccurate results, especially when Lactobacillus is present in mixed bacterial complex communities. Our study aimed to accurately identify Lactobacillus spp. using a novel Peptide Nucleic ...

  8. Hybrid polymeric hydrogels via peptide nucleic acid (PNA)/DNA complexation.

    Science.gov (United States)

    Chu, Te-Wei; Feng, Jiayue; Yang, Jiyuan; Kopeček, Jindřich

    2015-12-28

    This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications.

  9. Hybrid polymeric hydrogels via peptide nucleic acid (PNA)/DNA complexation.

    Science.gov (United States)

    Chu, Te-Wei; Feng, Jiayue; Yang, Jiyuan; Kopeček, Jindřich

    2015-12-28

    This work presents a new concept in hybrid hydrogel design. Synthetic water-soluble N-(2-hydroxypropyl)methacrylamide (HPMA) polymers grafted with multiple peptide nucleic acids (PNAs) are crosslinked upon addition of the linker DNA. The self-assembly is mediated by the PNA-DNA complexation, which results in the formation of hydrophilic polymer networks. We show that the hydrogels can be produced through two different types of complexations. Type I hydrogel is formed via the PNA/DNA double-helix hybridization. Type II hydrogel utilizes a unique "P-form" oligonucleotide triple-helix that comprises two PNA sequences and one DNA. Microrheology studies confirm the respective gelation processes and disclose a higher critical gelation concentration for the type I gel when compared to the type II design. Scanning electron microscopy reveals the interconnected microporous structure of both types of hydrogels. Type I double-helix hydrogel exhibits larger pore sizes than type II triple-helix gel. The latter apparently contains denser structure and displays greater elasticity as well. The designed hybrid hydrogels have potential as novel biomaterials for pharmaceutical and biomedical applications. PMID:26394062

  10. A Sensitive Peptide Nucleic Acid Probe Assay for Detection of BRAF V600 Mutations in Melanoma.

    Science.gov (United States)

    Chen, Tai-Long; Chang, John Wen-Cheng; Hsieh, Jia-Juan; Cheng, Hsin-Yi; Chiou, Chiuan-Chian

    Mutated v-Raf murine sarcoma viral oncogene homolog B (BRAF) is an important biomarker for the prediction of therapeutic efficacy of several anticancer drugs. The detection of BRAF mutation faces two challenges: Firstly, there are multiple types of mutations, and secondly, tumor samples usually contain various amounts of wild-type, normal tissues. Here, we describe a newly established method for sensitive detection of multiple types of BRAF V600 mutations in excess wild-type background. The method introduced a fluorophore-tagged peptide nucleic acid (PNA) to serve as both polymerase chain reaction (PCR) clamp and sensor probe, which inhibited the amplification of wild-type templates during PCR and revealed multiple types of mutant signals during melting analysis. We demonstrated the design and optimization process of the method, and applied it in the detection of BRAF mutations in 49 melanoma samples. This PNA probe assay method detected three types of mutations in 17 samples, and was much more sensitive than conventional PCR plus Sanger sequencing. PMID:27566656

  11. Application of peptide nucleic acids containing azobenzene self-assembled electrochemical biosensors in detecting DNA sequences

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Hybridization of peptide nucleic acids probe containing azobenzene (NH2-TNT4, N-PNAs) with DNA was performed by covalently immobilizing of NH2-TNT4 in sequence on the 3-mercaptopropionic acid self-assembled monolayer modified gold electrode with the helps of N-(3-dimethylaminopropy1)-N’-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS), and the hybrid was coded as N-PNAs/DNA. Using [Fe(CN)6]4-/3- (1:1) as the electrochemical indicator, the electrochemical properties of the N-PNAs self-assembled monolayer (N-PNAs-SAMs) and N-PNAs/DNA hybridization system under the conditions of before and after UV light irradiation were characterized with cyclic voltammetry (CV), differential pulse voltammetry (DPV), and electrochemical impedance spectra (EIS). Results showed that the redox currents decreased with the increase of irradiation time, suggesting that the ability of the charge transfer on the electrode surface was weakened and the conformation of hybrid system had been changed, and the control of PNAs/DNA hybridization could be realized by UV light irradiation.

  12. Modulation of mdm2 pre-mRNA splicing by 9-aminoacridine-PNA (peptide nucleic acid) conjugates targeting intron-exon junctions

    International Nuclear Information System (INIS)

    Modulation of pre-mRNA splicing by antisense molecules is a promising mechanism of action for gene therapeutic drugs. In this study, we have examined the potential of peptide nucleic acid (PNA) 9-aminoacridine conjugates to modulate the pre-mRNA splicing of the mdm2 human cancer gene in JAR cells. We screened 10 different 15 mer PNAs targeting intron2 at both the 5' - and the 3'-splice site for their effects on the splicing of mdm2 using RT-PCR analysis. We also tested a PNA (2512) targeting the 3'-splice site of intron3 with a complementarity of 4 bases to intron3 and 11 bases to exon4 for its splicing modulation effect. This PNA2512 was further tested for the effects on the mdm2 protein level as well as for inhibition of cell growth in combination with the DNA damaging agent camptothecin (CPT). We show that several of these PNAs effectively inhibit the splicing thereby producing a larger mRNA still containing intron2, while skipping of exon3 was not observed by any of these PNAs. The most effective PNA (PNA2406) targeting the 3'-splice site of intron2 had a complementarity of 4 bases to intron2 and 11 bases to exon3. PNA (2512) targeting the 3'-splice site of intron3 induced both splicing inhibition (intron3 skipping) and skipping of exon4. Furthermore, treatment of JAR cells with this PNA resulted in a reduction in the level of MDM2 protein and a concomitant increase in the level of tumor suppressor p53. In addition, a combination of this PNA with CPT inhibited cell growth more than CPT alone. We have identified several PNAs targeting the 5'- or 3'-splice sites in intron2 or the 3'-splice site of intron3 of mdm2 pre-mRNA which can inhibit splicing. Antisense targeting of splice junctions of mdm2 pre-mRNA may be a powerful method to evaluate the cellular function of MDM2 splice variants as well as a promising approach for discovery of mdm2 targeted anticancer drugs

  13. Efficiency of cellular delivery of antisense peptide nucleic acid by electroporation depends on charge and electroporation geometry

    DEFF Research Database (Denmark)

    Joergensen, Mette; Agerholm-Larsen, Birgit; Nielsen, Peter E;

    2011-01-01

    Electroporation is potentially a very powerful technique for both in vitro cellular and in vivo drug delivery, particularly relating to oligonucleotides and their analogs for genetic therapy. Using a sensitive and quantitative HeLa cell luciferase RNA interference mRNA splice correction assay wit......, polymerase chain reaction, and confocal microscopy. In conclusion, we have found that the charge of PNA and electroporation system combination greatly influences the transfer efficiency, thereby illustrating the complexity of the electroporation mechanism....

  14. Cationic liposomes loaded with pro-apoptotic peptide D-(KLAKLAK)2 and bcl-2 antisense oligodeoxynucleotide G3139 for enhanced anti-cancer therapy

    Science.gov (United States)

    Ko, Young Tag; Falcao, Claudio; Torchilin, Vladimir P.

    2009-01-01

    The treatment of cancer using macromolecular therapeutics such as oligonucleotides or peptides requires efficient delivery systems capable of intracellular penetration and may also benefit from use of a combination of therapeutics with different mechanisms of action. With this possibility in mind, we constructed cationic liposome loaded with the proapoptotic peptide, D-(KLAKLAK)2 and the bcl-2 antisense oligodeoxynucleotide, G3139, and determined whether the combination of the proapoptotic macromolecules in a single cationic liposome can enhance antitumor efficacy. Advantage was taken of alternating charge interaction to entrap macromolecules of opposite charge. The polycationic pepetide D-(KLAKLAK)2 was first condensed with the polyanionic oligodeoxynucleotide G3139 to obtain overall negatively charged peptide/oligodeoxynucleotide complexes. The complexes were then entrapped into DOTAP/DOPE cationic liposomes (CL). This sequential charge interaction ensured efficient entrapment of D-(KLAKLAK)2 and G3139 with a high loading efficiency (50 %) and capacity (7.5 wt%). In vitro treatment of mouse melanoma B16(F10) with CL loaded with D-(KLAKLAK)2/G3139 led to significantly enhanced antitumor efficacy, mediated by stimulated induction of apoptotic (Caspase 3/7) activity, when compared to CL loaded with G3139 alone. Intratumoral injection of CL loaded with D-(KLAKLAK)2/G3139 in B16(F10) mice xenograft also led to suppressed tumor growth associated with enhanced apoptotic activity. Thus, the combination of proapoptotic peptide D-(KLAKLAK)2 and antisense oligonucleotide G3139 in a cationic liposome led to enhanced apoptotic/antitumor efficacy and may provide a promising tool for cancer treatment. PMID:19317442

  15. Novel approaches to tumor imaging in mice: pre targeting with radiolabeled peptide nucleic acid

    International Nuclear Information System (INIS)

    Full text.Since targeting of tumour by conventional methods is not consistently favorable, we have considered pre targeting with separate administrations of anti tumour antibody and radiolabel. As an alternative to streptavidin and biotin for this application, we earlier considered single stranded peptide nucleic acid (PNA) bound to an irrelevant protein administered first and allowed to diffuse non specifically into tumour. This was followed later by the administration of 99 m Tc labeled complementary PNA. We now report on the first studies with PNA conjugated anti tumour antibody to allow specific binding. PNA was conjugated to the NRLU-10 IgG antibody while the complementary PNA (amine derivatized) was labeled with ((m Tc using MAG3. LS174T tumour-bearing nude mice received IV 200 ug of the PNA-antibody conjugate and 20 h later, received IV 100 ug (130 uCl) of 99m Tc- complementary PNA. Animals were imaged and sacrificed 5 h later. Because of rapid clearance, at sacrifice all tissue levels of 99 m Tc were low, the highest being kidneys at about 4%ID/gm. Tumour uptake was 0.55%ID/gm for the study animals vs. 0. 13 for controls and tumour/muscle ratios were 9.8 vs. 3.6 respectively. These values represent a 2.5-fold improvement in localization over the nonspecific study. The whole body images also reflected the superior targeting of study vs. control animals. We conclude that single-stranded PNAs should be a useful alternative to streptavidin and biotin for pre targeting studies

  16. Immobilization-free electrochemical DNA detection with anthraquinone-labeled pyrrolidinyl peptide nucleic acid probe.

    Science.gov (United States)

    Kongpeth, Jutatip; Jampasa, Sakda; Chaumpluk, Piyasak; Chailapakul, Orawon; Vilaivan, Tirayut

    2016-01-01

    Electrochemical detection provides a simple, rapid, sensitive and inexpensive method for DNA detection. In traditional electrochemical DNA biosensors, the probe is immobilized onto the electrode. Hybridization with the DNA target causes a change in electrochemical signal, either from the intrinsic signal of the probe/target or through a label or a redox indicator. The major drawback of this approach is the requirement for probe immobilization in a controlled fashion. In this research, we take the advantage of different electrostatic properties between PNA and DNA to develop an immobilization-free approach for highly sequence-specific electrochemical DNA sensing on a screen-printed carbon electrode (SPCE) using a square-wave voltammetric (SWV) technique. Anthraquinone-labeled pyrrolidinyl peptide nucleic acid (AQ-PNA) was employed as a probe together with an SPCE that was modified with a positively-charged polymer (poly quaternized-(dimethylamino-ethyl)methacrylate, PQDMAEMA). The electrostatic attraction between the negatively-charged PNA-DNA duplex and the positively-charged modified SPCE attributes to the higher signal of PNA-DNA duplex than that of the electrostatically neutral PNA probe, resulting in a signal change. The calibration curve of this proposed method exhibited a linear range between 0.35 and 50 nM of DNA target with a limit of detection of 0.13 nM (3SD(blank)/Slope). The sub-nanomolar detection limit together with a small sample volume required (20 μL) allowed detection of DNA. With the high specificity of the pyrrolidinyl PNA probe used, excellent discrimination between complementary and various single-mismatched DNA targets was obtained. An application of this new platform for a sensitive and specific detection of isothermally-amplified shrimp's white spot syndrome virus (WSSV) DNA was successfully demonstrated.

  17. Light-emitting self-assembled peptide nucleic acids exhibit both stacking interactions and Watson-Crick base pairing

    Science.gov (United States)

    Berger, Or; Adler-Abramovich, Lihi; Levy-Sakin, Michal; Grunwald, Assaf; Liebes-Peer, Yael; Bachar, Mor; Buzhansky, Ludmila; Mossou, Estelle; Forsyth, V. Trevor; Schwartz, Tal; Ebenstein, Yuval; Frolow, Felix; Shimon, Linda J. W.; Patolsky, Fernando; Gazit, Ehud

    2015-05-01

    The two main branches of bionanotechnology involve the self-assembly of either peptides or DNA. Peptide scaffolds offer chemical versatility, architectural flexibility and structural complexity, but they lack the precise base pairing and molecular recognition available with nucleic acid assemblies. Here, inspired by the ability of aromatic dipeptides to form ordered nanostructures with unique physical properties, we explore the assembly of peptide nucleic acids (PNAs), which are short DNA mimics that have an amide backbone. All 16 combinations of the very short di-PNA building blocks were synthesized and assayed for their ability to self-associate. Only three guanine-containing di-PNAs—CG, GC and GG—could form ordered assemblies, as observed by electron microscopy, and these di-PNAs efficiently assembled into discrete architectures within a few minutes. The X-ray crystal structure of the GC di-PNA showed the occurrence of both stacking interactions and Watson-Crick base pairing. The assemblies were also found to exhibit optical properties including voltage-dependent electroluminescence and wide-range excitation-dependent fluorescence in the visible region.

  18. Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells

    OpenAIRE

    Fang, Huafeng; Yue, Xuan; Li, Xiaoxu; Taylor, John-Stephen

    2005-01-01

    We have recently shown that an MCF-7 tumor can be imaged in a mouse by PET with 64Cu-labeled Peptide nucleic acids (PNAs) tethered to the permeation peptide Lys4 that recognize the uniquely overexpressed and very abundant upstream of N-ras or N-ras related gene (unr mRNA) expressed in these cells. Herein we describe how the high affinity antisense PNAs to the unr mRNA were identified and characterized. First, antisense binding sites on the unr mRNA were mapped by an reverse transcriptase rand...

  19. Biophysical and morphological studies on the dual interaction of non-octarepeat prion protein peptides with copper and nucleic acids.

    Science.gov (United States)

    Chaves, Juliana A P; Sanchez-López, Carolina; Gomes, Mariana P B; Sisnande, Tháyna; Macedo, Bruno; de Oliveira, Vanessa End; Braga, Carolina A C; Rangel, Luciana P; Silva, Jerson L; Quintanar, Liliana; Cordeiro, Yraima

    2014-08-01

    Conversion of prion protein (PrP) to an altered conformer, the scrapie PrP (PrP(Sc)), is a critical step in the development of transmissible spongiform encephalopathies. Both Cu(II) and nucleic acid molecules have been implicated in this conversion. Full-length PrP can bind up to six copper ions; four Cu(II) binding sites are located in the octarepeat domain (residues 60-91), and His-96 and His-111 coordinate two additional copper ions. Experimental evidence shows that PrP binds different molecules, resulting in diverse cellular signaling events. However, there is little information about the interaction of macromolecular ligands with Cu(II)-bound PrP. Both RNA and DNA sequences can bind PrP, and this interaction results in reciprocal conformational changes. Here, we investigated the interaction of Cu(II) and nucleic acids with amyloidogenic non-octarepeat PrP peptide models (comprising human PrP residues 106-126 and hamster PrP residues 109-149) that retain His-111 as the copper-anchoring residue. The effect of Cu(II) and DNA or RNA sequences in the aggregation, conformation, and toxicity of PrP domains was investigated at low and neutral pH. Circular dichroism and EPR spectroscopy data indicate that interaction of the PrP peptides with Cu(II) and DNA occurs at pH 7. This dual interaction induces conformational changes in the peptides, modulating their aggregation, and affecting the morphology of the aggregated species, resulting in different cytotoxic effects. These results provide new insights into the role of Cu(II) and nucleic acid sequences in the structural conversion and aggregation of PrP, which are both critical events related to prion pathogenesis.

  20. Short Peptide Nucleic Acids Bind Strongly to Homopurine Tract of Double Helical RNA at pH 5.5

    OpenAIRE

    Li, Ming; Zengeya, Thomas; Rozners, Eriks

    2010-01-01

    The important role that non-coding RNA plays in cell biology makes it an attractive target for molecular recognition. However, the discovery of small molecules that bind double helical RNA selectively and may serve as biochemical probes and potential drug leads has been relatively slow. Herein, we show that peptide nucleic acids, as short as six nucleobases, bind very strongly (Ka > 107) and sequence selectively to a homopurine tract of double helical RNA at pH 5.5. The isothermal titration c...

  1. Self-assembled multicompartment liquid crystalline lipid carriers for protein, peptide, and nucleic acid drug delivery.

    Science.gov (United States)

    Angelova, Angelina; Angelov, Borislav; Mutafchieva, Rada; Lesieur, Sylviane; Couvreur, Patrick

    2011-02-15

    Lipids and lipopolymers self-assembled into biocompatible nano- and mesostructured functional materials offer many potential applications in medicine and diagnostics. In this Account, we demonstrate how high-resolution structural investigations of bicontinuous cubic templates made from lyotropic thermosensitive liquid-crystalline (LC) materials have initiated the development of innovative lipidopolymeric self-assembled nanocarriers. Such structures have tunable nanochannel sizes, morphologies, and hierarchical inner organizations and provide potential vehicles for the predictable loading and release of therapeutic proteins, peptides, or nucleic acids. This Account shows that structural studies of swelling of bicontinuous cubic lipid/water phases are essential for overcoming the nanoscale constraints for encapsulation of large therapeutic molecules in multicompartment lipid carriers. For the systems described here, we have employed time-resolved small-angle X-ray scattering (SAXS) and high-resolution freeze-fracture electronic microscopy (FF-EM) to study the morphology and the dynamic topological transitions of these nanostructured multicomponent amphiphilic assemblies. Quasi-elastic light scattering and circular dichroism spectroscopy can provide additional information at the nanoscale about the behavior of lipid/protein self-assemblies under conditions that approximate physiological hydration. We wanted to generalize these findings to control the stability and the hydration of the water nanochannels in liquid-crystalline lipid nanovehicles and confine therapeutic biomolecules within these structures. Therefore we analyzed the influence of amphiphilic and soluble additives (e.g. poly(ethylene glycol)monooleate (MO-PEG), octyl glucoside (OG), proteins) on the nanochannels' size in a diamond (D)-type bicontinuous cubic phase of the lipid glycerol monooleate (MO). At body temperature, we can stabilize long-living swollen states, corresponding to a diamond cubic phase

  2. Efficient in vitro inhibition of HIV-1 gag reverse transcription by peptide nucleic acid (PNA) at minimal ratios of PNA/RNA

    DEFF Research Database (Denmark)

    Koppelhus, Uffe; Zachar, Vladimir; Nielsen, P.E.;

    1997-01-01

    We have tested the inhibitory potential of peptide nucleic acid (PNA) on in vitro reverse transcription of the HIV-1 gag gene. PNA was designed to target different regions of the HIV-1 gag gene and the effect on reverse transcription by HIV-1, MMLV and AMV reverse transcriptases (RTs) was investi......We have tested the inhibitory potential of peptide nucleic acid (PNA) on in vitro reverse transcription of the HIV-1 gag gene. PNA was designed to target different regions of the HIV-1 gag gene and the effect on reverse transcription by HIV-1, MMLV and AMV reverse transcriptases (RTs...

  3. 正义-反义多肽的相互作用%The Interaction between Sense and Antisense Peptides

    Institute of Scientific and Technical Information of China (English)

    吕凤林; 朱锡华

    1999-01-01

    @@ 遗传学中心法则指出,DNA有义链(即正义链,sense RNA strand)可转录为单链mRNA,其核苷酸序列决定蛋白质的氨基酸序列.虽然DNA无义链(即反义DNA链,antisense DNA strand)能以框转录方式转录成反义mRNA,甚至能以框翻译方式翻译成蛋白质,但在正常情况下,反义DNA在细胞中不表达具有功能意义的蛋白质.反义RNA能与mRNA特异地互补结合,从而抑制该mRNA的加工与翻译.

  4. Polyanionic Carboxyethyl Peptide Nucleic Acids (ce-PNAs: Synthesis and DNA Binding.

    Directory of Open Access Journals (Sweden)

    Yuliya Kirillova

    Full Text Available New polyanionic modifications of polyamide nucleic acid mimics were obtained. Thymine decamers were synthesized from respective chiral α- and γ-monomers, and their enantiomeric purity was assessed. Here, we present the decamer synthesis, purification and characterization by MALDI-TOF mass spectrometry and an investigation of the hybridization properties of the decamers. We show that the modified γ-S-carboxyethyl-T10 PNA forms a stable triplex with polyadenine DNA.

  5. Targeted correction of a thalassemia-associated beta-globin mutation induced by pseudo-complementary peptide nucleic acids

    DEFF Research Database (Denmark)

    Lonkar, Pallavi; Kim, Ki-Hyun; Kuan, Jean Y;

    2009-01-01

    Beta-thalassemia is a genetic disorder caused by mutations in the beta-globin gene. Triplex-forming oligonucleotides and triplex-forming peptide nucleic acids (PNAs) have been shown to stimulate recombination in mammalian cells via site-specific binding and creation of altered helical structures...... that provoke DNA repair. However, the use of these molecules for gene targeting requires homopurine tracts to facilitate triple helix formation. Alternatively, to achieve binding to mixed-sequence target sites for the induced gene correction, we have used pseudo-complementary PNAs (pcPNAs). Due to steric...... hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences via double duplex-invasion complexes. We demonstrate here that pcPNAs, when co-transfected with donor DNA fragments, can promote single base pair modification at the start of the second intron of the beta...

  6. Peptide Nucleic Acid with a Lysine Side Chain at the β-Position: Synthesis and Application for DNA Cleavage.

    Science.gov (United States)

    Sugiyama, Toru; Kuwata, Keiko; Imamura, Yasutada; Demizu, Yosuke; Kurihara, Masaaki; Takano, Masashi; Kittaka, Atsushi

    2016-01-01

    This paper reports the synthesis of new β-Lys peptide nucleic acid (PNA) monomers and their incorporation into a 10-residue PNA sequence. PNA containing β-Lys PNA units formed a stable hybrid duplex with DNA. However, incorporation of β-Lys PNA units caused destabilization of PNA-DNA duplexes to some extent. Electrostatic attractions between β-PNA and DNA could reduce this destabilization effect. Subsequently, bipyridine-conjugated β-Lys PNA was prepared and exhibited sequence selective cleavage of DNA. Based on the structures of the cleavage products and molecular modeling, we reasoned that bipyridine moiety locates within the minor groove of the PNA-DNA duplexes. The lysine side chain of β-PNA is a versatile handle for attaching various functional molecules. PMID:27373637

  7. 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...

  8. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

    OpenAIRE

    Chin, Joanna Y; Kuan, Jean Y.; Lonkar, Pallavi S.; Krause, Diane S.; Seidman, Michael M.; Peterson, Kenneth R.; Nielsen, Peter E.; Kole, Ryszard; Glazer, Peter M.

    2008-01-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the hu...

  9. Improved Synthesis Strategy for Peptide Nucleic Acids (PNA) appropriate for Cell-specific Fluorescence Imaging

    Science.gov (United States)

    Pipkorn, Rüdiger; Wiessler, Manfred; Waldeck, Waldemar; Hennrich, Ute; Nokihara, Kiyoshi; Beining, Marcel; Braun, Klaus

    2012-01-01

    Progress in genomics and proteomics attended to the door for better understanding the recent rapid expanding complex research field of metabolomics. This trend in biomedical research increasingly focuses to the development of patient-specific therapeutic approaches with higher efficiency and sustainability. Simultaneously undesired adverse reactions are avoided. In parallel, the development of molecules for molecular imaging is required not only for the imaging of morphological structures but also for the imaging of metabolic processes like the aberrant expression of the cysteine protease cathepsin B (CtsB) gene and the activity of the resulting product associated with metastasis and invasiveness of malign tumors. Finally the objective is to merge imaging and therapy at the same level. The design of molecules which fulfil these responsibilities is pivotal and requires proper chemical methodologies. In this context our modified solid phase peptide chemistry using temperature shifts during synthesis is considered as an appropriate technology. We generated highly variable conjugates which consist of molecules useful as diagnostically and therapeutically active molecules. As an example the modular PNA products with the complementary sequence to the CtsB mRNA and additionally with a cathepsin B cleavage site had been prepared as functional modules for distinction of cell lines with different CtsB gene expression. After ligation to the modular peptide-based BioShuttle carrier, which was utilized to facilitate the delivery of the functional modules into the cells' cytoplasm, the modules were scrutinized. PMID:22211082

  10. Improved Synthesis Strategy for Peptide Nucleic Acids (PNA appropriate for Cell-specific Fluorescence Imaging

    Directory of Open Access Journals (Sweden)

    Rüdiger Pipkorn, Manfred Wiessler, Waldemar Waldeck, Ute Hennrich, Kiyoshi Nokihara, Marcel Beining, Klaus Braun

    2012-01-01

    Full Text Available Progress in genomics and proteomics attended to the door for better understanding the recent rapid expanding complex research field of metabolomics. This trend in biomedical research increasingly focuses to the development of patient-specific therapeutic approaches with higher efficiency and sustainability. Simultaneously undesired adverse reactions are avoided. In parallel, the development of molecules for molecular imaging is required not only for the imaging of morphological structures but also for the imaging of metabolic processes like the aberrant expression of the cysteine protease cathepsin B (CtsB gene and the activity of the resulting product associated with metastasis and invasiveness of malign tumors. Finally the objective is to merge imaging and therapy at the same level. The design of molecules which fulfil these responsibilities is pivotal and requires proper chemical methodologies. In this context our modified solid phase peptide chemistry using temperature shifts during synthesis is considered as an appropriate technology. We generated highly variable conjugates which consist of molecules useful as diagnostically and therapeutically active molecules. As an example the modular PNA products with the complementary sequence to the CtsB mRNA and additionally with a cathepsin B cleavage site had been prepared as functional modules for distinction of cell lines with different CtsB gene expression. After ligation to the modular peptide-based BioShuttle carrier, which was utilized to facilitate the delivery of the functional modules into the cells' cytoplasm, the modules were scrutinized.

  11. Peptide nucleic acid fluorescence in situ hybridization for identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii.

    Science.gov (United States)

    Zhang, Xiaofeng; Wu, Shan; Li, Ke; Shuai, Jiangbing; Dong, Qiang; Fang, Weihuan

    2012-07-01

    A fluorescent in situ hybridization (FISH) method in conjunction with fluorescin-labeled peptide nucleic acid (PNA) probes (PNA-FISH) for detection of Listeria species was developed. In silico analysis showed that three PNA probes Lis-16S-1, Lm-16S-2 and Liv-16S-5 were suitable for specific identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii, respectively. These probes were experimentally verified by their reactivity against 19 strains of six Listeria species (excluding newly described species Listeria marthii and Listeria rocourtiae) and eight other bacterial species. The PNA-FISH method was optimized as 30 min of hybridization with 0.2% Triton X-100 in the solution and used to identify 85 Listeria strains from individual putative Listeria colonies on PALCAM agar plates streaked from selectively enriched cultures of 780 food or food-related samples. Of the 85 Listeria strains, thirty-seven were identified as L. monocytogenes with the probe Lm-16S-2 and two as L. ivanovii with the probe Liv-16S-5 which was in agreement with the results obtained by the API LISTERIA method. Thus, the PNA-FISH protocol has the potential for identification of pathogenic Listeria spp. from food or food-related samples.

  12. Nanoparticles that deliver triplex-forming peptide nucleic acid molecules correct F508del CFTR in airway epithelium.

    Science.gov (United States)

    McNeer, Nicole Ali; Anandalingam, Kavitha; Fields, Rachel J; Caputo, Christina; Kopic, Sascha; Gupta, Anisha; Quijano, Elias; Polikoff, Lee; Kong, Yong; Bahal, Raman; Geibel, John P; Glazer, Peter M; Saltzman, W Mark; Egan, Marie E

    2015-01-01

    Cystic fibrosis (CF) is a lethal genetic disorder most commonly caused by the F508del mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. It is not readily amenable to gene therapy because of its systemic nature and challenges including in vivo gene delivery and transient gene expression. Here we use triplex-forming peptide nucleic acids and donor DNA in biodegradable polymer nanoparticles to correct F508del. We confirm modification with sequencing and a functional chloride efflux assay. In vitro correction of chloride efflux occurs in up to 25% of human cells. Deep-sequencing reveals negligible off-target effects in partially homologous sites. Intranasal delivery of nanoparticles in CF mice produces changes in the nasal epithelium potential difference assay, consistent with corrected CFTR function. Also, gene correction is detected in the nasal and lung tissue. This work represents facile genome engineering in vivo with oligonucleotides using a nanoparticle system to achieve clinically relevant levels of gene editing without off-target effects. PMID:25914116

  13. Site-directed gene mutation at mixed sequence targets by psoralen-conjugated pseudo-complementary peptide nucleic acids.

    Science.gov (United States)

    Kim, Ki-Hyun; Nielsen, Peter E; Glazer, Peter M

    2007-01-01

    Sequence-specific DNA-binding molecules such as triple helix-forming oligonucleotides (TFOs) provide a means for inducing site-specific mutagenesis and recombination at chromosomal sites in mammalian cells. However, the utility of TFOs is limited by the requirement for homopurine stretches in the target duplex DNA. Here, we report the use of pseudo-complementary peptide nucleic acids (pcPNAs) for intracellular gene targeting at mixed sequence sites. Due to steric hindrance, pcPNAs are unable to form pcPNA-pcPNA duplexes but can bind to complementary DNA sequences by Watson-Crick pairing via double duplex-invasion complex formation. We show that psoralen-conjugated pcPNAs can deliver site-specific photoadducts and mediate targeted gene modification within both episomal and chromosomal DNA in mammalian cells without detectable off-target effects. Most of the induced psoralen-pcPNA mutations were single-base substitutions and deletions at the predicted pcPNA-binding sites. The pcPNA-directed mutagenesis was found to be dependent on PNA concentration and UVA dose and required matched pairs of pcPNAs. Neither of the individual pcPNAs alone had any effect nor did complementary PNA pairs of the same sequence. These results identify pcPNAs as new tools for site-specific gene modification in mammalian cells without purine sequence restriction, thereby providing a general strategy for designing gene targeting molecules. PMID:17977869

  14. Investigation on natural diets of larval marine animals using peptide nucleic acid-directed polymerase chain reaction clamping.

    Science.gov (United States)

    Chow, Seinen; Suzuki, Sayaka; Matsunaga, Tadashi; Lavery, Shane; Jeffs, Andrew; Takeyama, Haruko

    2011-04-01

    The stomach contents of the larvae of marine animals are usually very small in quantity and amorphous, especially in invertebrates, making morphological methods of identification very difficult. Nucleotide sequence analysis using polymerase chain reaction (PCR) is a likely approach, but the large quantity of larval (host) DNA present may mask subtle signals from the prey genome. We have adopted peptide nucleic acid (PNA)-directed PCR clamping to selectively inhibit amplification of host DNA for this purpose. The Japanese spiny lobster (Panulirus japonicus) and eel (Anguilla japonica) were used as model host and prey organisms, respectively. A lobster-specific PNA oligomer (20 bases) was designed to anneal to the sequence at the junction of the 18 S rDNA gene and the internal transcribed spacer 1 (ITS1) of the lobster. PCR using eukaryote universal primers for amplifying the ITS1 region used in conjunction with the lobster-specific PNA on a mixed DNA template of lobster and eel demonstrated successful inhibition of lobster ITS1 amplification while allowing efficient amplification of eel ITS1. This method was then applied to wild-caught lobster larvae of P. japonicus and P. longipes bispinosus collected around Ryukyu Archipelago, Japan. ITS1 sequences of a wide variety of animals (Ctenophora, Cnidaria, Crustacea, Teleostei, Mollusca, and Chaetognatha) were detected. PMID:20535520

  15. Effects of Hypoxanthine Substitution in Peptide Nucleic Acids Targeting KRAS2 Oncogenic mRNA Molecules: Theory and Experiment

    Science.gov (United States)

    Sanders, Jeffrey M.; Wampole, Matthew E.; Chen, Chang-Po; Sethi, Dalip; Singh, Amrita; Dupradeau, François-Yves; Wang, Fan; Gray, Brian D.; Thakur, Mathew L.; Wickstrom, Eric

    2013-01-01

    Genetic disorders can arise from single base substitutions in a single gene. A single base substitution for wild type guanine in the twelfth codon of KRAS2 mRNA occurs frequently to initiate lung, pancreatic, and colon cancer. We have observed single base mismatch specificity in radioimaging of mutant KRAS2 mRNA in tumors in mice by in vivo hybridization with radiolabeled peptide nucleic acid (PNA) dodecamers. We hypothesized that multi-mutant specificity could be achieved with a PNA dodecamer incorporating hypoxanthine, which can form Watson-Crick basepairs with adenine, cytosine, thymine, and uracil. Using molecular dynamics simulations and free energy calculations, we show that hypoxanthine substitutions in PNAs are tolerated in KRAS2 RNA-PNA duplexes where wild type guanine is replaced by mutant uracil or adenine in RNA. To validate our predictions, we synthesized PNA dodecamers with hypoxanthine, and then measured the thermal stability of RNA-PNA duplexes. Circular dichroism thermal melting results showed that hypoxanthine-containing PNAs are more stable in duplexes where hypoxanthine-adenine and hypoxanthine-uracil base pairs are formed than single mismatch duplexes or duplexes containing hypoxanthine-guanine opposition. PMID:23972113

  16. Variable coordination of cotranscribed genes in Escherichia coli following antisense repression

    Directory of Open Access Journals (Sweden)

    Kulyté Agne

    2006-11-01

    Full Text Available Abstract Background A majority of bacterial genes belong to tight clusters and operons, which complicates gene functional studies using conventional knock-out methods. Antisense agents can down-regulate the expression of genes without disrupting the genome because they bind mRNA and block its expression. However, it is unclear how antisense inhibition affects expression from genes that are cotranscribed with the target. Results To examine the effects of antisense inhibition on cotranscribed genes, we constructed a plasmid expressing the two reporter genes gfp and DsRed as one transcriptional unit. Incubation with antisense peptide nucleic acid (PNA targeted to the mRNA start codon region of either the upstream gfp or the downstream DsRed gene resulted in a complete expression discoordination from this artificial construct. The same approach was applied to the three cotranscribed genes in the endogenously expressed lac-operon (lacZ, Y and A and partial downstream expression coordination was seen when the lacZ start codon was targeted with antisense PNA. Targeting the lacY mRNA start codon region showed no effect on the upstream lacZ gene expression whereas expression from the downstream lacA gene was affected as strongly as the lacY gene. Determination of lacZ and lacY mRNA levels revealed a pattern of reduction that was similar to the Lac-proteins, indicating a relation between translation inhibition and mRNA degradation as a response to antisense PNA treatment. Conclusion The results show that antisense mediated repression of genes within operons affect cotranscribed genes to a variable degree. Target transcript stability appears to be closely related to inhibition of translation and presumably depends on translating ribosomes protecting the mRNA from intrinsic decay mechanisms. Therefore, for genes within operons and clusters it is likely that the nature of the target transcript will determine the inhibitory effects on cotranscribed genes

  17. Orientation Preferences of Backbone Secondary Amide Functional Groups in Peptide Nucleic Acid Complexes: Quantum Chemical Calculations Reveal an Intrinsic Preference of Cationic D-Amino Acid-Based Chiral PNA Analogues for the P-form

    OpenAIRE

    Topham, Christopher M.; Smith, Jeremy C.

    2006-01-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like basepair stacking. Quantum chemical calculations on the bindin...

  18. Antisense Therapy in Neurology

    OpenAIRE

    Lee, Joshua J.A.; Toshifumi Yokota

    2013-01-01

    Antisense therapy is an approach to fighting diseases using short DNA-like molecules called antisense oligonucleotides. Recently, antisense therapy has emerged as an exciting and promising strategy for the treatment of various neurodegenerative and neuromuscular disorders. Previous and ongoing pre-clinical and clinical trials have provided encouraging early results. Spinal muscular atrophy (SMA), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), Duchenne muscular dystrophy (DMD)...

  19. Fluorous Peptide Nucleic Acids: PNA Analogues with Fluorine in Backbone (γ-CF2-apg-PNA) Enhance Cellular Uptake.

    Science.gov (United States)

    Ellipilli, Satheesh; Ganesh, Krishna N

    2015-09-18

    Fluorous PNA analogues possessing fluorine as inherent part of aminopropylglycine (apg) backbone (γ-CF2-apg PNA) have been synthesized and evaluated for biophysical and cell penetrating properties. These form duplexes of higher thermal stability with cRNA than cDNA, although destabilized compared to duplexes of standard aeg-PNA. Cellular uptake of the fluorinated γ-CF2-apg PNAs in NIH 3T3 and HeLa cells was 2-3-fold higher compared to that of nonfluorinated apg PNA, with NIH 3T3 cells showing better permeability compared to HeLa cells. The backbone fluorinated PNAs, which are first in this class, when combined with other chemical modifications may have potential for future PNA-based antisense agents. PMID:26322827

  20. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids

    DEFF Research Database (Denmark)

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S;

    2008-01-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian...... DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent...... cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor...

  1. Identification and characterization of high affinity antisense PNAs for the human unr (upstream of N-ras) mRNA which is uniquely overexpressed in MCF-7 breast cancer cells.

    Science.gov (United States)

    Fang, Huafeng; Yue, Xuan; Li, Xiaoxu; Taylor, John-Stephen

    2005-01-01

    We have recently shown that an MCF-7 tumor can be imaged in a mouse by PET with 64Cu-labeled Peptide nucleic acids (PNAs) tethered to the permeation peptide Lys4 that recognize the uniquely overexpressed and very abundant upstream of N-ras or N-ras related gene (unr mRNA) expressed in these cells. Herein we describe how the high affinity antisense PNAs to the unr mRNA were identified and characterized. First, antisense binding sites on the unr mRNA were mapped by an reverse transcriptase random oligonucleotide library (RT-ROL) method that we have improved, and by a serial analysis of antisense binding sites (SAABS) method that we have developed which is similar to another recently described method. The relative binding affinities of oligodeoxynucleotides (ODNs) complementary to the antisense binding sites were then qualitatively ranked by a new Dynabead-based dot blot assay. Dissociation constants for a subset of the ODNs were determined by a new Dynabead-based solution assay and were found to be 300 pM for the best binders in 1 M salt. PNAs corresponding to the ODNs with the highest affinities were synthesized with an N-terminal CysTyr and C-terminal Lys4 sequence. Dissociation constants of these hybrid PNAs were determined by the Dynabead-based solution assay to be about 10 pM for the highest affinity binders. PMID:16314303

  2. A FRET-enabled molecular peptide beacon with a significant red shift for the ratiometric detection of nucleic acids.

    Science.gov (United States)

    Maity, Debabrata; Jiang, Juanjuan; Ehlers, Martin; Wu, Junchen; Schmuck, Carsten

    2016-05-01

    A cationic molecular peptide beacon NAP1 functionalized with a fluorescence resonance energy transfer-pair at its ends allows the ratiometric detection of ds-DNA with a preference for AT rich sequences. NAP1 most likely binds in a folded form into the minor groove of ds-DNA, which results in a remarkable change in its fluorescence properties. As NAP1 exhibits quite low cytotoxicity, it can also be used for imaging of nuclear DNA in cells. PMID:27071707

  3. A FRET-enabled molecular peptide beacon with a significant red shift for the ratiometric detection of nucleic acids.

    Science.gov (United States)

    Maity, Debabrata; Jiang, Juanjuan; Ehlers, Martin; Wu, Junchen; Schmuck, Carsten

    2016-05-01

    A cationic molecular peptide beacon NAP1 functionalized with a fluorescence resonance energy transfer-pair at its ends allows the ratiometric detection of ds-DNA with a preference for AT rich sequences. NAP1 most likely binds in a folded form into the minor groove of ds-DNA, which results in a remarkable change in its fluorescence properties. As NAP1 exhibits quite low cytotoxicity, it can also be used for imaging of nuclear DNA in cells.

  4. Preparation of 99Tcm labeled survivin mRNA antisense PNA and gene imaging in nude mice bearing lung carcinoma A549 xenografts

    International Nuclear Information System (INIS)

    Objective: To prepare the 99Tcm-survivin mRNA antisense peptide nucleic acid (PNA)and investigate its value as a gene imaging agent in tumor bearing mice and early diagnosis in tumor. Methods: Survivin mRNA antisense PNA and mismatch PNA were synthesized. Four amino acids (Gly- (D)Ala-Gly-Gly) and Aba (4-aminobutyric acid) were linked to the 5' end of PNA. Gly- (D)Ala-Gly-Gly served as a chelating moiety for strong chelation of 99Tcm and Aba acted as a spacer to minimize the steric hindrance. PNAs were labeled with 99Tcm by the ligand-exchange method. The labeling efficiency and radiochemical purity were measured by HPLC and ITLC methods. There were five BALB/c nude mice bearing human lung carcinoma (A549) in each of antisense PNA and mismatch PNA groups. Gene imaging of 99Tcm-survivin mRNA antisense and mismatch PNAs were performed at 1, 2 and 4 h post the injection, respectively, and the T/NT ratio was measured by the method of ROI. The statistical comparisons of average values were performed with the two-group t-test for independent sample by SPSS 13.0. Results: The product kept stable in vitro. The labeling efficiency of 99Tcm-survivin mRNA antisense PNA was (95.48 ±1.92)% and more than 85% after the incubation for 24 h in serum. The radiochemical purity was >95%. The labeling efficiency of mismatch PNA was similar to the antisense PNA. 99Tcm-survivin mRNA antisense PNA was especially uptaken by tumor lesion, and its accumulation reached the top at 4 h post the injection. T/NT ratios at 1, 2, and 4 h were 2.70 ± 0.28, 3.44 ± 0.35,4.21 ± 0.63, respectively. In the comparison, the T/NT ratio of 99Tcm-survivin mRNA mismatch PNA at 4 h (3.12 ±0.50) was significantly lower (t=2.918, P=0.019). Conclusions: 99Tcm-survivin mRNA antisense PNA has high labeling efficiency,good stability and no need of purification. Its characteristic of especial uptake by tumor lesion provides the potential value in early diagnosis of tumor. (authors)

  5. Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye.

    Science.gov (United States)

    Hnedzko, Dziyana; McGee, Dennis W; Rozners, Eriks

    2016-09-15

    Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines.

  6. Unravelling the Bacterial Vaginosis-Associated Biofilm: A Multiplex Gardnerella vaginalis and Atopobium vaginae Fluorescence In Situ Hybridization Assay Using Peptide Nucleic Acid Probes.

    Directory of Open Access Journals (Sweden)

    Liselotte Hardy

    Full Text Available Bacterial vaginosis (BV, a condition defined by increased vaginal discharge without significant inflammation, is characterized by a change in the bacterial composition of the vagina. Lactobacillus spp., associated with a healthy vaginal microbiome, are outnumbered by BV-associated organisms. These bacteria could form a polymicrobial biofilm which allows them to persist in spite of antibiotic treatment. In this study, we examined the presence of Gardnerella vaginalis and Atopobium vaginae in vaginal biofilms using Peptide Nucleic Acid (PNA probes targeting these bacteria. For this purpose, we developed three new PNA probes for A. vaginae. The most specific A. vaginae probe, AtoITM1, was selected and then used in an assay with two existing probes, Gard162 and BacUni-1, to evaluate multiplex FISH on clinical samples. Using quantitative polymerase chain reaction (qPCR as the gold standard, we demonstrated a sensitivity of 66.7% (95% confidence interval: 54.5% - 77.1% and a specificity of 89.4% (95% confidence interval: 76.1% - 96% of the new AtoITM1 probe. FISH enabled us to show the presence of a polymicrobial biofilm in bacterial vaginosis, in which Atopobium vaginae is part of a Gardnerella vaginalis-dominated biofilm. We showed that the presence of this biofilm is associated with high bacterial loads of A. vaginae and G. vaginalis.

  7. Correction of a splice-site mutation in the beta-globin gene stimulated by triplex-forming peptide nucleic acids.

    Science.gov (United States)

    Chin, Joanna Y; Kuan, Jean Y; Lonkar, Pallavi S; Krause, Diane S; Seidman, Michael M; Peterson, Kenneth R; Nielsen, Peter E; Kole, Ryszard; Glazer, Peter M

    2008-09-01

    Splice-site mutations in the beta-globin gene can lead to aberrant transcripts and decreased functional beta-globin, causing beta-thalassemia. Triplex-forming DNA oligonucleotides (TFOs) and peptide nucleic acids (PNAs) have been shown to stimulate recombination in reporter gene loci in mammalian cells via site-specific binding and creation of altered helical structures that provoke DNA repair. We have designed a series of triplex-forming PNAs that can specifically bind to sequences in the human beta-globin gene. We demonstrate here that these PNAs, when cotransfected with recombinatory donor DNA fragments, can promote single base-pair modification at the start of the second intron of the beta-globin gene, the site of a common thalassemia-associated mutation. This single base pair change was detected by the restoration of proper splicing of transcripts produced from a green fluorescent protein-beta-globin fusion gene. The ability of these PNAs to induce recombination was dependent on dose, sequence, cell-cycle stage, and the presence of a homologous donor DNA molecule. Enhanced recombination, with frequencies up to 0.4%, was observed with use of the lysomotropic agent chloroquine. Finally, we demonstrate that these PNAs were effective in stimulating the modification of the endogenous beta-globin locus in human cells, including primary hematopoietic progenitor cells. This work suggests that PNAs can be effective tools to induce heritable, site-specific modification of disease-related genes in human cells. PMID:18757759

  8. Synthesis and properties of peptide nucleic acid labeled at the N-terminus with HiLyte Fluor 488 fluorescent dye.

    Science.gov (United States)

    Hnedzko, Dziyana; McGee, Dennis W; Rozners, Eriks

    2016-09-15

    Fluorescently labeled peptide nucleic acids (PNAs) are important tools in fundamental research and biomedical applications. However, synthesis of labeled PNAs, especially using modern and expensive dyes, is less explored than similar preparations of oligonucleotide dye conjugates. Herein, we present a simple procedure for labeling of the PNA N-terminus with HiLyte Fluor 488 as the last step of solid phase PNA synthesis. A minimum excess of 1.25equiv of activated carboxylic acid achieved labeling yields close to 90% providing a good compromise between the price of dye and the yield of product and significant improvement over previous literature procedures. The HiLyte Fluor 488-labeled PNAs retained the RNA binding ability and in live cell fluorescence microscopy experiments were brighter and significantly more photostable than PNA labeled with carboxyfluorescein. In contrast to fluorescein-labeled PNA, the fluorescence of PNAs labeled with HiLyte Fluor 488 was independent of pH in the biologically relevant range of 5-8. The potential of HiLyte Fluor 488-labeling for studies of PNA cellular uptake and distribution was demonstrated in several cell lines. PMID:27430566

  9. Interaction of α-Melanocortin and Its Pentapeptide Antisense LVKAT: Effects on Hepatoprotection in Male CBA Mice

    Directory of Open Access Journals (Sweden)

    Paško Konjevoda

    2011-08-01

    Full Text Available The genetic code defines nucleotide patterns that code for individual amino acids and their complementary, i.e., antisense, pairs. Peptides specified by the complementary mRNAs often bind to each other with a higher specificity and efficacy. Applications of this genetic code property in biomedicine are related to the modulation of peptide and hormone biological function, selective immunomodulation, modeling of discontinuous and linear epitopes, modeling of mimotopes, paratopes and antibody mimetics, peptide vaccine development, peptidomimetic and drug design. We have investigated sense-antisense peptide interactions and related modulation of the peptide function by modulating the effects of a-MSH on hepatoprotection with its antisense peptide LVKAT. First, transcription of complementary mRNA sequence of a-MSH in 3’→5’ direction was used to design antisense peptide to the central motif that serves as a-MSH pharmacophore for melanocortin receptors. Second, tryptophan spectrofluorometric titration was applied to evaluate the binding of a-MSH and its central pharmacophore motif to the antisense peptide, and it was concluded that this procedure represents a simple and efficient method to evaluate sense-antisense peptide interaction in vitro. Third, we showed that antisense peptide LVKAT abolished potent hepatoprotective effects of a-MSH in vivo.

  10. Amyloid-associated nucleic acid hybridisation.

    Directory of Open Access Journals (Sweden)

    Sebastian Braun

    Full Text Available Nucleic acids promote amyloid formation in diseases including Alzheimer's and Creutzfeldt-Jakob disease. However, it remains unclear whether the close interactions between amyloid and nucleic acid allow nucleic acid secondary structure to play a role in modulating amyloid structure and function. Here we have used a simplified system of short basic peptides with alternating hydrophobic and hydrophilic amino acid residues to study nucleic acid - amyloid interactions. Employing biophysical techniques including X-ray fibre diffraction, circular dichroism spectroscopy and electron microscopy we show that the polymerized charges of nucleic acids concentrate and enhance the formation of amyloid from short basic peptides, many of which would not otherwise form fibres. In turn, the amyloid component binds nucleic acids and promotes their hybridisation at concentrations below their solution K(d, as shown by time-resolved FRET studies. The self-reinforcing interactions between peptides and nucleic acids lead to the formation of amyloid nucleic acid (ANA fibres whose properties are distinct from their component polymers. In addition to their importance in disease and potential in engineering, ANA fibres formed from prebiotically-produced peptides and nucleic acids may have played a role in early evolution, constituting the first entities subject to Darwinian evolution.

  11. Optimization of peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) for the detection of bacteria: The effect of pH, dextran sulfate and probe concentration.

    Science.gov (United States)

    Rocha, Rui; Santos, Rita S; Madureira, Pedro; Almeida, Carina; Azevedo, Nuno F

    2016-05-20

    Fluorescence in situ hybridization (FISH) is a molecular technique widely used for the detection and characterization of microbial populations. FISH is affected by a wide variety of abiotic and biotic variables and the way they interact with each other. This is translated into a wide variability of FISH procedures found in the literature. The aim of this work is to systematically study the effects of pH, dextran sulfate and probe concentration in the FISH protocol, using a general peptide nucleic acid (PNA) probe for the Eubacteria domain. For this, response surface methodology was used to optimize these 3 PNA-FISH parameters for Gram-negative (Escherichia coli and Pseudomonas fluorescens) and Gram-positive species (Listeria innocua, Staphylococcus epidermidis and Bacillus cereus). The obtained results show that a probe concentration higher than 300nM is favorable for both groups. Interestingly, a clear distinction between the two groups regarding the optimal pH and dextran sulfate concentration was found: a high pH (approx. 10), combined with lower dextran sulfate concentration (approx. 2% [w/v]) for Gram-negative species and near-neutral pH (approx. 8), together with higher dextran sulfate concentrations (approx. 10% [w/v]) for Gram-positive species. This behavior seems to result from an interplay between pH and dextran sulfate and their ability to influence probe concentration and diffusion towards the rRNA target. This study shows that, for an optimum hybridization protocol, dextran sulfate and pH should be adjusted according to the target bacteria. PMID:27021959

  12. Stimuli-Responsive Codelivery of Oligonucleotides and Drugs by Self-Assembled Peptide Nanoparticles.

    Science.gov (United States)

    Sigg, Severin J; Postupalenko, Viktoriia; Duskey, Jason T; Palivan, Cornelia G; Meier, Wolfgang

    2016-03-14

    Ever more emerging combined treatments exploiting synergistic effects of drug combinations demand smart, responsive codelivery carriers to reveal their full potential. In this study, a multifunctional stimuli-responsive amphiphilic peptide was designed and synthesized to self-assemble into nanoparticles capable of co-bearing and -releasing hydrophobic drugs and antisense oligonucleotides for combined therapies. The rational design was based on a hydrophobic l-tryptophan-d-leucine repeating unit derived from a truncated sequence of gramicidin A (gT), to entrap hydrophobic cargo, which is combined with a hydrophilic moiety of histidines to provide electrostatic affinity to nucleotides. Stimuli-responsiveness was implemented by linking the hydrophobic and hydrophilic sequence through an artificial amino acid bearing a disulfide functional group (H3SSgT). Stimuli-responsive peptides self-assembled in spherical nanoparticles in sizes (100-200 nm) generally considered as preferable for drug delivery applications. Responsive peptide nanoparticles revealed notable nucleotide condensing abilities while maintaining the ability to load hydrophobic cargo. The disulfide cleavage site introduced in the peptide sequence induced responsiveness to physiological concentrations of reducing agent, serving to release the incorporated molecules. Furthermore, the peptide nanoparticles, singly loaded or coloaded with boron-dipyrromethene (BODIPY) and/or antisense oligonucleotides, were efficiently taken up by cells. Such amphiphilic peptides that led to noncytotoxic, reduction-responsive nanoparticles capable of codelivering hydrophobic and nucleic acid payloads simultaneously provide potential toward combined treatment strategies to exploit synergistic effects. PMID:26871486

  13. 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.

  14. Translational Inhibition of CTX-M Extended Spectrum β-Lactamase in Clinical Strains of Escherichia coli by Synthetic Antisense Oligonucleotides Partially Restores Sensitivity to Cefotaxime.

    Science.gov (United States)

    Readman, John B; Dickson, George; Coldham, Nick G

    2016-01-01

    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 bla CTX-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 (CTX) 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 (CPP; (KFF)3K) and both significantly (P < 0.05) increased sensitivity to CTX in a dose dependent manner (0-40 nM) in field and clinical isolates harboring CTX-M group 1 β-lactamases. Antisense oligonucleotides targeted to the translational initiation site and Shine-Dalgarno region of bla CTX-M-15 inhibited gene expression, and when conjugated to a cell penetrating delivery vehicle, partially restored antibiotic sensitivity to both field and clinical isolates. PMID:27047482

  15. [Evaluation of peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method in the identifi cation of Candida species isolated from blood cultures].

    Science.gov (United States)

    Aydemir, Gonca; Koç, Ayşe Nedret; Atalay, Mustafa Altay

    2016-04-01

    In recent years, increased number of patients who are hospitalized in intensive care units, received immunosuppressive therapy and treated with broad-spectrum antibiotics that can lead an increase in the incidence of systemic candidiasis. In these patients, the most common clinical manifestation is candidemia. Since the identification of Candida species isolated from blood cultures is time consuming by conventional (morphological and biochemical) methods, rapid, reliable and accurate methods are needed. For this purpose novel systems have been developed to identify the agent directly. The aim of this study was to evaluate the peptide nucleic acid fluorescent in situ hybridization (PNA FISH) method for the identification of Candida species by comparing with the conventional methods. A total of 50 patients who were admitted to Erciyes University Medical Faculty Hospital clinics and followed with prediagnosis of systemic fungal infections whose blood cultures were positive for the yeasts between July 2011 and July 2012 were included in the study. The conventional identification of Candida isolates was performed by considering macroscopic and microscopic morphology, germ tube test, cycloheximide sensitivity, urease activity and carbohydrate assimilation patterns with API 20C AUX (bioMerieux, France) test. PNA FISH method was conducted by the use of a commercial kit namely Yeast Traffic Light(®) PNA FISH (AdvanDx, USA). According to morphological and biochemical characteristics (conventional methods), 19 (38%) out of 50 Candida isolates were identified as C.albicans, 12 (24%) as C.glabrata, five (10%) as C.parapsilosis, five (10%) as C.kefyr, four (8%) as C.krusei, two (4%) as C.guilliermondii, two (4%) as C.tropicalis and one (2%) as C.lusitaniae. On the other hand, 24 (48%) of the isolates were identified as C.albicans/C.parapsilosis (with green fluorescence), 16 (32%) as C.glabrata/C.krusei (with red fluorescence) and one (%2) as C.tropicalis (with yellow

  16. Sense antisense DNA strand?

    Science.gov (United States)

    Boldogkói, Z; Kaliman, A V; Murvai, J; Fodor, I

    1994-01-01

    Recent evidence indicates that alphaherpesviruses express latency associated transcripts (LATs) from the antisense strand of immediate-early (IE) genes of the viral genome. It has been suggested that LATs containing extended open reading frames (ORFs), might be translated into (a) protein product(s). We found that a salient feature of some herpesvirus DNAs is a high GC preference at the third codon positions. The consequence of this feature is that the probability of a stop-codon appearing at two of the six reading frames of the DNA strand is very low. Therefore, the presence of an extended ORF does not necessarily mean that it is relevant to real translation. PMID:7810418

  17. 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...... applied. LNA oligonucleotides are commercially available, can be transfected using standard techniques, are non-toxic, lead to increased target accessibility, can be designed to activate RNase H, and function in steric block approaches. LNA-Antisense, including gapmer LNA containing a central DNA...... or phosphorothioate-DNA segment flanked by LNA gaps, rivals siRNA as the technology of choice for target validation and therapeutic applications....

  18. Orientation Preferences of Backbone Secondary Amide Functional Groups in Peptide Nucleic Acid Complexes: Quantum Chemical Calculations Reveal an Intrinsic Preference of Cationic D-Amino Acid-Based Chiral PNA Analogues for the P-form

    Energy Technology Data Exchange (ETDEWEB)

    Smith, Jeremy C [ORNL; Topham, Christopher [University of Heidelberg

    2007-01-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like base pair stacking. Quantum chemical calculations on the binding of a water molecule to an O2 base atom in glycine-based PNA thymine dimers indicate that junctions modeled with P-form backbone conformations are lower in energy than a dimer comprising the predominant conformation observed in A-like helices. It is further shown in model systems that PNA analogs based on D-lysine are better able to preorganize in a conformation exclusive to P-form helices than is glycine-based PNA. An intrinsic preference for this conformation is also exhibited by positively charged chiral PNA dimers carrying 3-amino-D-alanine or 4-aza-D-leucine residue units that provide for additional rigidity by side-chain hydrogen bonding to the backbone carbonyl oxygen. Structural modifications stabilizing P-form helices may obviate the need for large heterocycles to target DNA pyrimidine bases via PNADNA-PNA triplex formation. Quantum chemical modeling methods are used to propose candidate PNA Hoogsteen strand designs.

  19. Orientation preferences of backbone secondary amide functional groups in peptide nucleic acid complexes: quantum chemical calculations reveal an intrinsic preference of cationic D-amino acid-based chiral PNA analogues for the P-form.

    Science.gov (United States)

    Topham, Christopher M; Smith, Jeremy C

    2007-02-01

    Geometric descriptions of nonideal interresidue hydrogen bonding and backbone-base water bridging in the minor groove are established in terms of polyamide backbone carbonyl group orientation from analyses of residue junction conformers in experimentally determined peptide nucleic acid (PNA) complexes. Two types of interresidue hydrogen bonding are identified in PNA conformers in heteroduplexes with nucleic acids that adopt A-like basepair stacking. Quantum chemical calculations on the binding of a water molecule to an O2 base atom in glycine-based PNA thymine dimers indicate that junctions modeled with P-form backbone conformations are lower in energy than a dimer comprising the predominant conformation observed in A-like helices. It is further shown in model systems that PNA analogs based on D-lysine are better able to preorganize in a conformation exclusive to P-form helices than is glycine-based PNA. An intrinsic preference for this conformation is also exhibited by positively charged chiral PNA dimers carrying 3-amino-D-alanine or 4-aza-D-leucine residue units that provide for additional rigidity by side-chain hydrogen bonding to the backbone carbonyl oxygen. Structural modifications stabilizing P-form helices may obviate the need for large heterocycles to target DNA pyrimidine bases via PNA.DNA-PNA triplex formation. Quantum chemical modeling methods are used to propose candidate PNA Hoogsteen strand designs. PMID:17071666

  20. Exploitation of a Very Small Peptide Nucleic Acid as a New Inhibitor of miR-509-3p Involved in the Regulation of Cystic Fibrosis Disease-Gene Expression

    Directory of Open Access Journals (Sweden)

    Felice Amato

    2014-01-01

    Full Text Available Computational techniques, and in particular molecular dynamics (MD simulations, have been successfully used as a complementary technique to predict and analyse the structural behaviour of nucleic acids, including peptide nucleic acid- (PNA- RNA hybrids. This study shows that a 7-base long PNA complementary to the seed region of miR-509-3p, one of the miRNAs involved in the posttranscriptional regulation of the CFTR disease-gene of Cystic Fibrosis, and bearing suitable functionalization at its N- and C-ends aimed at improving its resistance to nucleases and cellular uptake, is able to revert the expression of the luciferase gene containing the 3′UTR of the gene in A549 human lung cancer cells, in agreement with the MD results that pointed at the formation of a stable RNA/PNA heteroduplex notwithstanding the short sequence of the latter. The here reported results widen the interest towards the use of small PNAs as effective anti-miRNA agents.

  1. Amplification-Free Detection of Circulating microRNA Biomarkers from Body Fluids Based on Fluorogenic Oligonucleotide-Templated Reaction between Engineered Peptide Nucleic Acid Probes: Application to Prostate Cancer Diagnosis.

    Science.gov (United States)

    Metcalf, Gavin A D; Shibakawa, Akifumi; Patel, Hinesh; Sita-Lumsden, Ailsa; Zivi, Andrea; Rama, Nona; Bevan, Charlotte L; Ladame, Sylvain

    2016-08-16

    Highly abundant in cells, microRNAs (or miRs) play a key role as regulators of gene expression. A proportion of them are also detectable in biofluids making them ideal noninvasive biomarkers for pathologies in which miR levels are aberrantly expressed, such as cancer. Peptide nucleic acids (PNAs) are engineered uncharged oligonucleotide analogues capable of hybridizing to complementary nucleic acids with high affinity and high specificity. Herein, novel PNA-based fluorogenic biosensors have been designed and synthesized that target miR biomarkers for prostate cancer (PCa). The sensing strategy is based on oligonucleotide-templated reactions where the only miR of interest serves as a matrix to catalyze an otherwise highly unfavorable fluorogenic reaction. Validated in vitro using synthetic RNAs, these newly developed biosensors were then shown to detect endogenous concentrations of miR in human blood samples without the need for any amplification step and with minimal sample processing. This low-cost, quantitative, and versatile sensing technology has been technically validated using gold-standard RT-qPCR. Compared to RT-qPCR however, this enzyme-free, isothermal blood test is amenable to incorporation into low-cost portable devices and could therefore be suitable for widespread public screening. PMID:27498854

  2. Does Active Learning through an Antisense Jigsaw Make Sense?

    Science.gov (United States)

    Seetharaman, Mahadevan; Musier-Forsyth, Karin

    2003-12-01

    Three journal articles on nucleic acid antisense modification strategies were assigned to 12 students as part of an active learning "jigsaw" exercise for a graduate-level chemistry course on nucleic acids. Each student was required to read one of the three articles. This assignment was preceded by an hour-long lecture on the basic concepts in antisense antigene technology. On the day of the jigsaw, the students with the same article (three groups of four students) discussed their article briefly, and then formed four new groups where no one had read the same article. Each student spent about five minutes teaching his or her article to the other group members, using specific questions provided to guide the discussion. This exercise laid the foundation for bringing the discussion to the entire class, where most of the students actively participated. To test the students' comprehension of the reading materials, a problem set was designed that required not only an understanding of the three articles, but also application of the concepts learned. The effectiveness of this active learning strategy and its applicability to other topics are discussed in this article.

  3. Unlocked nucleic acid - an RNA modification with broad potential

    DEFF Research Database (Denmark)

    Pasternak, Anna; Wengel, Jesper

    2011-01-01

    The first unlocked nucleic acid (UNA) monomer was described more than a decade ago, but only recent reports have revealed the true potential applications of this acyclic RNA mimic. UNA monomers enable the modulation of the thermodynamic stability of various nucleic acid structures such as RNA...... and DNA duplexes, quadruplexes or i-motifs. Moreover, UNA monomers were found to be compatible with RNase H activity, a property which is important for single stranded antisense constructs. Notably, UNA monomers can be applied in the design of superior siRNAs, combining potent gene silencing...

  4. Antisense precision polymer micelles require less poly(ethylenimine) for efficient gene knockdown

    Science.gov (United States)

    Fakhoury, Johans J.; Edwardson, Thomas G.; Conway, Justin W.; Trinh, Tuan; Khan, Farhad; Barłóg, Maciej; Bazzi, Hassan S.; Sleiman, Hanadi F.

    2015-12-01

    Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable therapeutics. We have synthesized antisense-polymer conjugates, where the polymeric block is completely monodisperse and sequence-controlled. Depending on the polymer sequence, these can self-assemble to produce micelles of very low polydispersity. The introduction of linear poly(ethylenimine) to these micelles leads to aggregation into size-defined PEI-mediated superstructures. Subsequently, both cellular uptake and gene silencing are greatly enhanced over extended periods compared to antisense alone, while at the same time cellular cytotoxicity remains very low. In contrast, gene silencing is not enhanced with antisense polymer conjugates that are not able to self-assemble into micelles. Thus, using antisense precision micelles, we are able to achieve significant transfection and knockdown with minimal cytotoxicity at much lower concentrations of linear PEI then previously reported. Consequently, a conceptual solution to the problem of antisense or siRNA delivery is to self-assemble these molecules into `gene-like' micelles with high local charge and increased stability, thus reducing the amount of transfection agent needed for effective gene silencing.Therapeutic nucleic acids are powerful molecules for shutting down protein expression. However, their cellular uptake is poor and requires transport vectors, such as cationic polymers. Of these, poly(ethylenimine) (PEI) has been shown to be an efficient vehicle for nucleic acid transport into cells. However, cytotoxicity has been a major hurdle in the development of PEI-DNA complexes as clinically viable

  5. Antisense approaches in prostate cancer.

    Science.gov (United States)

    Chi, Kim N; Gleave, Martin E

    2004-06-01

    Patients with hormone refractory prostate cancer have limited treatment options and new therapies are urgently needed. Advances in the understanding of the molecular mechanisms implicated in prostate cancer progression have identified many potential therapeutic gene targets that are involved in apoptosis, growth factors, cell signalling and the androgen receptor (AR). Antisense oligonucleotides are short sequences of synthetic modified DNA that are designed to be complimentary to a selected gene's mRNA and thereby specifically inhibit expression of that gene. The antisense approach continues to hold promise as a therapeutic modality to target genes involved in cancer progression, especially those in which the gene products are not amenable to small molecule inhibition or antibodies. The current status and future direction of a number of antisense oligonucleotides targeting several genes, including BCL-2, BCL-XL, clusterin, the inhibitors of apoptosis (IAP) family, MDM2, protein kinase C-alpha, c-raf, insulin-like growth factor binding proteins and the AR, that have potential clinical use in prostate cancer are reviewed. PMID:15174974

  6. Cleavage of nucleic acids

    Science.gov (United States)

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    2000-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  7. Imaging Functional Nucleic Acid Delivery to Skin.

    Science.gov (United States)

    Kaspar, Roger L; Hickerson, Robyn P; González-González, Emilio; Flores, Manuel A; Speaker, Tycho P; Rogers, Faye A; Milstone, Leonard M; Contag, Christopher H

    2016-01-01

    Monogenic skin diseases arise from well-defined single gene mutations, and in some cases a single point mutation. As the target cells are superficial, these diseases are ideally suited for treatment by nucleic acid-based therapies as well as monitoring through a variety of noninvasive imaging technologies. Despite the accessibility of the skin, there remain formidable barriers for functional delivery of nucleic acids to the target cells within the dermis and epidermis. These barriers include the stratum corneum and the layered structure of the skin, as well as more locally, the cellular, endosomal and nuclear membranes. A wide range of technologies for traversing these barriers has been described and moderate success has been reported for several approaches. The lessons learned from these studies include the need for combinations of approaches to facilitate nucleic acid delivery across these skin barriers and then functional delivery across the cellular and nuclear membranes for expression (e.g., reporter genes, DNA oligonucleotides or shRNA) or into the cytoplasm for regulation (e.g., siRNA, miRNA, antisense oligos). The tools for topical delivery that have been evaluated include chemical, physical and electrical methods, and the development and testing of each of these approaches has been greatly enabled by imaging tools. These techniques allow delivery and real time monitoring of reporter genes, therapeutic nucleic acids and also triplex nucleic acids for gene editing. Optical imaging is comprised of a number of modalities based on properties of light-tissue interaction (e.g., scattering, autofluorescence, and reflectance), the interaction of light with specific molecules (e.g., absorbtion, fluorescence), or enzymatic reactions that produce light (bioluminescence). Optical imaging technologies operate over a range of scales from macroscopic to microscopic and if necessary, nanoscopic, and thus can be used to assess nucleic acid delivery to organs, regions, cells

  8. Imaging Functional Nucleic Acid Delivery to Skin.

    Science.gov (United States)

    Kaspar, Roger L; Hickerson, Robyn P; González-González, Emilio; Flores, Manuel A; Speaker, Tycho P; Rogers, Faye A; Milstone, Leonard M; Contag, Christopher H

    2016-01-01

    Monogenic skin diseases arise from well-defined single gene mutations, and in some cases a single point mutation. As the target cells are superficial, these diseases are ideally suited for treatment by nucleic acid-based therapies as well as monitoring through a variety of noninvasive imaging technologies. Despite the accessibility of the skin, there remain formidable barriers for functional delivery of nucleic acids to the target cells within the dermis and epidermis. These barriers include the stratum corneum and the layered structure of the skin, as well as more locally, the cellular, endosomal and nuclear membranes. A wide range of technologies for traversing these barriers has been described and moderate success has been reported for several approaches. The lessons learned from these studies include the need for combinations of approaches to facilitate nucleic acid delivery across these skin barriers and then functional delivery across the cellular and nuclear membranes for expression (e.g., reporter genes, DNA oligonucleotides or shRNA) or into the cytoplasm for regulation (e.g., siRNA, miRNA, antisense oligos). The tools for topical delivery that have been evaluated include chemical, physical and electrical methods, and the development and testing of each of these approaches has been greatly enabled by imaging tools. These techniques allow delivery and real time monitoring of reporter genes, therapeutic nucleic acids and also triplex nucleic acids for gene editing. Optical imaging is comprised of a number of modalities based on properties of light-tissue interaction (e.g., scattering, autofluorescence, and reflectance), the interaction of light with specific molecules (e.g., absorbtion, fluorescence), or enzymatic reactions that produce light (bioluminescence). Optical imaging technologies operate over a range of scales from macroscopic to microscopic and if necessary, nanoscopic, and thus can be used to assess nucleic acid delivery to organs, regions, cells

  9. Decoy oligodeoxyribonucleotides and peptide nucleic acids-DNA chimeras targeting nuclear factor kappa-B: inhibition of IL-8 gene expression in cystic fibrosis cells infected with Pseudomonas aeruginosa.

    Science.gov (United States)

    Gambari, Roberto; Borgatti, Monica; Bezzerri, Valentino; Nicolis, Elena; Lampronti, Ilaria; Dechecchi, Maria Cristina; Mancini, Irene; Tamanini, Anna; Cabrini, Giulio

    2010-12-15

    Cystic fibrosis (CF) is characterized by a deep inflammatory process, with production and release of cytokines and chemokines, among which interleukin 8 (IL-8) represents one of the most important. Accordingly, there is a growing interest in developing therapies against IL-8, with the aim of reducing the excessive inflammatory response in the airways of CF patients. Since transcription factor NF-kappaB plays a critical role in IL-8 expression, the transcription factor decoy (TFD) strategy might be of interest. TFD is based on biomolecules mimicking the target sites of transcription factors (TFs) and able to interfere with TF activity when delivered to target cells. Here, we review the inhibitory effects of decoy oligodeoxyribonucleotides (ODNs) on expression of IL-8 gene and secretion of IL-8 by cystic fibrosis cells infected by Pseudomonas aeruginosa. In addition, the effects of decoy molecules based on peptide nucleic acids (PNAs) are discussed. In this respect PNA-DNA-PNA (PDP) chimeras are interesting: (a) unlike PNAs, they can be complexed with liposomes and microspheres; (b) unlike oligodeoxyribonucleotides (ODNs), they are resistant to DNAses, serum and cytoplasmic extracts; (c) unlike PNA/PNA and PNA/DNA hybrids, they are potent decoy molecules. Interestingly, PDP/PDP NF-kappaB decoy chimeras inhibit accumulation of pro-inflammatory mRNAs (including IL-8 mRNA) in P. aeruginosa infected IB3-1, cells reproducing the effects of decoy oligonucleotides. The effects of PDP/PDP chimeras, unlike ODN-based decoys, are observed even in absence of protection with lipofectamine. Since IL-8 is pivotal in pro-inflammatory processes affecting cystic fibrosis, inhibition of its functions might have a clinical relevance. PMID:20615393

  10. Comparison of peptide nucleic acid fluorescence in situ hybridization assays with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry for the identification of bacteria and yeasts from blood cultures and cerebrospinal fluid cultures.

    Science.gov (United States)

    Calderaro, A; Martinelli, M; Motta, F; Larini, S; Arcangeletti, M C; Medici, M C; Chezzi, C; De Conto, F

    2014-08-01

    Peptide nucleic acid fluorescence in situ hybridization (PNA FISH) is a molecular diagnostic tool for the rapid detection of pathogens directly from liquid media. The aim of this study was to prospectively evaluate PNA FISH assays in comparison with culture-based matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) identification, as a reference method, for both blood and cerebrospinal fluid (CSF) cultures, during a 1-year investigation. On the basis of the Gram stain microscopy results, four different PNA FISH commercially available assays were used ('Staphylococcus aureus/CNS', 'Enterococcus faecalis/OE', 'GNR Traffic Light' and 'Yeasts Traffic Light' PNA FISH assays, AdvanDx). The four PNA FISH assays were applied to 956 positive blood cultures (921 for bacteria and 35 for yeasts) and 11 CSF cultures. Among the 921 blood samples positive for bacteria, PNA FISH gave concordant results with MALDI-TOF MS in 908/921 (98.64%) samples, showing an agreement of 99.4% in the case of monomicrobial infections. As regards yeasts, the PNA FISH assay showed a 100% agreement with the result obtained by MALDI-TOF MS. When PNA FISH assays were tested on the 11 CSF cultures, the results agreed with the reference method in all cases (100%). PNA FISH assays provided species identification at least one work-day before the MALDI-TOF MS culture-based identification. PNA FISH assays showed an excellent efficacy in the prompt identification of main pathogens, yielding a significant reduction in reporting time and leading to more appropriate patient management and therapy in cases of sepsis and severe infections.

  11. Development of Antisense Therapeutic and Imaging Agents to Detect and Suppress Inducible Nitric Oxide Synthase (iNOS) Expression in Acute Lung Injury (ALI)

    Science.gov (United States)

    Shen, Yuefei

    This dissertation focuses on the development and investigation of antisense imaging and therapeutic agents, combined with nanotechnology, to detect and suppress inducible nitric oxide synthase (iNOS) expression for the diagnosis and treatment of acute lung injury (ALI). To achieve this goal, several efforts were made. The first effort was the identification and characterization of high binding affinity antisense peptide nucleic acids (PNAs) and shell-crosslinked knedel-like nanoparticle (SCK)-PNA conjugates to the iNOS mRNA. Antisense binding sites on the iNOS mRNA were first mapped by a procedure for rapidly generating a library of antisense accessible sites on native mRNAs (MASL) which involves reverse transcription of whole cell mRNA extracts with a random oligodeoxynucleotide primer followed by mRNA-specific PCR. Antisense PNAs against the antisense accessible sites were accordingly synthesized and characterized. The second effort was the investigation of cationic shell crosslinked knedel-like nanoparticle (cSCK)-mediated siRNA delivery to suppress iNOS expression for the treatment of ALI. siRNA with its unique gene-specific properties could serve as a promising therapeutic agent, however success in this area has been challenged by a lack of efficient biocompatible transfection agents. cSCK with its nanometer size and positive charge previously showed efficient cellular delivery of phosphorothioate ODNs (oligodeoxynucleotides), plasmid DNA and PNA. Herein, cSCK showed good siRNA binding and facilitated efficient siRNA transfection in HeLa, a mouse macrophage cell line and other human cell lines. cSCK led to greater silencing efficiency than Lipofectamine 2000 in HeLa cells as determined by the viability following transfection with cytotoxic and non-cytotoxic siRNAs, as well in 293T and HEK cells, and was comparable in BEAS-2B and MCF10a cells. The third effort was the preparation of an iNOS imaging probe through electrostatic complexation between a radiolabeled

  12. Furan oxidation based cross-linking: a new approach for the study and targeting of nucleic acid and protein interactions.

    Science.gov (United States)

    Carrette, L L G; Gyssels, E; De Laet, N; Madder, A

    2016-01-28

    Furan mediated nucleic acid cross-linking, initially developed for DNA interstrand duplex cross-linking, has matured into a versatile tool for the study of protein and nucleic acid interactions, ready to face its applications. The methodology was initially developed for easy and clean chemical generation of DNA interstrand cross-linked duplexes, but has been further expanded for use with other probes, targets and triggers, now allowing mild biologically significant cross-linking with potential therapeutic benefit. It was shown that the methodology could be repurposed for RNA interstrand cross-linking, which is very relevant in today's antisense approaches or miRNA target identification endeavors. This further illustrates the furan oxidation method's generality and mildness, especially when using red light for oxidation. A complementary antigene approach has been explored through duplex targeting with furan modified triplex forming oligonucleotides (TFOs) and DNA binding proteins. Also targeting of peptides and proteins by furan-modified DNA and peptides has been explored. Thorough methodology examination exploring variable reaction conditions in combination with a series of different furan-modified building blocks and application of different activation signals resulted in a detailed understanding of the mechanisms involved and factors influencing the yield and selectivity of the reaction. In order to draw the bigger picture of the scope and limitations of furan-oxidation cross-linking, we here provide a unique side by side comparison and discussion of our published data, supplemented with unpublished results, providing a clear performance report of the currently established furan toolbox and its application potential in various biomacromolecular complexes. PMID:26679922

  13. Functional correction by antisense therapy of a splicing mutation in the GALT gene.

    Science.gov (United States)

    Coelho, Ana I; Lourenço, Sílvia; Trabuco, Matilde; Silva, Maria João; Oliveira, Anabela; Gaspar, Ana; Diogo, Luísa; Tavares de Almeida, Isabel; Vicente, João B; Rivera, Isabel

    2015-04-01

    In recent years, antisense therapy has emerged as an increasingly important therapeutic approach to tackle several genetic disorders, including inborn errors of metabolism. Intronic mutations activating cryptic splice sites are particularly amenable to antisense therapy, as the canonical splice sites remain intact, thus retaining the potential for restoring constitutive splicing. Mutational analysis of Portuguese galactosemic patients revealed the intronic variation c.820+13A>G as the second most prevalent mutation, strongly suggesting its pathogenicity. The aim of this study was to functionally characterize this intronic variation, to elucidate its pathogenic molecular mechanism(s) and, ultimately, to correct it by antisense therapy. Minigene splicing assays in two distinct cell lines and patients' transcript analyses showed that the mutation activates a cryptic donor splice site, inducing an aberrant splicing of the GALT pre-mRNA, which in turn leads to a frameshift with inclusion of a premature stop codon (p.D274Gfs*17). Functional-structural studies of the recombinant wild-type and truncated GALT showed that the latter is devoid of enzymatic activity and prone to aggregation. Finally, two locked nucleic acid oligonucleotides, designed to specifically recognize the mutation, successfully restored the constitutive splicing, thus establishing a proof of concept for the application of antisense therapy as an alternative strategy for the clearly insufficient dietary treatment in classic galactosemia. PMID:25052314

  14. Electroporation increases antitumoral efficacy of the bcl-2 antisense G3139 and chemotherapy in a human melanoma xenograft

    OpenAIRE

    Baldi Alfonso; D'Angelo Carmen; Scarsella Marco; De Mori Roberta; Biroccio Annamaria; Spugnini Enrico P; Leonetti Carlo

    2011-01-01

    Abstract Background Nucleic acids designed to modulate the expression of target proteins remain a promising therapeutic strategy in several diseases, including cancer. However, clinical success is limited by the lack of efficient intracellular delivery. In this study we evaluated whether electroporation could increase the delivery of antisense oligodeoxynucleotides against bcl-2 (G3139) as well as the efficacy of combination chemotherapy in human melanoma xenografts. Methods Melanoma-bearing ...

  15. Method for isolating nucleic acids

    Science.gov (United States)

    Hurt, Jr., Richard Ashley; Elias, Dwayne A.

    2015-09-29

    The current disclosure provides methods and kits for isolating nucleic acid from an environmental sample. The current methods and compositions further provide methods for isolating nucleic acids by reducing adsorption of nucleic acids by charged ions and particles within an environmental sample. The methods of the current disclosure provide methods for isolating nucleic acids by releasing adsorbed nucleic acids from charged particles during the nucleic acid isolation process. The current disclosure facilitates the isolation of nucleic acids of sufficient quality and quantity to enable one of ordinary skill in the art to utilize or analyze the isolated nucleic acids for a wide variety of applications including, sequencing or species population analysis.

  16. Nucleic Acid Aptamers: Research Tools in Disease Diagnostics and Therapeutics

    OpenAIRE

    Baby Santosh; Pramod K. Yadava

    2014-01-01

    Aptamers are short sequences of nucleic acid (DNA or RNA) or peptide molecules which adopt a conformation and bind cognate ligands with high affinity and specificity in a manner akin to antibody-antigen interactions. It has been globally acknowledged that aptamers promise a plethora of diagnostic and therapeutic applications. Although use of nucleic acid aptamers as targeted therapeutics or mediators of targeted drug delivery is a relatively new avenue of research, one aptamer-based drug “Ma...

  17. C5a反义肽对脓毒症凝血异常及其急性肺损伤发病的影响%Effects of C5a anti-sense peptide on sepsis-induced abnormal coagulation and acute lung injury

    Institute of Scientific and Technical Information of China (English)

    郭振辉; 姜巧; 陈蕊; 孙杰; 俞宙; 肖飞; 熊日成; 吕凤林

    2011-01-01

    目的 观察C5a反义肽对脓毒症凝血异常及ALI发病的影响。方法 盲肠结扎穿孔复制小鼠脓毒症ALI模型,随机(随机数字法)分为健康组、假手术组、造模组和C5a反义肽干预组,分别在2,4,8,12 h时点观察肺组织损伤的病理改变和凝血指标;并采用ELISA检测血浆TF质量浓度的变化。结果 盲肠结扎穿孔后,2h后出现肺组织炎症细胞浸润、蛋白渗出和毛细血管内弥漫血栓形成,并进行性加重;而反义肽干预后的不同时间点上述病理改变均显著改善。造模组和C5a反义肽干预组,PT,TT,APTT进行性延长和DD进行性升高,而Fbg和PLT计数进行性降低,与健康组和假手术组比较P<0.05;而与造模组比较,反义肽干预后相应时间点的各指标明显改善(P<0.05)。TF质量浓度在健康组、假手术组分别为(42.75 ±1.25) pg/mL和(60.73±1.43) pg/mL;造模组2,4,6,12 h时点其浓度进行性升高,其中,12 h组最高为(139.81±6.17) pg/mL;而与造模组相比,反义肽组各相应时间点明显降低,12 h组为(121.41±7.21)pg/mL,各组比较有统计学意义(P<0.05)。结论 CSa反义肽能改善脓毒症的促凝亢进与ALI/ARDS的病理改变。%Objective To investigate the effects of C 5a anti-sense peptide on the pathogenesis of sepsis -induced abnormal coagulation and acute lung injury (ALI) in the early stage of sepsis. Methods The method of cecal ligature and puncture (CLP) was used to establish sepsis modeL Mice were randomly (random number) divided into control group, sham operation group, model group and C5a anti-sense peptide treatment group. In each group, pathological changes of the lung tissue and coagulation marker of mice were observed at intervals of 2 h, 4 h, 8 h and 12 hours after CLP. Meanwhile, the blood levels of tissue factor (TF) were detected by using ELISA at various intervals. Results The microscopicexamination showed that inflammatory

  18. Human Immunodeficiency Virus-Type 1 LTR DNA contains an intrinsic gene producing antisense RNA and protein products

    Directory of Open Access Journals (Sweden)

    Hsiao Chiu-Bin

    2006-11-01

    Full Text Available Abstract Background While viruses have long been shown to capitalize on their limited genomic size by utilizing both strands of DNA or complementary DNA/RNA intermediates to code for viral proteins, it has been assumed that human retroviruses have all their major proteins translated only from the plus or sense strand of RNA, despite their requirement for a dsDNA proviral intermediate. Several studies, however, have suggested the presence of antisense transcription for both HIV-1 and HTLV-1. More recently an antisense transcript responsible for the HTLV-1 bZIP factor (HBZ protein has been described. In this study we investigated the possibility of an antisense gene contained within the human immunodeficiency virus type 1 (HIV-1 long terminal repeat (LTR. Results Inspection of published sequences revealed a potential transcription initiator element (INR situated downstream of, and in reverse orientation to, the usual HIV-1 promoter and transcription start site. This antisense initiator (HIVaINR suggested the possibility of an antisense gene responsible for RNA and protein production. We show that antisense transcripts are generated, in vitro and in vivo, originating from the TAR DNA of the HIV-1 LTR. To test the possibility that protein(s could be translated from this novel HIV-1 antisense RNA, recombinant HIV antisense gene-FLAG vectors were designed. Recombinant protein(s were produced and isolated utilizing carboxy-terminal FLAG epitope (DYKDDDDK sequences. In addition, affinity-purified antisera to an internal peptide derived from the HIV antisense protein (HAP sequences identified HAPs from HIV+ human peripheral blood lymphocytes. Conclusion HIV-1 contains an antisense gene in the U3-R regions of the LTR responsible for both an antisense RNA transcript and proteins. This antisense transcript has tremendous potential for intrinsic RNA regulation because of its overlap with the beginning of all HIV-1 sense RNA transcripts by 25 nucleotides. The

  19. Therapeutic nucleic acids: current clinical status.

    Science.gov (United States)

    Sridharan, Kannan; Gogtay, Nithya Jaideep

    2016-09-01

    Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are simple linear polymers that have been the subject of considerable research in the last two decades and have now moved into the realm of being stand-alone therapeutic agents. Much of this has stemmed from the appreciation that they carry out myriad functions that go beyond mere storage of genetic information and protein synthesis. Therapy with nucleic acids either uses unmodified DNA or RNA or closely related compounds. From both a development and regulatory perspective, they fall somewhere between small molecules and biologics. Several of these compounds are in clinical development and many have received regulatory approval for human use. This review addresses therapeutic uses of DNA based on antisense oligonucleotides, DNA aptamers and gene therapy; and therapeutic uses of RNA including micro RNAs, short interfering RNAs, ribozymes, RNA decoys and circular RNAs. With their specificity, functional diversity and limited toxicity, therapeutic nucleic acids hold enormous promise. However, challenges that need to be addressed include targeted delivery, mass production at low cost, sustaining efficacy and minimizing off-target toxicity. Technological developments will hold the key to this and help accelerate drug approvals in the years to come. PMID:27111518

  20. Nucleic acid-based approaches to STAT inhibition.

    Science.gov (United States)

    Sen, Malabika; Grandis, Jennifer R

    2012-10-01

    Silencing of abnormally activated genes can be accomplished in a highly specific manner using nucleic acid based approaches. The focus of this review includes the different nucleic acid based inhibition strategies such as antisense oligodeoxynucleotides, small interfering RNA (siRNA), dominant-negative constructs, G-quartet oligonucleotides and decoy oligonucleotides, their mechanism of action and the effectiveness of these approaches to targeting the STAT (signal transducer and activator of transcription) proteins in cancer. Among the STAT proteins, especially STAT3, followed by STAT5, are the most frequently activated oncogenic STATs, which have emerged as plausible therapeutic cancer targets. Both STAT3 and STAT5 have been shown to regulate numerous oncogenic signaling pathways including proliferation, survival, angiogenesis and migration/invasion. PMID:24058785

  1. Analysis and Diagnosis of Nucleic Acid Based and Protein Peptide in Aged Patients with Coronary Heart Disease%老龄冠心病患者的核酸基与蛋白多肽分析及诊疗

    Institute of Scientific and Technical Information of China (English)

    陈龙皓; 严金龙

    2015-01-01

    Objective:In aged patients with coronary heart disease (CHD) nucleic acid and protein polypeptide has different characteristics, prevention of coronary heart disease. To achieve the effective diagnosis and treatment of aged patients with coronary heart disease (CHD). Methods:To a specialized hospital of 33 cases of senile patients with coronary heart disease for subjects and for patients with clinical follow-up observation and biochemical index test, in aged patients with coronary heart disease in vivo nucleic acid bases and protein peptide feature. Effect on coronary heart disease of TSH suppression. In the polymerization of high density lipoprotein cholesterol, contrast rate was 3 ml/s, the Valsalva test, the kinase glo lu-minescent kinase assay kit determination of protein lipid body phospholipid and protein content, using SPSS12.0 software was used for statistical analysis and treatment scheme. Results:the carbon carbon triple bond series transformation, when free radicals is added to the reaction system caused agent (AIBN, 10 mol%), formation of optical coherence tomography (OCT), combined with auxiliary examination found evidence of myocardial ischemia and coronary occlusion, coronary angi-ography was performed, statistical analysis and treatment for aged patients with coronary heart disease. By adjusting the dif-ferent time protein polypeptide and nucleic acid bases. Conclusion:Check up method in aged patients with coronary heart disease (CHD) including routine electrocardiogram (ECG) and electrocardiogram stress test, nuclide myocardial imaging, and screening of high-risk patients with coronary artery disease, using the method of stable coronary artery disease and acute coronary artery syndrome has significant curative effect and preventive.%目的:老龄冠心病患者体内的核酸基与蛋白多肽具有差异性特征,通过对该类核酸基与蛋白多肽差异性特征进行分析,了解并干预危险因素,进行冠心病预防,实现对老龄

  2. Antiviral Effects of Antisense Morpholino Oligomers in Murine Coronavirus Infection Models▿

    OpenAIRE

    Burrer, Renaud; Neuman, Benjamin W.; Ting, Joey P.C.; Stein, David A.; Moulton, Hong M.; Iversen, Patrick L.; Kuhn, Peter; Michael J Buchmeier

    2007-01-01

    The recent emergence of novel pathogenic human and animal coronaviruses has highlighted the need for antiviral therapies that are effective against a spectrum of these viruses. We have used several strains of murine hepatitis virus (MHV) in cell culture and in vivo in mouse models to investigate the antiviral characteristics of peptide-conjugated antisense phosphorodiamidate morpholino oligomers (P-PMOs). Ten P-PMOs directed against various target sites in the viral genome were tested in cell...

  3. Neutron Nucleic Acid Crystallography.

    Science.gov (United States)

    Chatake, Toshiyuki

    2016-01-01

    The hydration shells surrounding nucleic acids and hydrogen-bonding networks involving water molecules and nucleic acids are essential interactions for the structural stability and function of nucleic acids. Water molecules in the hydration shells influence various conformations of DNA and RNA by specific hydrogen-bonding networks, which often contribute to the chemical reactivity and molecular recognition of nucleic acids. However, X-ray crystallography could not provide a complete description of structural information with respect to hydrogen bonds. Indeed, X-ray crystallography is a powerful tool for determining the locations of water molecules, i.e., the location of the oxygen atom of H2O; however, it is very difficult to determine the orientation of the water molecules, i.e., the orientation of the two hydrogen atoms of H2O, because X-ray scattering from the hydrogen atom is very small.Neutron crystallography is a specialized tool for determining the positions of hydrogen atoms. Neutrons are not diffracted by electrons, but are diffracted by atomic nuclei; accordingly, neutron scattering lengths of hydrogen and its isotopes are comparable to those of non-hydrogen atoms. Therefore, neutron crystallography can determine both of the locations and orientations of water molecules. This chapter describes the current status of neutron nucleic acid crystallographic research as well as the basic principles of neutron diffraction experiments performed on nucleic acid crystals: materials, crystallization, diffraction experiments, and structure determination.

  4. PCSK9 LNA antisense oligonucleotides induce sustained reduction of LDL cholesterol in nonhuman primates.

    Science.gov (United States)

    Lindholm, Marie W; Elmén, Joacim; Fisker, Niels; Hansen, Henrik F; Persson, Robert; Møller, Marianne R; Rosenbohm, Christoph; Ørum, Henrik; Straarup, Ellen M; Koch, Troels

    2012-02-01

    Proprotein convertase subtilisin/kexin type 9 (PCSK9) has emerged as a therapeutic target for the reduction of low-density lipoprotein cholesterol (LDL-C). PCSK9 increases the degradation of the LDL receptor, resulting in high LDL-C in individuals with high PCSK9 activity. Here, we show that two locked nucleic acid (LNA) antisense oligonucleotides targeting PCSK9 produce sustained reduction of LDL-C in nonhuman primates after a loading dose (20 mg/kg) and four weekly maintenance doses (5 mg/kg). PCSK9 messenger RNA (mRNA) and serum PCSK9 protein were reduced by 85% which resulted in a 50% reduction in circulating LDL-C. Serum total cholesterol (TC) levels were reduced to the same extent as LDL-C with no reduction in high-density lipoprotein levels, demonstrating a specific pharmacological effect on LDL-C. The reduction in hepatic PCSK9 mRNA correlated with liver LNA oligonucleotide content. This verified that anti-PCSK9 LNA oligonucleotides regulated LDL-C through an antisense mechanism. The compounds were well tolerated with no observed effects on toxicological parameters (liver and kidney histology, alanine aminotransferase, aspartate aminotransferase, urea, and creatinine). The pharmacologic evidence and initial safety profile of the compounds used in this study indicate that LNA antisense oligonucleotides targeting PCSK9 provide a viable therapeutic strategy and are potential complements to statins in managing high LDL-C.

  5. Re-sensitizing drug-resistant bacteria to antibiotics by designing Antisense Therapeutics

    Science.gov (United States)

    Courtney, Colleen; Chatterjee, Anushree

    2014-03-01

    ``Super-bugs'' or ``multi-drug resistant organisms'' are a serious international health problem, with devastating consequences to patient health care. The Center for Disease Control has identified antibiotic resistance as one of the world's most pressing public health problems as a significant fraction of bacterial infections contracted are drug resistant. Typically, antibiotic resistance is encoded by ``resistance-genes'' which express proteins that carryout the resistance causing functions inside the bacterium. We present a RNA based therapeutic strategy for designing antimicrobials capable of re-sensitizing resistant bacteria to antibiotics by targeting labile regions of messenger RNAs encoding for resistance-causing proteins. We perform in silico RNA secondary structure modeling to identify labile target regions in an mRNA of interest. A synthetic biology approach is then used to administer antisense nucleic acids to our model system of ampicillin resistant Escherichia coli. Our results show a prolonged lag phase and decrease in viability of drug-resistant E. colitreated with antisense molecules. The antisense strategy can be applied to alter expression of other genes in antibiotic resistance pathways or other pathways of interest.

  6. Calorimetry of Nucleic Acids.

    Science.gov (United States)

    Rozners, Eriks; Pilch, Daniel S; Egli, Martin

    2015-12-01

    This unit describes the application of calorimetry to characterize the thermodynamics of nucleic acids, specifically, the two major calorimetric methodologies that are currently employed: differential scanning (DSC) and isothermal titration calorimetry (ITC). DSC is used to study thermally induced order-disorder transitions in nucleic acids. A DSC instrument measures, as a function of temperature (T), the excess heat capacity (C(p)(ex)) of a nucleic acid solution relative to the same amount of buffer solution. From a single curve of C(p)(ex) versus T, one can derive the following information: the transition enthalpy (ΔH), entropy (ΔS), free energy (ΔG), and heat capacity (ΔCp); the state of the transition (two-state versus multistate); and the average size of the molecule that melts as a single thermodynamic entity (e.g., the duplex). ITC is used to study the hybridization of nucleic acid molecules at constant temperature. In an ITC experiment, small aliquots of a titrant nucleic acid solution (strand 1) are added to an analyte nucleic acid solution (strand 2), and the released heat is monitored. ITC yields the stoichiometry of the association reaction (n), the enthalpy of association (ΔH), the equilibrium association constant (K), and thus the free energy of association (ΔG). Once ΔH and ΔG are known, ΔS can also be derived. Repetition of the ITC experiment at a number of different temperatures yields the ΔCp for the association reaction from the temperature dependence of ΔH.

  7. Functionalization of an Antisense Small RNA

    Science.gov (United States)

    Rodrigo, Guillermo; Prakash, Satya; Cordero, Teresa; Kushwaha, Manish; Jaramillo, Alfonso

    2016-01-01

    In order to explore the possibility of adding new functions to preexisting genes, we considered a framework of riboregulation. We created a new riboregulator consisting of the reverse complement of a known riboregulator. Using computational design, we engineered a cis-repressing 5′ untranslated region that can be activated by this new riboregulator. As a result, both RNAs can orthogonally trans-activate translation of their cognate, independent targets. The two riboregulators can also repress each other by antisense interaction, although not symmetrically. Our work highlights that antisense small RNAs can work as regulatory agents beyond the antisense paradigm and that, hence, they could be interfaced with other circuits used in synthetic biology. PMID:26756967

  8. Invasive cleavage of nucleic acids

    Science.gov (United States)

    Prudent, James R.; Hall, Jeff G.; Lyamichev, Victor I.; Brow, Mary Ann D.; Dahlberg, James E.

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof.

  9. 评估人类精子中原位非整倍体:引物原位杂交和缩氨酸核酸荧光原位杂交技术的使用%In situ aneuploidy assessment in human sperm: the use of primed in situ and peptide nucleic acid-fluorescence in situ hybridization techniques

    Institute of Scientific and Technical Information of China (English)

    F.Pellestor

    2006-01-01

    引物原位杂交(PRINS)和缩氨酸核酸荧光原位杂交(PNA-FISH)技术都可替代传统的荧光原位杂交(FISH)技术,用于染色体的研究.PRINS反应的基本物是DNA聚合酶和引物原位扩展反应中的标引核苷.缩氨酸核酸探针是合成的不带电聚酰胺主链的DNA类似物.这两种技术特异、快速、识别能力强的优点使它们细胞遗传学研究中很受欢迎.它们在人类精子研究中的应用使男性配子的染色体筛选过程得到新的快速发展,成为男性配子非整倍体原位评估中的FISH技术的有效补充.%Both the primed in situ (PRINS) and the peptide nucleic acid-fluorescence in situ hybridization (PNA-FISH)techniques constitute alternatives to the conventional (fluorescence in situ hybridization, FISH) procedure for chromosomal investigations. The PRINS reaction is based on the use of a DNA polymerase and labeled nucleotide in an in situ primer extension reaction. Peptide nucleic acid probes are synthetic DNA analogs with uncharged polyamide backbones. The two procedures present several advantages (specificity, rapidity and discriminating ability) that make them very attractive for cytogenetic purposes. Their adaptation to human spermatozoa has allowed the development of new and fast procedures for the chromosomal screening of male gametes and has provided efficient complements to FISH for in situ assessment of aneuploidy in male gametes.

  10. Chemosensitization by antisense oligonucleotides targeting MDM2.

    Science.gov (United States)

    Bianco, Roberto; Ciardiello, Fortunato; Tortora, Giampaolo

    2005-02-01

    The MDM2 oncogene is overexpressed in many human cancers, including sarcomas, certain hematologic malignancies, and breast, colon and prostate cancers. The p53-MDM2 interaction pathway has been suggested as a novel target for cancer therapy. To that end, several strategies have been explored, including the use of small polypeptides targeted to the MDM2-p53 binding domain, anti-MDM2 antisense oligonucleotides, and natural agents. Different generations of anti-human-MDM2 oligonucleotides have been tested in in vitro and in vivo human cancer models, revealing specific inhibition of MDM2 expression and significant antitumor activity. Use of antisense oligos potentiated the effects of growth inhibition, p53 activation and p21 induction by several chemotherapeutic agents. Increased therapeutic effectiveness of chemotherapeutic drugs in human cancer cell lines carrying p53 mutations or deletions have shown the ability of MDM2 inhibitors to act as chemosensitizers in various types of tumors through both p53-dependent and p53-independent mechanisms. Inhibiting MDM2 appears to also have a role in radiation therapy for human cancer, regardless of p53 status, providing a rationale for the development of a new class of radiosensitizers. Moreover, MDM2 antisense oligonucleotides potentiate the effect of epidermal growth factor receptor (EGFR) inhibitors by affecting in vitro and in vivo proliferation, apoptosis and protein expression in hormone-refractory and hormone-dependent human prostate cancer cells. These data support the development, among other MDM2 inhibitors, of anti-MDM2 antisense oligonucleotides as a novel class of anticancer agents, and suggest a potentially relevant role for the oligonucleotides when integrated with conventional treatments and/or other signaling inhibitors in novel therapeutic strategies.

  11. Biopolymers: protein and nucleic acids. Annual report, 15 September 1987-14 September 1988

    Energy Technology Data Exchange (ETDEWEB)

    Richards, J.H.; Abelson, J.N.; Dervan, P.B.; Hood, L.H.; Simon, M.I.

    1988-09-15

    The work focuses on learning the principles that govern interactions between proteins and nucleic acids, both DNA and RNA (specifically tRNA). With these principles as guides peptides (of about 50 amino acids) that bind to specific regions of DNA are being synthesized. Various reactive functionalities are being attached to the synthetic peptides to generate reagents that cleave DNA specifically at the site to which the peptide binds. The work also involves biophysical studies of the protein/nucleic acid complexes in order to expand our understanding of the principles of protein binding to nucleic acids. Development of improved procedures for the chemical synthesis of peptides forms another important aspect of the program.

  12. The prevalence and regulation of antisense transcripts in Schizosaccharomyces pombe.

    Directory of Open Access Journals (Sweden)

    Ting Ni

    Full Text Available A strand-specific transcriptome sequencing strategy, directional ligation sequencing or DeLi-seq, was employed to profile antisense transcriptome of Schizosaccharomyces pombe. Under both normal and heat shock conditions, we found that polyadenylated antisense transcripts are broadly expressed while distinct expression patterns were observed for protein-coding and non-coding loci. Dominant antisense expression is enriched in protein-coding genes involved in meiosis or stress response pathways. Detailed analyses further suggest that antisense transcripts are independently regulated with respect to their sense transcripts, and diverse mechanisms might be potentially involved in the biogenesis and degradation of antisense RNAs. Taken together, antisense transcription may have profound impacts on global gene regulation in S. pombe.

  13. Using both strands: The fundamental nature of antisense transcription.

    Science.gov (United States)

    Murray, Struan C; Mellor, Jane

    2016-01-01

    Non-coding transcription across the antisense strands of genes is an abundant, pervasive process in eukaryotes from yeast to humans, however its biological function remains elusive. Here, we provide commentary on a recent study of ours, which demonstrates a genome-wide role for antisense transcription: establishing a unique, dynamic chromatin architecture over genes. Antisense transcription increases the level of nucleosome occupancy and histone acetylation at the promoter and body of genes, without necessarily modulating the level of protein-coding sense transcription. It is also associated with high levels of histone turnover. By allowing genes to sample a wider range of chromatin configurations, antisense transcription could serve to make genes more sensitive to changing signals, priming them for responses to developmental programs or stressful cellular environments. Given the abundance of antisense transcription and the breadth of these chromatin changes, we propose that antisense transcription represents a fundamental, canonical feature of eukaryotic genes.

  14. Nucleic Acid Vaccines

    Institute of Scientific and Technical Information of China (English)

    LU Shan

    2004-01-01

    @@ Anew method of immunization was discovered in the early 1990s. Several research groups independently demonstrated that direct inoculation of DNA plasmids coding for a specific protein antigen could elicit immune responses against that antigen[1-4].Since in theory the mRNA molecules also have the potential to be translated into the protein antigen, this vaccination approach was officially named by WHO as the nucleic acid vaccination even though the term DNA vaccine has been used more commonly in the literature. This novel approach is considered the fourth generation of vaccines after live attenuated vaccines, killed or inactivated vaccines and recombinant protein based subunit vaccines.

  15. Inhibition, Escape, and Attenuated Growth of Severe Acute Respiratory Syndrome Coronavirus Treated with Antisense Morpholino Oligomers†

    OpenAIRE

    Neuman, Benjamin W.; Stein, David A.; Kroeker, Andrew D.; Churchill, Michael J.; Kim, Alice M.; Kuhn, Peter; Dawson, Philip; Moulton, Hong M.; Bestwick, Richard K.; Iversen, Patrick L.; Michael J Buchmeier

    2005-01-01

    The recently emerged severe acute respiratory syndrome coronavirus (SARS-CoV) is a potent pathogen of humans and is capable of rapid global spread. Peptide-conjugated antisense morpholino oligomers (P-PMO) were designed to bind by base pairing to specific sequences in the SARS-CoV (Tor2 strain) genome. The P-PMO were tested for their capacity to inhibit production of infectious virus as well as to probe the function of conserved viral RNA motifs and secondary structures. Several virus-targete...

  16. Progress toward therapy with antisense-mediated splicing modulation

    OpenAIRE

    Du, Liutao; Gatti, Richard A.

    2009-01-01

    Antisense oligonucleotides (AO) or antisense RNA can complementarily bind to a target site in pre-mRNA and regulate gene splicing, either to restore gene function by reprogramming gene splicing or to inhibit gene expression by disrupting splicing. These two applications represent novel therapeutic strategies for several types of diseases such as genetic disorders, cancers and infectious diseases. In this review, the recent developments and applications of antisense-mediated splicing modulatio...

  17. Diversification of antisense research and development: review of the Ringberg meeting, April 1994. Mechanisms of antisense-mediated gene silencing.

    Science.gov (United States)

    Hawkins, J W; Nellen, W

    1994-01-01

    Antisense technology has established itself as a new and vibrant entrant into the discipline of molecular biology. As such, it has contributed to basic research by providing tools for the molecular dissection of diverse experimental systems. In applied research, antisense approaches have contributed to development of agricultural products (D. Grierson) now coming to market and to the design of a number of oligonucleotide drugs, now in clinical trials. However, few activities to date have focused on the study of antisense per se. Further, few conceptual perspectives have regarded antisense as an integral part of cellular function and genetic regulation. The Ringberg conference showcased a number of systems that would seem unrelated if we regard antisense as a superficial tool to be imposed on nature. On the other hand, if we want to begin to regard antisense as a field of its own with deeper biological and genetic rationales, the Ringberg meeting provided much tantalizing evidence to do so.

  18. Antisense treatment of caliciviridae: an emerging disease agent of animals and humans.

    Science.gov (United States)

    Smith, Alvin W; Matson, David O; Stein, David A; Skilling, Douglas E; Kroeker, Andrew D; Berke, Tamas; Iversen, Patrick L

    2002-04-01

    The Earth's oceans are the primary reservoir for an emerging family of RNA viruses, the Caliciviridae, which can cause a spectrum of diseases in marine animals, wildlife, farm animals, pets and humans. Certain members of this family have unusually broad host ranges, and some are zoonotic (transmissible from animals to humans). The RNA virus replicative processes lack effective genetic repair mechanisms, and, therefore, virtually every calicivirus replicate is a mutant. Hence, traditional therapeutics dependent on specific nucleic acid sequences or protein epitopes lack the required diversity of sequence or conformational specificity that would be required to reliably detect, prevent or treat infections from these mutant clusters (quasi-species) of RNA viruses, including the Caliciviridae. Antisense technology using phosphorodiamidate morpholino oligomers shows promise in overcoming these current diagnostic and therapeutic problems inherent with newly emerging viral diseases. PMID:12044040

  19. Design and synthesis of dendritic molecular transporter that achieves efficient in vivo delivery of morpholino antisense oligo.

    Science.gov (United States)

    Li, Yong-Fu; Morcos, Paul A

    2008-07-01

    Safe and efficient in vivo delivery of Morpholino antisense oligos was probably the last and most difficult challenge for the broad application of antisense in animal research and therapeutics. Several arginine-rich peptides effective for in vivo delivery of Morpholino antisense oligos require rather complex and expensive procedures for synthesis and conjugation. This work describes the design and synthesis of a dendritic transporter in a most concise manner where the selection of the core scaffold, functional group multiplication, orthogonal protecting group manipulation, solid phase conjugation, and off-resin perguanidinylation of the transporter structure are all orchestrated for efficient assembly. We utilized triazine as a core to provide a site for on-column conjugation to the Morpholino oligo and to anchor functional side arms which, after extension, multiplication, and deprotection, are subsequently converted from primary amines to the eight guanidinium headgroups that serve for transport across cell membranes. Intravenous administration of the delivery-enabled Morpholino into a splice-reporter strain of transgenic living mice results in de novo expression of splice-corrected green fluorescent protein in a broad range of tissues and organs in those treated mice. This rigorously demonstrates that this new dendritic transporter achieves effective delivery of a Morpholino oligo into the cytosol/nuclear compartment of cells systemically in vivo. The practical conjugation process may overcome any availability limitation for routine use by the scientific community, and the efficient delivery ability of this transporter may advance the application of Morpholino antisense technology in animals.

  20. Nucleic Acid Charge Transfer: Black, White and Gray

    OpenAIRE

    Venkatramani, Ravindra; Keinan, Shahar; Balaeff, Alexander; Beratan, David N.

    2011-01-01

    Theoretical studies of charge transport in deoxyribonucleic acid (DNA) and peptide nucleic acid (PNA) indicate that structure and dynamics modulate the charge transfer rates, and that different members of a structural ensemble support different charge transport mechanisms. Here, we review the influences of nucleobase geometry, electronic structure, solvent environment, and thermal conformational fluctuations on the charge transfer mechanism. We describe an emerging framework for understanding...

  1. Lipophilic nucleic acids--a flexible construction kit for organization and functionalization of surfaces.

    Science.gov (United States)

    Schade, Matthias; Berti, Debora; Huster, Daniel; Herrmann, Andreas; Arbuzova, Anna

    2014-06-01

    Lipophilic nucleic acids have become a versatile tool for structuring and functionalization of lipid bilayers and biological membranes as well as cargo vehicles to transport and deliver bioactive compounds, like interference RNA, into cells by taking advantage of reversible hybridization with complementary strands. This contribution reviews the different types of conjugates of lipophilic nucleic acids, and their physicochemical and self-assembly properties. Strategies for choosing a nucleic acid, lipophilic modification, and linker are discussed. Interaction with lipid membranes and its stability, dynamic structure and assembly of lipophilic nucleic acids upon embedding into biological membranes are specific points of the review. A large diversity of conjugates including lipophilic peptide nucleic acid and siRNA provides tailored solutions for specific applications in bio- and nanotechnology as well as in cell biology and medicine, as illustrated through some selected examples. PMID:24650567

  2. Voltage-gated calcium channel and antisense oligonucleotides thereto

    Science.gov (United States)

    Hruska, Keith A. (Inventor); Friedman, Peter A. (Inventor); Barry, Elizabeth L. R. (Inventor); Duncan, Randall L. (Inventor)

    1998-01-01

    An antisense oligonucleotide of 10 to 35 nucleotides in length that can hybridize with a region of the .alpha..sub.1 subunit of the SA-Cat channel gene DNA or mRNA is provided, together with pharmaceutical compositions containing and methods utilizing such antisense oligonucleotide.

  3. The landscape of antisense gene expression in human cancers.

    Science.gov (United States)

    Balbin, O Alejandro; Malik, Rohit; Dhanasekaran, Saravana M; Prensner, John R; Cao, Xuhong; Wu, Yi-Mi; Robinson, Dan; Wang, Rui; Chen, Guoan; Beer, David G; Nesvizhskii, Alexey I; Chinnaiyan, Arul M

    2015-07-01

    High-throughput RNA sequencing has revealed more pervasive transcription of the human genome than previously anticipated. However, the extent of natural antisense transcripts' (NATs) expression, their regulation of cognate sense genes, and the role of NATs in cancer remain poorly understood. Here, we use strand-specific paired-end RNA sequencing (ssRNA-seq) data from 376 cancer samples covering nine tissue types to comprehensively characterize the landscape of antisense expression. We found consistent antisense expression in at least 38% of annotated transcripts, which in general is positively correlated with sense gene expression. Investigation of sense/antisense pair expressions across tissue types revealed lineage-specific, ubiquitous and cancer-specific antisense loci transcription. Comparisons between tumor and normal samples identified both concordant (same direction) and discordant (opposite direction) sense/antisense expression patterns. Finally, we provide OncoNAT, a catalog of cancer-related genes with significant antisense transcription, which will enable future investigations of sense/antisense regulation in cancer. Using OncoNAT we identified several functional NATs, including NKX2-1-AS1 that regulates the NKX2-1 oncogene and cell proliferation in lung cancer cells. Overall, this study provides a comprehensive account of NATs and supports a role for NATs' regulation of tumor suppressors and oncogenes in cancer biology. PMID:26063736

  4. Antisense transcription as a tool to tune gene expression.

    Science.gov (United States)

    Brophy, Jennifer A N; Voigt, Christopher A

    2016-01-14

    A surprise that has emerged from transcriptomics is the prevalence of genomic antisense transcription, which occurs counter to gene orientation. While frequent, the roles of antisense transcription in regulation are poorly understood. We built a synthetic system in Escherichia coli to study how antisense transcription can change the expression of a gene and tune the response characteristics of a regulatory circuit. We developed a new genetic part that consists of a unidirectional terminator followed by a constitutive antisense promoter and demonstrate that this part represses gene expression proportionally to the antisense promoter strength. Chip-based oligo synthesis was applied to build a large library of 5,668 terminator-promoter combinations that was used to control the expression of three repressors (PhlF, SrpR, and TarA) in a simple genetic circuit (NOT gate). Using the library, we demonstrate that antisense promoters can be used to tune the threshold of a regulatory circuit without impacting other properties of its response function. Finally, we determined the relative contributions of antisense RNA and transcriptional interference to repressing gene expression and introduce a biophysical model to capture the impact of RNA polymerase collisions on gene repression. This work quantifies the role of antisense transcription in regulatory networks and introduces a new mode to control gene expression that has been previously overlooked in genetic engineering.

  5. Cycloadditions for Studying Nucleic Acids.

    Science.gov (United States)

    Kath-Schorr, Stephanie

    2016-02-01

    Cycloaddition reactions for site-specific or global modification of nucleic acids have enabled the preparation of a plethora of previously inaccessible DNA and RNA constructs for structural and functional studies on naturally occurring nucleic acids, the assembly of nucleic acid nanostructures, therapeutic applications, and recently, the development of novel aptamers. In this chapter, recent progress in nucleic acid functionalization via a range of different cycloaddition (click) chemistries is presented. At first, cycloaddition/click chemistries already used for modifying nucleic acids are summarized, ranging from the well-established copper(I)-catalyzed alkyne-azide cycloaddition reaction to copper free methods, such as the strain-promoted azide-alkyne cycloaddition, tetrazole-based photoclick chemistry and the inverse electron demand Diels-Alder cycloaddition reaction between strained alkenes and tetrazine derivatives. The subsequent sections contain selected applications of nucleic acid functionalization via click chemistry; in particular, site-specific enzymatic labeling in vitro, either via DNA and RNA recognizing enzymes or by introducing unnatural base pairs modified for click reactions. Further sections report recent progress in metabolic labeling and fluorescent detection of DNA and RNA synthesis in vivo, click nucleic acid ligation, click chemistry in nanostructure assembly and click-SELEX as a novel method for the selection of aptamers. PMID:27572987

  6. Vector insert-targeted integrative antisense expression system for plasmid stabilization.

    Science.gov (United States)

    Luke, Jeremy M; Carnes, Aaron E; Hodgson, Clague P; Williams, James A

    2011-01-01

    Some DNA vaccine and gene therapy vector-encoded transgenes are toxic to the E. coli plasmid production host resulting in poor production yields. For plasmid products undergoing clinical evaluation, sequence modification to eliminate toxicity is undesirable because an altered vector is a new chemical entity. We hypothesized that: (1) insert-encoded toxicity is mediated by unintended expression of a toxic insert-encoded protein from spurious bacterial promoters; and (2) that toxicity could be eliminated with antisense RNA-mediated translation inhibition. We developed the pINT PR PL vector, a chromosomally integrable RNA expression vector, and utilized it to express insert-complementary (anti-insert) RNA from a single defined site in the bacterial chromosome. Anti-insert RNA eliminated leaky fluorescent protein expression from a target plasmid. A toxic retroviral gag pol helper plasmid produced in a gag pol anti-insert strain had fourfold improved plasmid fermentation yields. Plasmid fermentation yields were also fourfold improved when a DNA vaccine plasmid containing a toxic Influenza serotype H1 hemagglutinin transgene was grown in an H1 sense strand anti-insert production strain, suggesting that in this case toxicity was mediated by an antisense alternative reading frame-encoded peptide. This anti-insert chromosomal RNA expression technology is a general approach to improve production yields with plasmid-based vectors that encode toxic transgenes, or toxic alternative frame peptides. PMID:20607625

  7. Nucleic acid interactions with pyrite surfaces

    Science.gov (United States)

    Mateo-Martí, E.; Briones, C.; Rogero, C.; Gomez-Navarro, C.; Methivier, Ch.; Pradier, C. M.; Martín-Gago, J. A.

    2008-09-01

    The study of the interaction of nucleic acid molecules with mineral surfaces is a field of growing interest in organic chemistry, origin of life, material science and biotechnology. We have characterized the adsorption of single-stranded peptide nucleic acid (ssPNA) on a natural pyrite surface, as well as the further adsorption of ssDNA on a PNA-modified pyrite surface. The characterization has been performed by means of reflection absorption infrared spectroscopy (RAIRS), atomic force microscopy (AFM) and X-ray photoemission spectroscopy (XPS) techniques. The N(1s) and S(2p) XPS core level peaks of PNA and PNA + DNA have been decomposed in curve-components that we have assigned to different chemical species. RAIRS spectra recorded for different concentrations show the presence of positive and negative adsorption bands, related to the semiconducting nature of the surface. The combination of the information gathered by these techniques confirms that PNA adsorbs on pyrite surface, interacting through nitrogen-containing groups of the nucleobases and the iron atoms of the surface, instead of the thiol group of the molecule. The strong PNA/pyrite interaction inhibits further hybridization of PNA with complementary ssDNA, contrary to the behavior reported on gold surfaces.

  8. Nucleic Acid Drugs for Prevention of Cardiac Rejection

    Directory of Open Access Journals (Sweden)

    Jun-ichi Suzuki

    2009-01-01

    Full Text Available Heart transplantation has been broadly performed in humans. However, occurrence of acute and chronic rejection has not yet been resolved. Several inflammatory factors, such as cytokines and adhesion molecules, enhance the rejection. The graft arterial disease (GAD, which is a type of chronic rejection, is characterized by intimal thickening comprised of proliferative smooth muscle cells. Specific treatments that target the attenuation of acute rejection and GAD formation have not been well studied in cardiac transplantation. Recent progress in the nucleic acid drugs, such as antisense oligodeoxynucleotides (ODNs to regulate the transcription of disease-related genes, has important roles in therapeutic applications. Transfection of cis-element double-stranded DNA, named as “decoy,” has been also reported to be a useful nucleic acid drug. This decoy strategy has been not only a useful method for the experimental studies of gene regulation but also a novel clinical strategy. In this paper, we reviewed the experimental results of NF-κB, E2F, AP-1, and STAT-1 decoy and other ODNs using the experimental heart transplant models.

  9. Origin of nucleic acids

    International Nuclear Information System (INIS)

    The appearance of nucleic acids is the first event after the birth of membranes which made it possible to assure the perenniality of information. The complexity of these molecules has led some scientists to propose that they were not prebiotic but rather derived a more simple and achiral primitive ancestor. This hypothesis suggests that ribose possesses properties that allowed the formation of certain polysaccharides which evolved to RNA. The first step of the hypothesis is the selection and concentration of ribofuranose. This sugar has chelating properties and its alpha-ribofuranose is favoured in the chelating position. The density of the sugar with a heavy cation is greater than water and thus the complex can escape the UV radiation at the surface of the ocean. The particularity of ribose is to be able to form a homochiral regular array of these basic chelating structures with pyrophosphite. These arrays evolve towards the formation of polysaccharides (poly ribose phosphate) which have a very organized structure. These polysaccharides in turn evolve to RNA by binding of adenine and deoxyguanine which are HCN derivatives that can react with the polysaccharides. The primitive RNA is methylated and oxidized to form prebiotic RNA with adenosine, cytidine, 7methyl-guanosine and ribothymidine as nucleic bases. The pathway of biosynthesis of DNA form RNA will be studied. I suggest that the appearance of DNA results form the interaction between prebiotic double stranded RNA and proteins. DNA could be a product of RNA degradation by proteins. The catabolism of RNA to DNA requires a source of free radicals, protons and hydrides. RNA cannot produce free radicals, which are provided by the phenol group of the amino acid tyrosien. Protons are provided by the medium and hydrides are provided by 7-methyl-guanosine which can fix hydrides coming from hydrogen gas and donate them for the transformation of a riboside to a deoxyriboside. This pathway suggests that DNA appeared at

  10. Serial incorporation of a monovalent GalNAc phosphoramidite unit into hepatocyte-targeting antisense oligonucleotides.

    Science.gov (United States)

    Yamamoto, Tsuyoshi; Sawamura, Motoki; Wada, Fumito; Harada-Shiba, Mariko; Obika, Satoshi

    2016-01-01

    The targeting of abundant hepatic asialoglycoprotein receptors (ASGPR) with trivalent N-acetylgalactosamine (GalNAc) is a reliable strategy for efficiently delivering antisense oligonucleotides (ASOs) to the liver. We here experimentally demonstrate the high systemic potential of the synthetically-accessible, phosphodiester-linked monovalent GalNAc unit when tethered to the 5'-terminus of well-characterised 2',4'-bridged nucleic acid (also known as locked nucleic acid)-modified apolipoprotein B-targeting ASO via a bio-labile linker. Quantitative analysis of the hepatic disposition of the ASOs revealed that phosphodiester is preferable to phosphorothioate as an interunit linkage in terms of ASGPR binding of the GalNAc moiety, as well as the subcellular behavior of the ASO. The flexibility of this monomeric unit was demonstrated by attaching up to 5 GalNAc units in a serial manner and showing that knockdown activity improves as the number of GalNAc units increases. Our study suggests the structural requirements for efficient hepatocellular targeting using monovalent GalNAc and could contribute to a new molecular design for suitably modifying ASO.

  11. Optimizing RNA/ENA chimeric antisense oligonucleotides using in vitro splicing.

    Science.gov (United States)

    Takeshima, Yasuhiro; Yagi, Mariko; Matsuo, Masafumi

    2012-01-01

    A molecular therapy for Duchenne muscular dystrophy (DMD) that converts dystrophin mRNA from out-of-frame to in-frame transcripts by inducing exon skipping with antisense oligonucleotides (AOs) is now approaching clinical application. To exploit the broad therapeutic applicability of exon skipping therapy, it is necessary to identify AOs that are able to induce efficient and specific exon skipping. To optimize AOs, we have established an in vitro splicing system using cultured DMD myocytes. Here, we describe the process of identifying the best AO.Cultured DMD myocytes are established from a biopsy sample and the target exon is chosen. A series of AOs are designed to cover the whole target exon sequence. As AOs, we use 15-20-mer chimeric oligonucleotides consisting of 2'-O-methyl RNA and modified nucleic acid (2'-O, 4'-C-ethylene-bridged nucleic acid). Each AO is transfected individually into cultured DMD myocytes, and the resulting mRNA is analyzed by reverse transcription-PCR. The ability of each AO to induce exon skipping is examined by comparing the amount of cDNA with and without exon skipping. If necessary, having roughly localized the target region, another set of AOs are designed and the exon skipping abilities of the new AOs are examined. Finally, one AO is determined as the best for the molecular therapy.Our simple and reliable methods using an in vitro splicing system have enabled us to identify optimized AOs against many exons of the DMD gene.

  12. Interaction of nucleic acids with carbon nanotubes and dendrimers

    Indian Academy of Sciences (India)

    Bidisha Nandy; Mogurampelly Santosh; Prabal K Maiti

    2012-07-01

    Nucleic acid interaction with nanoscale objects like carbon nanotubes (CNTs) and dendrimers is of fundamental interest because of their potential application in CNT separation, gene therapy and antisense therapy. Combining nucleic acids with CNTs and dendrimers also opens the door towards controllable self-assembly to generate various supra-molecular and nano-structures with desired morphologies. The interaction between these nanoscale objects also serve as a model system for studying DNA compaction, which is a fundamental process in chromatin organization. By using fully atomistic simulations, here we report various aspects of the interactions and binding modes of DNA and small interfering RNA (siRNA) with CNTs, graphene and dendrimers. Our results give a microscopic picture and mechanism of the adsorption of single- and double-strand DNA (ssDNA and dsDNA) on CNT and graphene. The nucleic acid–CNT interaction is dominated by the dispersive van der Waals (vdW) interaction. In contrast, the complexation of DNA (both ssDNA and dsDNA) and siRNA with various generations of poly-amido-amine (PAMAM) dendrimers is governed by electrostatic interactions. Our results reveal that both the DNA and siRNA form stable complex with the PAMAM dendrimer at a physiological pH when the dendrimer is positively charged due to the protonation of the primary amines. The size and binding energy of the complex increase with increase in dendrimer generation. We also give a summary of the current status in these fields and discuss future prospects.

  13. Study on peptide-peptide interaction using high-performance affinity chromatography and quartz crystal microbalance biosensor

    Institute of Scientific and Technical Information of China (English)

    LUO Jia; HUANG YanYan; XIONG ShaoXiang; LIU GuoQuan; ZHAO Rui

    2007-01-01

    The specific interaction between sense and antisense peptides was studied by high-performance affinity chromatography (HPAC) and quartz crystal microbalance (QCM) biosensor. Fragment 1-14 of human interferon-β (hlFN-β) was chosen as sense peptide and its three antisense peptides (AS-IFN 1,AS-IFN 2, and AS-IFN 3) were designed according to the degeneracy of genetic codes. The affinity column was prepared with sense peptide as ligand and the affinity chromatographic behavior was evaluated. Glu-substituted antisense peptide (AS-IFN 3) showed the strongest binding to immobilized sense peptide at pH 7.5. A quartz crystal microbalance-flow injection analysis (QCM-FIA) system was introduced to investigate the recognition process in real-time. The equilibrium dissociation constants between sense peptide and AS-IFN 1, AS-IFN 2 and AS-IFN 3 measured 2.08×10-4, 1.31×10-4 and 2.22×10-5 mol/L, respectively. The mechanism study indicated that the specific recognition between sense peptide and AS-IFN 3 was due to sequence-dependent and multi-modal affinity interaction.

  14. Undetected antisense tRNAs in mitochondrial genomes?

    Directory of Open Access Journals (Sweden)

    Seligmann Hervé

    2010-06-01

    Full Text Available Abstract Background The hypothesis that both mitochondrial (mt complementary DNA strands of tRNA genes code for tRNAs (sense-antisense coding is explored. This could explain why mt tRNA mutations are 6.5 times more frequently pathogenic than in other mt sequences. Antisense tRNA expression is plausible because tRNA punctuation signals mt sense RNA maturation: both sense and antisense tRNAs form secondary structures potentially signalling processing. Sense RNA maturation processes by default 11 antisense tRNAs neighbouring sense genes. If antisense tRNAs are expressed, processed antisense tRNAs should have adapted more for translational activity than unprocessed ones. Four tRNA properties are examined: antisense tRNA 5' and 3' end processing by sense RNA maturation and its accuracy, cloverleaf stability and misacylation potential. Results Processed antisense tRNAs align better with standard tRNA sequences with the same cognate than unprocessed antisense tRNAs, suggesting less misacylations. Misacylation increases with cloverleaf fragility and processing inaccuracy. Cloverleaf fragility, misacylation and processing accuracy of antisense tRNAs decrease with genome-wide usage of their predicted cognate amino acid. Conclusions These properties correlate as if they adaptively coevolved for translational activity by some antisense tRNAs, and to avoid such activity by other antisense tRNAs. Analyses also suggest previously unsuspected particularities of aminoacylation specificity in mt tRNAs: combinations of competition between tRNAs on tRNA synthetases with competition between tRNA synthetases on tRNAs determine specificities of tRNA amino acylations. The latter analyses show that alignment methods used to detect tRNA cognates yield relatively robust results, even when they apparently fail to detect the tRNA's cognate amino acid and indicate high misacylation potential. Reviewers This article was reviewed by Dr Juergen Brosius, Dr Anthony M Poole and

  15. CCR5反义肽核酸联合低剂量雷帕霉素对同种异体胰岛移植存活的影响%Effect of combination of CCR5 antisense peptide nucleic acid and lose-dose rapamycin on islet allograft survival

    Institute of Scientific and Technical Information of China (English)

    杨蕾; 刘永锋; 李铁民; 赵国华; 程颖; 李弘

    2007-01-01

    目的 研究CCR5反义肽核酸(PNA CCR5)及联合应用低剂量雷帕霉素对同种异体胰岛移植急性排斥反应的影响.方法 胰岛移植受体随机分4组:PNA CCR5组,低剂量雷帕霉素组(0.2 mg/kg),PNA CCR5联合应用低剂量雷帕霉素组,注射生理盐水组为对照组.监测术后IL-2、IFN-γ、IL-10 mRNA水平和CCR5蛋白水平,检测T淋巴细胞增殖能力.同时,移植物标本进行组织形态学观察.结果 联合应用组与单独PNA CCR5组、低剂量雷帕霉素组相比,移植物存活时间明显延长(P<0.05).与对照组相比,低剂量雷帕霉素组、单独PNA CCR5组和联合治疗组IL-2 mRNA表达水平降低(P<0.05,P<0.01).联合应用组IL-10 mRNA水平较PNA CCR5组、低剂量雷帕霉素组升高,差异有统计学意义(P<0.01).低剂量雷帕霉素组和联合应用组淋巴细胞增殖能力降低,同PNA CCR5组和对照组比较,差异均有统计学意义.结论 PNA CCR5能够延长胰岛移植物存活时间,同时降低雷帕霉素用量,联合应用具有协同效应,进一步减轻排斥反应,延长胰岛移植物存活.

  16. Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

    Science.gov (United States)

    Vickers, Timothy A; Crooke, Stanley T

    2016-01-01

    Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells. PMID:27571227

  17. The landscape of antisense gene expression in human cancers

    OpenAIRE

    Balbin, O. Alejandro; Malik, Rohit; Dhanasekaran, Saravana M.; Prensner, John R.; Cao, Xuhong; Wu, Yi-Mi; Robinson, Dan; Wang, Rui; Chen, Guoan; Beer, David G.; NesvizhskiI, Alexey I.; Arul M Chinnaiyan

    2015-01-01

    High-throughput RNA sequencing has revealed more pervasive transcription of the human genome than previously anticipated. However, the extent of natural antisense transcripts’ (NATs) expression, their regulation of cognate sense genes, and the role of NATs in cancer remain poorly understood. Here, we use strand-specific paired-end RNA sequencing (ssRNA-seq) data from 376 cancer samples covering nine tissue types to comprehensively characterize the landscape of antisense expression. We found c...

  18. Spherical Nucleic Acids as Intracellular Agents for Nucleic Acid Based Therapeutics

    Science.gov (United States)

    Hao, Liangliang

    Recent functional discoveries on the noncoding sequences of human genome and transcriptome could lead to revolutionary treatment modalities because the noncoding RNAs (ncRNAs) can be applied as therapeutic agents to manipulate disease-causing genes. To date few nucleic acid-based therapeutics have been translated into the clinic due to challenges in the delivery of the oligonucleotide agents in an effective, cell specific, and non-toxic fashion. Unmodified oligonucleotide agents are destroyed rapidly in biological fluids by enzymatic degradation and have difficulty crossing the plasma membrane without the aid of transfection reagents, which often cause inflammatory, cytotoxic, or immunogenic side effects. Spherical nucleic acids (SNAs), nanoparticles consisting of densely organized and highly oriented oligonucleotides, pose one possible solution to circumventing these problems in both the antisense and RNA interference (RNAi) pathways. The unique three dimensional architecture of SNAs protects the bioactive oligonucleotides from unspecific degradation during delivery and supports their targeting of class A scavenger receptors and endocytosis via a lipid-raft-dependent, caveolae-mediated pathway. Owing to their unique structure, SNAs are able to cross cell membranes and regulate target genes expression as a single entity, without triggering the cellular innate immune response. Herein, my thesis has focused on understanding the interactions between SNAs and cellular components and developing SNA-based nanostructures to improve therapeutic capabilities. Specifically, I developed a novel SNA-based, nanoscale agent for delivery of therapeutic oligonucleotides to manipulate microRNAs (miRNAs), the endogenous post-transcriptional gene regulators. I investigated the role of SNAs involving miRNAs in anti-cancer or anti-inflammation responses in cells and in in vivo murine disease models via systemic injection. Furthermore, I explored using different strategies to construct

  19. [Circulating nucleic acids and infertility].

    Science.gov (United States)

    Scalici, E; Mullet, T; Ferrières Hoa, A; Gala, A; Loup, V; Anahory, T; Belloc, S; Hamamah, S

    2015-09-01

    Circulating nucleic acids (cell-free DNA and microRNAs) have for particularity to be easily detectable in the biological fluids of the body. Therefore, they constitute biomarkers of interest in female and male infertility care. Indeed, in female, they can be used to detect ovarian reserve disorders (polycystic ovary syndrome and low functional ovarian reserve) as well as to assess follicular microenvironment quality. Moreover, in men, their expression levels can vary in case of spermatogenesis abnormalities. Finally, circulating nucleic acids have also the ability to predict successfully the quality of in vitro embryo development. Their multiple contributions during assisted reproductive technology (ART) make of them biomarkers of interest, for the development of new diagnostic and/or prognostic tests, applied to our specialty. Circulating nucleic acids would so offer the possibility of personalized medical care for infertile couples in ART. PMID:26298813

  20. Nucleic Acid Aptamers Against Proteases

    DEFF Research Database (Denmark)

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø;

    2011-01-01

    Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing......-specifically, for instance with vastly different affinities to zymogen and active enzyme forms. Furthermore, aptamers can be selected to inhibit the enzyme activity of the target proteases, but also to inhibit functionally important exosite interactions, for instance cofactor binding. Several protease-inhibiting aptamers...... strategies and of new principles for regulating the activity of the inhibitory action of aptamers of general interest to researchers working with nucleic acid aptamers...

  1. Nucleic acid programmed polymeric nanomaterials for biological communication

    Science.gov (United States)

    Rush, Anthony Michael

    A number of nucleic acid-polymer conjugates were synthesized, resulting in amphiphilic polymer-nucleic acid conjugates with the capability to self-assemble into a range of discrete nanoscale architectures. These nanomaterials, termed DNA-polymer amphiphile nanoparticles (DPA NPs), were studied with respect to their enzymatic processing by both endo- and exonucleases and further deployed as antisense genetic regulatory elements in live cultured human cells. DPA NPs were designed to act as substrates for both non sequence-specific exonucleases and a sequence-specific endonuclease. In all cases, nucleic acids arranged in the corona of spherical nanoparticles exhibited increased resistance to nucleolytic cleavage as compared to native single- or double-stranded analogues. For the exonucleases studied (Exonuclease III from E. Coli and phosphodiesterase I from Crotalus adamanteus), nanoparticle display retarded enzymatic processing by roughly a factor of five. For the endonuclease studied (Nt.CviPII), nanoparticle display prohibited virtually all enzyme activity on oligonucleotides within the nanoparticle shell. To test the ability of these materials to regulate mRNA levels in live cultured human cells, LPA (LNA-polymer amphiphile) NPs were designed to be perfectly complementary to a 20-base region of mRNA encoding the anti-apoptosis protein survivin. In this study two key observations were made. The first observation is that packaging LNA into spherical micellar nanoparticles serves to dramatically enhance cellular uptake of LNA based on flow cytometry and fluorescence microscopy data. The second observation is that LPA NPs are capable of regulating mRNA levels by what is hypothesized to be activation of target mRNA for catalytic RNase H-mediated degradation. These materials represent a unique class of DNA delivery system capable of rendering nucleic acids with natural backbone chemistry resistant to nuclease degradation and further serving to deliver DNA into cells to

  2. Antisense Oligonucleotide Therapy for Inherited Retinal Dystrophies.

    Science.gov (United States)

    Gerard, Xavier; Garanto, Alejandro; Rozet, Jean-Michel; Collin, Rob W J

    2016-01-01

    Inherited retinal dystrophies (IRDs) are an extremely heterogeneous group of genetic diseases for which currently no effective treatment strategies exist. Over the last decade, significant progress has been made utilizing gene augmentation therapy for a few genetic subtypes of IRD, although several technical challenges so far prevent a broad clinical application of this approach for other forms of IRD. Many of the mutations leading to these retinal diseases affect pre-mRNA splicing of the mutated genes . Antisense oligonucleotide (AON)-mediated splice modulation appears to be a powerful approach to correct the consequences of such mutations at the pre-mRNA level , as demonstrated by promising results in clinical trials for several inherited disorders like Duchenne muscular dystrophy, hypercholesterolemia and various types of cancer. In this mini-review, we summarize ongoing pre-clinical research on AON-based therapy for a few genetic subtypes of IRD , speculate on other potential therapeutic targets, and discuss the opportunities and challenges that lie ahead to translate splice modulation therapy for retinal disorders to the clinic. PMID:26427454

  3. Optimizing antisense oligonucleotides using phosphorodiamidate morpholino oligomers.

    Science.gov (United States)

    Popplewell, Linda J; Malerba, Alberto; Dickson, George

    2012-01-01

    Duchenne muscular dystrophy (DMD) is caused by mutations that disrupt the reading frame of the human DMD gene. Selective removal of exons flanking an out-of-frame DMD mutation can result in an in-frame mRNA transcript that may be translated into an internally deleted Becker muscular dystrophy-like functionally active dystrophin protein with therapeutic activity. Antisense oligonucleotides (AOs) can be designed to bind to complementary sequences in the targeted mRNA and modify pre-mRNA splicing to correct the reading frame of a mutated transcript. AO-induced exon skipping resulting in functional truncated dystrophin has been demonstrated in animal models of DMD both in vitro and in vivo, in DMD patient cells in vitro in culture, and in DMD muscle explants. The recent advances made in this field suggest that it is likely that AO-induced exon skipping will be the first gene therapy for DMD to reach the clinic. However, it should be noted that personalized molecular medicine may be necessary, since the various reading frame-disrupting mutations are spread across the DMD gene. The different deletions that cause DMD would require skipping of different exons, which would require the optimization and clinical trial workup of many specific AOs. This chapter describes the methodologies available for the optimization of AOs, in particular phosphorodiamidate morpholino oligomers, for the targeted skipping of specific exons on the DMD gene. PMID:22454060

  4. Electroporation increases antitumoral efficacy of the bcl-2 antisense G3139 and chemotherapy in a human melanoma xenograft

    Directory of Open Access Journals (Sweden)

    Baldi Alfonso

    2011-07-01

    Full Text Available Abstract Background Nucleic acids designed to modulate the expression of target proteins remain a promising therapeutic strategy in several diseases, including cancer. However, clinical success is limited by the lack of efficient intracellular delivery. In this study we evaluated whether electroporation could increase the delivery of antisense oligodeoxynucleotides against bcl-2 (G3139 as well as the efficacy of combination chemotherapy in human melanoma xenografts. Methods Melanoma-bearing nude mice were treated i.v. with G3139 and/or cisplatin (DDP followed by the application of trains of electric pulses to tumors. Western blot, immunohistochemistry and real-time PCR were performed to analyze protein and mRNA expression. The effect of electroporation on muscles was determined by histology, while tumor apoptosis and the proliferation index were analyzed by immunohistochemistry. Antisense oligodeoxynucleotides tumor accumulation was measured by FACS and confocal microscopy. Results The G3139/Electroporation combined therapy produced a significant inhibition of tumor growth (TWI, more than 50% accompanied by a marked tumor re-growth delay (TRD, about 20 days. The efficacy of this treatment was due to the higher G3139 uptake in tumor cells which led to a marked down-regulation of bcl-2 protein expression. Moreover, the G3139/EP combination treatment resulted in an enhanced apoptotic index and a decreased proliferation rate of tumors. Finally, an increased tumor response was observed after treatment with the triple combination G3139/DDP/EP, showing a TWI of about 75% and TRD of 30 days. Conclusions These results demonstrate that electroporation is an effective strategy to improve the delivery of antisense oligodeoxynucleotides within tumor cells in vivo and it may be instrumental in optimizing the response of melanoma to chemotherapy. The high response rate observed in this study suggest to apply this strategy for the treatment of melanoma patients.

  5. Effects of 125I-Labeled Peptide Nuclear Acid Targeting Ki67 on the Growth of Implanted Human Renal Cell Carcinoma in Nude Mice

    Institute of Scientific and Technical Information of China (English)

    JiacunChen; JunnianZheng; SongWu; HaibiaoLai; XiaoqingSun; JunjieLiu

    2004-01-01

    OBJECTIVE To investigate the potential of 125I-labeled anti-sense peptide nucleic acids (125I-AS-PNAs) to inhibit the expression of the Ki-67 gene and growth of implanted human renal carcinoma cells in nude mice.METHODS Anti-sense peptide nucleic acids (AS-PNAs) targeting the Ki67 gene were synthesized and labeled with 125I by the Chloraseptine-T method. Drugs including PNAs and 125I-AS-PNAs capsulated by cationic lipid were directly injected into tumors in nude mice. The Ki67 expression in tumors was detected by an immunohistochemical technique and Western blot. The apoptosis of tumor cells was detected by a TUNEL assay. Tumor volumes were measured every 3 days and tumor suppression rates were calculated at 12 days after treatment. Control groups were treated with AS-PNA, MMPNAs (mismatch PNAs) and 125I-Na.RESULTS The Ki67 expression rate of tumors treated by 125I-AS-PNAs [(15.3±1.8)%] was lower than that treated by AS-PNAs [(23.0±2.4)%] (P<0.01). The Ki67 protein production rate of tumors treated by 125I-AS-PNAs [(43.6±3.5)%] was lower than that treated by AS-PNAs [(59.7±2.3)%] (P<0.01 ). The apoptosis rate of tumors treated by 125I-AS-PNAs [(40.3±2.4)%] was higher than that treated by AS-PNAs [(31.1 ±2.0)%] (P<0.01). The volume of tumors treated by 125I-AS-PNAs [(330.4±57.8) mm3 ]was smallerthan that treated by AS- PNAs [(513.2±64.2 ) mm3] (P<0.01 ).CONCLUSION 125I-AS-PNAs targeted against the Ki67 gene have a greater inhibitory effect on the expression of the Ki67 gene and a larger apoptotic action on human renal carcinoma cells and can more efficiently inhibit tumor growth than AS-PNAs. 125I-AS-PNAs targeting the Ki67 gene may be a promising anti-sense/anti-gene radiotherapy method for treating renal cell carcinoma.

  6. Hsp90 protein interacts with phosphorothioate oligonucleotides containing hydrophobic 2'-modifications and enhances antisense activity.

    Science.gov (United States)

    Liang, Xue-Hai; Shen, Wen; Sun, Hong; Kinberger, Garth A; Prakash, Thazha P; Nichols, Joshua G; Crooke, Stanley T

    2016-05-01

    RNase H1-dependent antisense oligonucleotides (ASOs) are chemically modified to enhance pharmacological properties. Major modifications include phosphorothioate (PS) backbone and different 2'-modifications in 2-5 nucleotides at each end (wing) of an ASO. Chemical modifications can affect protein binding and understanding ASO-protein interactions is important for better drug design. Recently we identified many intracellular ASO-binding proteins and found that protein binding could affect ASO potency. Here, we analyzed the structure-activity-relationships of ASO-protein interactions and found 2'-modifications significantly affected protein binding, including La, P54nrb and NPM. PS-ASOs containing more hydrophobic 2'-modifications exhibit higher affinity for proteins in general, although certain proteins, e.g. Ku70/Ku80 and TCP1, are less affected by 2'-modifications. We found that Hsp90 protein binds PS-ASOs containing locked-nucleic-acid (LNA) or constrained-ethyl-bicyclic-nucleic-acid ((S)-cEt) modifications much more avidly than 2'-O-methoxyethyl (MOE). ASOs bind the mid-domain of Hsp90 protein. Hsp90 interacts with more hydrophobic 2' modifications, e.g. (S)-cEt or LNA, in the 5'-wing of the ASO. Reduction of Hsp90 protein decreased activity of PS-ASOs with 5'-LNA or 5'-cEt wings, but not with 5'-MOE wing. Together, our results indicate Hsp90 protein enhances the activity of PS/LNA or PS/(S)-cEt ASOs, and imply that altering protein binding of ASOs using different chemical modifications can improve therapeutic performance of PS-ASOs. PMID:26945041

  7. Nucleic acid delivery with microbubbles and ultrasound.

    Science.gov (United States)

    Rychak, Joshua J; Klibanov, Alexander L

    2014-06-01

    Nucleic acid-based therapy is a growing field of drug delivery research. Although ultrasound has been suggested to enhance transfection decades ago, it took a combination of ultrasound with nucleic acid carrier systems (microbubbles, liposomes, polyplexes, and viral carriers) to achieve reasonable nucleic acid delivery efficacy. Microbubbles serve as foci for local deposition of ultrasound energy near the target cell, and greatly enhance sonoporation. The major advantage of this approach is in the minimal transfection in the non-insonated non-target tissues. Microbubbles can be simply co-administered with the nucleic acid carrier or can be modified to carry nucleic acid themselves. Liposomes with embedded gas or gas precursor particles can also be used to carry nucleic acid, release and deliver it by the ultrasound trigger. Successful testing in a wide variety of animal models (myocardium, solid tumors, skeletal muscle, and pancreas) proves the potential usefulness of this technique for nucleic acid drug delivery. PMID:24486388

  8. A model for protocellular coordination of nucleic acid and protein syntheses

    Science.gov (United States)

    Fox, S. W.

    1981-01-01

    The proteinoid model for the coordination of protein synthesis with nucleic acid coding within the evolving protocell is discussed. Evidence for the self-ordering of amino acid chains, which would enhance the catalytic activity of a lysine-rich proteinoid, is presented, along with that for the preferential formation of microparticles, particularly proteinoid microparticles, in various solutions. Demonstrations of the catalytic activity of lysine-rich proteinoids in the synthesis of peptide and internucleotide bonds are pointed out. The view of evolution as a two stage sequence in which the geological synthesis of peptides evolved to the protocellular synthesis of peptides and oligonucleotides is discussed, and contrasted with the alternative view, in accord with the central dogma, that nucleic acids arose first then governed the production of proteins and protocells.

  9. The zebrafish progranulin gene family and antisense transcripts

    Directory of Open Access Journals (Sweden)

    Baranowski David

    2005-11-01

    Full Text Available Abstract Background Progranulin is an epithelial tissue growth factor (also known as proepithelin, acrogranin and PC-cell-derived growth factor that has been implicated in development, wound healing and in the progression of many cancers. The single mammalian progranulin gene encodes a glycoprotein precursor consisting of seven and one half tandemly repeated non-identical copies of the cystine-rich granulin motif. A genome-wide duplication event hypothesized to have occurred at the base of the teleost radiation predicts that mammalian progranulin may be represented by two co-orthologues in zebrafish. Results The cDNAs encoding two zebrafish granulin precursors, progranulins-A and -B, were characterized and found to contain 10 and 9 copies of the granulin motif respectively. The cDNAs and genes encoding the two forms of granulin, progranulins-1 and -2, were also cloned and sequenced. Both latter peptides were found to be encoded by precursors with a simplified architecture consisting of one and one half copies of the granulin motif. A cDNA encoding a chimeric progranulin which likely arises through the mechanism of trans-splicing between grn1 and grn2 was also characterized. A non-coding RNA gene with antisense complementarity to both grn1 and grn2 was identified which may have functional implications with respect to gene dosage, as well as in restricting the formation of the chimeric form of progranulin. Chromosomal localization of the four progranulin (grn genes reveals syntenic conservation for grna only, suggesting that it is the true orthologue of mammalian grn. RT-PCR and whole-mount in situ hybridization analysis of zebrafish grns during development reveals that combined expression of grna and grnb, but not grn1 and grn2, recapitulate many of the expression patterns observed for the murine counterpart. This includes maternal deposition, widespread central nervous system distribution and specific localization within the epithelial

  10. In vitro selection of functional nucleic acids

    Science.gov (United States)

    Wilson, D. S.; Szostak, J. W.

    1999-01-01

    In vitro selection allows rare functional RNA or DNA molecules to be isolated from pools of over 10(15) different sequences. This approach has been used to identify RNA and DNA ligands for numerous small molecules, and recent three-dimensional structure solutions have revealed the basis for ligand recognition in several cases. By selecting high-affinity and -specificity nucleic acid ligands for proteins, promising new therapeutic and diagnostic reagents have been identified. Selection experiments have also been carried out to identify ribozymes that catalyze a variety of chemical transformations, including RNA cleavage, ligation, and synthesis, as well as alkylation and acyl-transfer reactions and N-glycosidic and peptide bond formation. The existence of such RNA enzymes supports the notion that ribozymes could have directed a primitive metabolism before the evolution of protein synthesis. New in vitro protein selection techniques should allow for a direct comparison of the frequency of ligand binding and catalytic structures in pools of random sequence polynucleotides versus polypeptides.

  11. Nucleic acid based logical systems.

    Science.gov (United States)

    Han, Da; Kang, Huaizhi; Zhang, Tao; Wu, Cuichen; Zhou, Cuisong; You, Mingxu; Chen, Zhuo; Zhang, Xiaobing; Tan, Weihong

    2014-05-12

    Researchers increasingly visualize a significant role for artificial biochemical logical systems in biological engineering, much like digital logic circuits in electrical engineering. Those logical systems could be utilized as a type of servomechanism to control nanodevices in vitro, monitor chemical reactions in situ, or regulate gene expression in vivo. Nucleic acids (NA), as carriers of genetic information with well-regulated and predictable structures, are promising materials for the design and engineering of biochemical circuits. A number of logical devices based on nucleic acids (NA) have been designed to handle various processes for technological or biotechnological purposes. This article focuses on the most recent and important developments in NA-based logical devices and their evolution from in vitro, through cellular, even towards in vivo biological applications.

  12. Nucleic Acid Aptamers Against Proteases

    OpenAIRE

    Dupont, D M; Andersen, L M; Bøtkjær, Kenneth Alrø; Andreasen, P A

    2011-01-01

    Proteases are potential or realized therapeutic targets in a wide variety of pathological conditions. Moreover, proteases are classical subjects for studies of enzymatic and regulatory mechanisms. We here review the literature on nucleic acid aptamers selected with proteases as targets. Designing small molecule protease inhibitors of sufficient specificity has proved a daunting task. Aptamers seem to represent a promising alternative. In our review, we concentrate on biochemical mechanisms of...

  13. Inhibition of retroviral replication by anti-sense RNA.

    OpenAIRE

    To, R Y; Booth, S C; Neiman, P E

    1986-01-01

    We tested the effect of anti-sense RNA on the replication of avian retroviruses in cultured cells. The replication of a recombinant retrovirus carrying a neomycin resistance gene (neor) in the anti-sense orientation was blocked when the cells expressed high steady-state levels of RNA molecules with neor in sequence in the sense was blocked when the cells expressed high steady-state levels of RNA molecules with neor sequences in the sense orientation, i.e., complementary to the viral sequence....

  14. Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD)

    DEFF Research Database (Denmark)

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

    Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most...... promising methods for restoration of dystrophin expression. This approach has been tested extensively targeting different exons in numerous models both in vitro and in vivo. During the past 10 years, there has been a considerable progress by using DMD animal models involving three types of antisense...

  15. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters

    Science.gov (United States)

    Lavender, Christopher A.; Hoffman, Jackson A.; Trotter, Kevin W.; Gilchrist, Daniel A.; Bennett, Brian D.; Burkholder, Adam B.; Fargo, David C.; Archer, Trevor K.

    2016-01-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  16. Downstream Antisense Transcription Predicts Genomic Features That Define the Specific Chromatin Environment at Mammalian Promoters.

    Science.gov (United States)

    Lavender, Christopher A; Cannady, Kimberly R; Hoffman, Jackson A; Trotter, Kevin W; Gilchrist, Daniel A; Bennett, Brian D; Burkholder, Adam B; Burd, Craig J; Fargo, David C; Archer, Trevor K

    2016-08-01

    Antisense transcription is a prevalent feature at mammalian promoters. Previous studies have primarily focused on antisense transcription initiating upstream of genes. Here, we characterize promoter-proximal antisense transcription downstream of gene transcription starts sites in human breast cancer cells, investigating the genomic context of downstream antisense transcription. We find extensive correlations between antisense transcription and features associated with the chromatin environment at gene promoters. Antisense transcription downstream of promoters is widespread, with antisense transcription initiation observed within 2 kb of 28% of gene transcription start sites. Antisense transcription initiates between nucleosomes regularly positioned downstream of these promoters. The nucleosomes between gene and downstream antisense transcription start sites carry histone modifications associated with active promoters, such as H3K4me3 and H3K27ac. This region is bound by chromatin remodeling and histone modifying complexes including SWI/SNF subunits and HDACs, suggesting that antisense transcription or resulting RNA transcripts contribute to the creation and maintenance of a promoter-associated chromatin environment. Downstream antisense transcription overlays additional regulatory features, such as transcription factor binding, DNA accessibility, and the downstream edge of promoter-associated CpG islands. These features suggest an important role for antisense transcription in the regulation of gene expression and the maintenance of a promoter-associated chromatin environment. PMID:27487356

  17. Stability of free and mineral-protected nucleic acids: Implications for the RNA world

    Science.gov (United States)

    Swadling, Jacob B.; Coveney, Peter V.; Christopher Greenwell, H.

    2012-04-01

    Using molecular dynamics simulations we study the structural stability of three different nucleic acids intercalated within a magnesium aluminium layered double hydroxide (LDH) mineral, at varying degrees of hydration, and free in aqueous solution. The nucleotides investigated are ribose nucleic acid (RNA), deoxyribose nucleic acid (DNA) and peptide nucleic acid (PNA), all in duplex form. Our simulations show that DNA has enhanced Watson-Crick hydrogen-bonding when intercalated within the LDH clay interlayers, compared with intercalated RNA and PNA, whilst the reverse trend is found for the nucleic acids in bulk water. The tendency for LDH to alter the stability of the three nucleic acids persists for higher temperature and pressure conditions. The uncharged protein backbone of PNA is found to have a detrimental effect on the overall stability of the duplex, as it experiences a greatly reduced electrostatic interaction with the charged LDH sheets compared to RNA and DNA. Assuming an RNA world, in which RNA preceded the DNA/protein world, at some point in time DNA must have taken over the role as the information storage molecule from RNA. These results suggest that a mineral based origin of life may have favoured DNA as the information-storage biomolecule over potentially competing RNA and PNA, providing a route to modern biology from the RNA world.

  18. 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...... improve biomolecular recognition by synthetic nucleic acid analogues. Circular dichroism (CD) measurements showed no distortion of the duplex structure by the incorporated peptide chains while studies in human serum indicated superior stability of the POCs compared to LNA/DNA mixmers and unmodified DNA...

  19. Sequence-specific nucleic acid detection from binary pore conductance measurement

    OpenAIRE

    Esfandiari, Leyla; Monbouquette, Harold G.; Jacob J. Schmidt

    2012-01-01

    We describe a platform for sequence-specific nucleic acid (NA) detection utilizing a micropipette tapered to a 2 μm diameter pore and 3 μm diameter polystyrene beads to which uncharged peptide nucleic acid (PNA) probe molecules have been conjugated. As the target NAs hybridize to the complementary PNA-beads, the beads acquire negative charge and become electrophoretically mobile. An applied electric field guides these NA-PNA-beads toward the pipette tip, which they obstruct, leading to an ind...

  20. In vitro inhibition of promyelocytic leukemia/retinoic acid receptor-alpha (PML/RARalpha) expression and leukemogenic activity by DNA/LNA chimeric antisense oligos.

    Science.gov (United States)

    Caprodossi, Sara; Galluzzi, Luca; Biagetti, Simona; Della Chiara, Giulia; Pelicci, Pier Giuseppe; Magnani, Mauro; Fanelli, Mirco

    2005-01-01

    Acute promyelocytic leukemia (APL) is a subtype of myeloid leukemia characterized by the chromosomal translocation t(15:17) that leads to the expression of promyelocytic leukemia/retinoic acid receptor-alpha (PML/ RARalpha) oncofusion protein. The block of differentiation at the promyelocytic stage of the blasts and their increased survival induced by PML/RARalpha are the principal biological features of the disease. Therapies based on pharmacological doses of retinoic acid (RA, 10(-6) M) are able to restore APL cell differentiation in most cases, but not to achieve complete hematological remission because retinoic acid resistance occurs in many patients. In order to elaborate alternative therapeutic approaches, we focused our attention on the use of antisense oligonucleotides as gene-specific drug directed to PML/RARalpha mRNA target. We used antisense molecules containing multiple locked nucleic acid (LNA) modifications. The LNAs are nucleotide analogues that are able to form duplexes with complementary DNA or RNA sequences with highly increased thermal stability and are resistant to 3'-exonuclease degradation in vitro. The DNA/LNA chimeric molecules were designed on the fusion sequence of PML and RARalpha genes to specifically target the oncofusion protein. Cell-free and in vitro experiments using U937-PR9-inducible cell line showed that DNA/LNA oligonucleotides were able to interfere with PML/RARalpha expression more efficiently than the corresponding unmodified DNA oligo. Moreover, the treatment of U937-PR9 cells with these chimeric antisense molecules was able to abrogate the block of differentiation induced by PML/RARalpha oncoprotein. These data suggest a possible application of oligonucleotides containing LNA in an antisense therapeutic strategy for APL.

  1. Design and Synthesis of Novel Peptide Nucleic Acid Monomers

    Institute of Scientific and Technical Information of China (English)

    白金泉; 李英; 刘克良

    2001-01-01

    All of the four nucleobases in DNA have replaced the 4-hydroxy group of N-[2-(tert-butoxycarbonylaminomethyl)-trams-4-hydroxy]tetrahydropyrrole acetic acid methyl ester with cis-stereochemistry. An efficient route for the synthesis of N-[2-(tert-butoxycarbonylaminomethyl)-trans-4-hydroxy]-tetrahydropyrrole acetic acid methyl ester has been developed.Starting with this intermediate, the protected monmers were synthesized by the Mitsunobu reaction or via its tosylate.

  2. Highly expressed genes are associated with inverse antisense transcription in mouse

    Indian Academy of Sciences (India)

    Andras Györffy; Pawel Surowiak; Zsolt Tulassay; Balazs Györffy

    2007-08-01

    There is a growing evidence, that antisense transcription might have a key role in a range of human diseases. Although predefined sense–antisense pairs were extensively studied, the antisense expression of the known sense genes is rarely investigated. We retrieved and correlated the expression of sense and antisense sequences of 1182 mouse transcripts to assess the prevalence and to find the characteristic pattern of antisense transcription. We contrasted three Affymetrix MGU74A version 1 mouse genome chips to six MGU74A version 2 chips. For these 1182 transcripts, the version 1 chips contain the antisense sequences of the transcripts presented on the version 2 chips. The original data was taken from the GEO database (GDS431 and GDS432). As the Affymetrix data are semiquantitative, the relative expression levels of antisense partners were analysed. We detected antisense transcription, although the average antisense expression is shifted towards smaller expression values (MGU74A version 1, 516; version 2, 1688). An inverse direct correlation between sense and antisense expression values could be observed at high expression values. At a very high relative expression—above 40,000—the Pearson correlation coefficient is getting closer to −1. Transcripts with high inverse expression ratio may be correlated to the investigated gene (major histocompatibility complex class II trans activator). The ratio of sense to antisense transcripts varied among different chromosomes; on chromosomes 14 and 1 the level of antisense expression was higher than that of sense. We conclude that antisense transcription is a common phenomenon in the mouse genome. The hypothesis of regulatory role of antisense transcripts is supported by the inverse antisense gene expression of highly expressed genes.

  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. Lysine metabolism in antisense C-hordein barley grains

    DEFF Research Database (Denmark)

    Schmidt, Daiana; Rizzi, Vanessa; Gaziola, Salete A;

    2015-01-01

    The grain proteins of barley are deficient in lysine and threonine due to their low concentrations in the major storage protein class, the hordeins, especially in the C-hordein subgroup. Previously produced antisense C-hordein transgenic barley lines have an improved amino acid composition, with ...

  5. Multiplexed microfluidic approach for nucleic acid enrichment

    Energy Technology Data Exchange (ETDEWEB)

    VanderNoot, Victoria A.; Langevin, Stanley Alan; Bent, Zachary; Renzi, Ronald F.; Ferko, Scott M.; Van De Vreugde, James L.; Lane, Todd; Patel, Kamlesh; Branda, Steven

    2016-04-26

    A system for enhancing a nucleic acid sample may include a one pump, a denaturing chamber; a microfluidic hydroxyapatite chromatography device configured for performing hydroxyapatite chromatography on the nucleic acid sample, a sample collector, and tubing connecting the pump with the denaturing chamber, the hydroxyapatite chromatography device and the sample collector such that the pump may be used to move the nucleic acid sample from the denaturing chamber to the hydroxyapatite chromatography device and then to the sample collector.

  6. Nucleic Acids in Human Glioma Treatment: Innovative Approaches and Recent Results

    Directory of Open Access Journals (Sweden)

    S. Catuogno

    2012-01-01

    Full Text Available Gliomas are the most common primary central nervous system tumors with a dismal prognosis. Despite recent advances in surgery, radiotherapy, and chemotherapy, current treatment regimens have a modest survival benefit. A crucial challenge is to deliver drugs effectively to invasive glioma cells residing in a sanctuary within the central nervous system. New therapies are essential, and oligonucleotide-based approaches, including antisense, microRNAs, small interfering RNAs, and nucleic acid aptamers, may provide a viable strategy. Thanks to their unique characteristics (low size, good affinity for the target, no immunogenicity, chemical structures that can be easily modified to improve their in vivo applications, these molecules may represent a valid alternative to antibodies particularly to overcome challenges presented by the blood-brain barrier. Here we will discuss recent results on the use of oligonucleotides that will hopefully provide new effective treatment for gliomas.

  7. The Contribution of the Activation Entropy to the Gas-Phase Stability of Modified Nucleic Acid Duplexes

    Science.gov (United States)

    Hari, Yvonne; Dugovič, Branislav; Istrate, Alena; Fignolé, Annabel; Leumann, Christian J.; Schürch, Stefan

    2016-07-01

    Tricyclo-DNA (tcDNA) is a sugar-modified analogue of DNA currently tested for the treatment of Duchenne muscular dystrophy in an antisense approach. Tandem mass spectrometry plays a key role in modern medical diagnostics and has become a widespread technique for the structure elucidation and quantification of antisense oligonucleotides. Herein, mechanistic aspects of the fragmentation of tcDNA are discussed, which lay the basis for reliable sequencing and quantification of the antisense oligonucleotide. Excellent selectivity of tcDNA for complementary RNA is demonstrated in direct competition experiments. Moreover, the kinetic stability and fragmentation pattern of matched and mismatched tcDNA heteroduplexes were investigated and compared with non-modified DNA and RNA duplexes. Although the separation of the constituting strands is the entropy-favored fragmentation pathway of all nucleic acid duplexes, it was found to be only a minor pathway of tcDNA duplexes. The modified hybrid duplexes preferentially undergo neutral base loss and backbone cleavage. This difference is due to the low activation entropy for the strand dissociation of modified duplexes that arises from the conformational constraint of the tc-sugar-moiety. The low activation entropy results in a relatively high free activation enthalpy for the dissociation comparable to the free activation enthalpy of the alternative reaction pathway, the release of a nucleobase. The gas-phase behavior of tcDNA duplexes illustrates the impact of the activation entropy on the fragmentation kinetics and suggests that tandem mass spectrometric experiments are not suited to determine the relative stability of different types of nucleic acid duplexes.

  8. Pulmonary delivery of nucleic acids.

    Science.gov (United States)

    Birchall, James

    2007-11-01

    The lung is an appropriate present and future target for gene therapy approaches designed to treat inherited monogenic diseases, eradicate bronchial tumours, transfer pharmacologically active products to the general circulation, express enzymes to catabolise toxins, manage pulmonary hypertension and lung injury and vaccinate against infection. Despite 35 years of gene therapy research and some significant milestones in molecular biology, the clinical potential of gene therapy has yet to be realised. In pulmonary gene therapy the nucleic acid cargo needs to be delivered to cells in the target region of the lung, and even in cases when these targets are well defined this is severely limited by the pulmonary architecture, clearance mechanisms, immune activation, the presence of respiratory mucus and the availability of a truly representative biological model. The challenge from a drug delivery perspective is to consider the suitability of conventional nebulisers and inhalers for delivering DNA to the lung and design and apply integrated formulation and device solutions specific to nucleic acid delivery. PMID:17970661

  9. High affinity nucleic acid aptamers for streptavidin incorporated into bi-specific capture ligands

    OpenAIRE

    Tahiri-Alaoui, Abdessamad; Frigotto, Laura; Manville, Nick; Ibrahim, Jamal; Romby, Pascale; James, William

    2002-01-01

    We have isolated 2′-Fluoro-substituted RNA aptamers that bind to streptavidin (SA) with an affinity around 7 ± 1.8 nM, comparable with that of recently described peptide aptamers. Binding to SA was not prevented by prior saturation with biotin, enabling nucleic acid aptamers to form useful ternary complexes. Mutagenesis, secondary structure analysis, ribonuclease footprinting and deletion analysis provided evidence for the essential structural features of SA-binding aptamers. In order to prov...

  10. Bacterial antisense RNAs are mainly the product of transcriptional noise

    Science.gov (United States)

    Lloréns-Rico, Verónica; Cano, Jaime; Kamminga, Tjerko; Gil, Rosario; Latorre, Amparo; Chen, Wei-Hua; Bork, Peer; Glass, John I.; Serrano, Luis; Lluch-Senar, Maria

    2016-01-01

    cis-Encoded antisense RNAs (asRNAs) are widespread along bacterial transcriptomes. However, the role of most of these RNAs remains unknown, and there is an ongoing discussion as to what extent these transcripts are the result of transcriptional noise. We show, by comparative transcriptomics of 20 bacterial species and one chloroplast, that the number of asRNAs is exponentially dependent on the genomic AT content and that expression of asRNA at low levels exerts little impact in terms of energy consumption. A transcription model simulating mRNA and asRNA production indicates that the asRNA regulatory effect is only observed above certain expression thresholds, substantially higher than physiological transcript levels. These predictions were verified experimentally by overexpressing nine different asRNAs in Mycoplasma pneumoniae. Our results suggest that most of the antisense transcripts found in bacteria are the consequence of transcriptional noise, arising at spurious promoters throughout the genome. PMID:26973873

  11. Antisense Oligonucleotides: Treating Neurodegeneration at the Level of RNA

    OpenAIRE

    DeVos, Sarah L.; Miller, Timothy M.

    2013-01-01

    Adequate therapies are lacking for Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, amyotrophic lateral sclerosis, and other neurodegenerative diseases. The ability to use antisense oligonucleotides (ASOs) to target disease-associated genes by means of RNA may offer a potent approach for the treatment of these, and other, neurodegenerative disorders. In modifying the basic backbone chemistry, chemical groups, and target sequence, ASOs can act through numerous mechanisms to decr...

  12. Antisense mediated exon skipping therapy for duchenne muscular dystrophy (DMD)

    OpenAIRE

    Brolin, Camilla; Shiraishi, Takehiko

    2011-01-01

    Duchenne Muscular Dystrophy (DMD) is a lethal disease caused by mutations in the dystrophin gene (DMD) that result in the absence of essential muscle protein dystrophin. Among many different approaches for DMD treatment, exon skipping, mediated by antisense oligonucleotides, is one of the most promising methods for restoration of dystrophin expression. This approach has been tested extensively targeting different exons in numerous models both in vitro and in vivo. During the past 10 years, th...

  13. Progress in therapeutic antisense applications for neuromuscular disorders

    OpenAIRE

    Aartsma-Rus, Annemieke; van Ommen, Gert-Jan B.

    2009-01-01

    Neuromuscular disorders are a frequent cause of chronic disability in man. They often result from mutations in single genes and are thus, in principle, well suited for gene therapy. However, the tissues involved (muscle and the central nervous system) are post-mitotic, which poses a challenge for most viral vectors. In some cases, alternative approaches may use small molecules, for example, antisense oligonucleotides (AONs). These do not deliver a new gene, but rather modulate existing gene p...

  14. Combination Antisense Treatment for Destructive Exon Skipping of Myostatin and Open Reading Frame Rescue of Dystrophin in Neonatal mdx Mice.

    Science.gov (United States)

    Lu-Nguyen, Ngoc B; Jarmin, Susan A; Saleh, Amer F; Popplewell, Linda; Gait, Michael J; Dickson, George

    2015-08-01

    The fatal X-linked Duchenne muscular dystrophy (DMD), characterized by progressive muscle wasting and muscle weakness, is caused by mutations within the DMD gene. The use of antisense oligonucleotides (AOs) modulating pre-mRNA splicing to restore the disrupted dystrophin reading frame, subsequently generating a shortened but functional protein has emerged as a potential strategy in DMD treatment. AO therapy has recently been applied to induce out-of-frame exon skipping of myostatin pre-mRNA, knocking-down expression of myostatin protein, and such an approach is suggested to enhance muscle hypertrophy/hyperplasia and to reduce muscle necrosis. Within this study, we investigated dual exon skipping of dystrophin and myostatin pre-mRNAs using phosphorodiamidate morpholino oligomers conjugated with an arginine-rich peptide (B-PMOs). Intraperitoneal administration of B-PMOs was performed in neonatal mdx males on the day of birth, and at weeks 3 and 6. At week 9, we observed in treated mice (as compared to age-matched, saline-injected controls) normalization of muscle mass, a recovery in dystrophin expression, and a decrease in muscle necrosis, particularly in the diaphragm. Our data provide a proof of concept for antisense therapy combining dystrophin restoration and myostatin inhibition for the treatment of DMD. PMID:25959011

  15. Do nucleic acids moonlight as molecular chaperones?

    Science.gov (United States)

    Docter, Brianne E.; Horowitz, Scott; Gray, Michael J.; Jakob, Ursula; Bardwell, James C.A.

    2016-01-01

    Organisms use molecular chaperones to combat the unfolding and aggregation of proteins. While protein chaperones have been widely studied, here we demonstrate that DNA and RNA exhibit potent chaperone activity in vitro. Nucleic acids suppress the aggregation of classic chaperone substrates up to 300-fold more effectively than the protein chaperone GroEL. Additionally, RNA cooperates with the DnaK chaperone system to refold purified luciferase. Our findings reveal a possible new role for nucleic acids within the cell: that nucleic acids directly participate in maintaining proteostasis by preventing protein aggregation. PMID:27105849

  16. Chip-based sequencing nucleic acids

    Science.gov (United States)

    Beer, Neil Reginald

    2014-08-26

    A system for fast DNA sequencing by amplification of genetic material within microreactors, denaturing, demulsifying, and then sequencing the material, while retaining it in a PCR/sequencing zone by a magnetic field. One embodiment includes sequencing nucleic acids on a microchip that includes a microchannel flow channel in the microchip. The nucleic acids are isolated and hybridized to magnetic nanoparticles or to magnetic polystyrene-coated beads. Microreactor droplets are formed in the microchannel flow channel. The microreactor droplets containing the nucleic acids and the magnetic nanoparticles are retained in a magnetic trap in the microchannel flow channel and sequenced.

  17. Development of PNA-Surfactant Systems for Nucleic Acid Separations

    Science.gov (United States)

    Vernille, James; Armitage, Bruce; Schneider, James

    2002-03-01

    We have been exploring the use of novel peptide nucleic acid (PNA) surfactants for use in sequence specific, scalable DNA separations. While the synthetic and physical characteristics of PNA make it a useful molecule for bioseparations, PNA shows limited water solubility. Here we describe a molecular design strategy to improve water solubility while maintaining sequence specificity. A candidate molecule has been identified which contains lysine residues and a short alkane tail. Melting temperature data show that lipid tail interactions with the DNA nucleobases have a small but significant effect on stability while the added lysines stabilize the complex in an ionic strength dependent way. We also discuss the incorporation of these surfactants into micellar systems for novel separations.

  18. Coiled coil interactions for the targeting of liposomes for nucleic acid delivery

    Science.gov (United States)

    Oude Blenke, Erik E.; van den Dikkenberg, Joep; van Kolck, Bartjan; Kros, Alexander; Mastrobattista, Enrico

    2016-04-01

    Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes

  19. Coiled coil interactions for the targeting of liposomes for nucleic acid delivery

    Science.gov (United States)

    Oude Blenke, Erik E.; van den Dikkenberg, Joep; van Kolck, Bartjan; Kros, Alexander; Mastrobattista, Enrico

    2016-04-01

    Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes encapsulating a splice correcting oligonucleotide or siRNA. These peptide-functionalized vesicles are highly stable in solution but start to cluster when vesicles modified with complementary peptides are mixed together, demonstrating that the peptides quickly coil and crosslink the vesicles. When one of the peptides was anchored to the cell membrane using a hydrophobic cholesterol anchor, vesicles functionalized with the complementary peptide could be docked to these cells, whereas non-functionalized cells did not show any vesicle tethering. Although the anchored peptides do not have a downstream signaling pathway, microscopy pictures revealed that after four hours, the majority of the docked vesicles were internalized by endocytosis. Finally, for the first time, it was shown that the coiled coil assembly at the interface between the vesicles and the cell membrane induces active uptake and leads to cytosolic delivery of the nucleic acid cargo. Both the siRNA and the splice correcting oligonucleotide were functionally delivered, resulting respectively in the silencing or recovery of luciferase expression in the appropriate cell lines. These results demonstrate that the docking to the cell by coiled coil interaction can induce active uptake and achieve the successful intracellular delivery of otherwise membrane impermeable nucleic acids in a highly specific manner.Coiled coil interactions are strong protein-protein interactions that are involved in many biological processes, including intracellular trafficking and membrane fusion. A synthetic heterodimeric coiled-coil forming peptide pair, known as E3 (EIAALEK)3 and K3 (KIAALKE)3 was used to functionalize liposomes

  20. Sense and antisense transcription are associated with distinct chromatin architectures across genes.

    Science.gov (United States)

    Murray, Struan C; Haenni, Simon; Howe, Françoise S; Fischl, Harry; Chocian, Karolina; Nair, Anitha; Mellor, Jane

    2015-09-18

    Genes from yeast to mammals are frequently subject to non-coding transcription of their antisense strand; however the genome-wide role for antisense transcription remains elusive. As transcription influences chromatin structure, we took a genome-wide approach to assess which chromatin features are associated with nascent antisense transcription, and contrast these with features associated with nascent sense transcription. We describe a distinct chromatin architecture at the promoter and gene body specifically associated with antisense transcription, marked by reduced H2B ubiquitination, H3K36 and H3K79 trimethylation and increased levels of H3 acetylation, chromatin remodelling enzymes, histone chaperones and histone turnover. The difference in sense transcription between genes with high or low levels of antisense transcription is slight; thus the antisense transcription-associated chromatin state is not simply analogous to a repressed state. Using mutants in which the level of antisense transcription is reduced at GAL1, or altered genome-wide, we show that non-coding transcription is associated with high H3 acetylation and H3 levels across the gene, while reducing H3K36me3. Set1 is required for these antisense transcription-associated chromatin changes in the gene body. We propose that nascent antisense and sense transcription have fundamentally distinct relationships with chromatin, and that both should be considered canonical features of eukaryotic genes.

  1. Scaffolding along Nucleic Acid Duplexes Using 2'-Amino-Locked Nucleic Acids

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Wengel, Jesper

    2014-01-01

    Conspectus Incorporation of chemically modified nucleotide scaffolds into nucleic acids to form assemblies rich in function is an innovative area with great promise for nanotechnology and biomedical and material science applications. The intrinsic biorecognition potential of nucleic acids combined...... of the biopolymers of Nature may solve the major challenges of the future material science and biomedicine. However, this requires strong scientific progress and efficient interdisciplinary research. Examples of this Account demonstrate that among other synthetic biopolymers, synthetic nucleic acids containing...

  2. Functional analysis of splicing mutations in the IDS gene and the use of antisense oligonucleotides to exploit an alternative therapy for MPS II.

    Science.gov (United States)

    Matos, Liliana; Gonçalves, Vânia; Pinto, Eugénia; Laranjeira, Francisco; Prata, Maria João; Jordan, Peter; Desviat, Lourdes R; Pérez, Belén; Alves, Sandra

    2015-12-01

    Mucopolysaccharidosis II is a lysosomal storage disorder caused by mutations in the IDS gene, including exonic alterations associated with aberrant splicing. In the present work, cell-based splicing assays were performed to study the effects of two splicing mutations in exon 3 of IDS, i.e., c.241C>T and c.257C>T, whose presence activates a cryptic splice site in exon 3 and one in exon 8, i.e., c.1122C>T that despite being a synonymous mutation is responsible for the creation of a new splice site in exon 8 leading to a transcript shorter than usual. Mutant minigene analysis and overexpression assays revealed that SRSF2 and hnRNP E1 might be involved in the use and repression of the constitutive 3' splice site of exon 3 respectively. For the c.1122C>T the use of antisense therapy to correct the splicing defect was explored, but transfection of patient fibroblasts with antisense morpholino oligonucleotides (n=3) and a locked nucleic acid failed to abolish the abnormal transcript; indeed, it resulted in the appearance of yet another aberrant splicing product. Interestingly, the oligonucleotides transfection in control fibroblasts led to the appearance of the aberrant transcript observed in patients' cells after treatment, which shows that the oligonucleotides are masking an important cis-acting element for 5' splice site regulation of exon 8. These results highlight the importance of functional studies for understanding the pathogenic consequences of mis-splicing and highlight the difficulty in developing antisense therapies involving gene regions under complex splicing regulation. PMID:26407519

  3. NMR studies of nucleic acid dynamics

    Science.gov (United States)

    Al-Hashimi, Hashim M.

    2013-12-01

    Nucleic acid structures have to satisfy two diametrically opposite requirements; on one hand they have to adopt well-defined 3D structures that can be specifically recognized by proteins; on the other hand, their structures must be sufficiently flexible to undergo very large conformational changes that are required during key biochemical processes, including replication, transcription, and translation. How do nucleic acids introduce flexibility into their 3D structure without losing biological specificity? Here, I describe the development and application of NMR spectroscopic techniques in my laboratory for characterizing the dynamic properties of nucleic acids that tightly integrate a broad set of NMR measurements, including residual dipolar couplings, spin relaxation, and relaxation dispersion with sample engineering and computational approaches. This approach allowed us to obtain fundamental new insights into directional flexibility in nucleic acids that enable their structures to change in a very specific functional manner.

  4. Efficient down-regulation of PKC-α gene expression in A549 lung cancer cells mediated by antisense oligodeoxynucleotides in dendrosomes.

    Science.gov (United States)

    Movassaghian, Sara; Moghimi, Hamid R; Shirazi, Farshad H; Koshkaryev, Alexander; Trivedi, Malav S; Torchilin, Vladimir P

    2013-01-30

    The completion of human genome project has increased our knowledge of the molecular mechanisms of many diseases, including cancer, thus providing new opportunities for gene therapy. Antisense oligodeoxynucleotides (AsODN) possess great potential as sequence-specific therapeutic agents, which in contrast to classic treatments provide more efficient and target-specific approach to modulate disease-related genes. To be therapeutically effective, sufficient concentrations of intact AsODN must bypass membrane barriers and access the site of action. In this study, a dendrosome delivery strategy was designed to improve the encapsulation of AsODN in non-cationic liposomes to target PKC-α in lung cancer cells in vitro. Subcellular trafficking of fluorescently labeled AsODN was visualized using confocal microscopy. Uptake and expression of mRNA and target protein after AsODN delivery was measured by flow cytometry, qRT-PCR and Western blot analysis, respectively. Dendrosomes showed favorable physicochemical parameters: high encapsulation efficiency and uptake in serum-containing medium with no apparent cytotoxicity. AsODN encapsulated in dendrosome efficiently and specifically suppress the target gene at both mRNA and protein levels. Additional in vivo studies on the application of dendrosome as a delivery system for nucleic acid molecules may lead to improvement of this technology and facilitate the development of therapeutic antisense techniques. PMID:23262426

  5. Two perspectives to consider Nucleic Acids

    OpenAIRE

    Sosic, A

    2013-01-01

    Since their discovery, nucleic acids have been the object of intense and thorough explorations, leading to the understanding of their structure and functions. Their role as genetic information carriers is well known but there are evidences that they are also involved in a series of other less known processes. No longer have nucleic acids to be considered passive structures, but they are dynamic and active macromolecules, able to assume a number of three-dimensional conformations. Precisely, t...

  6. Rapid nuclear import of short nucleic acids.

    Science.gov (United States)

    Kitagawa, Mai; Okamoto, Akimitsu

    2016-10-01

    Exogenous short-chain nucleic acids undergo rapid import into the nucleus. Fluorescence-labeled dT1-13 DNA microinjected into the cytoplasm domain of a HeLa cell was rapidly imported into the nucleus domain within 1min. This is much more rapid than what has been observed for intracellular diffusion of small molecules. In contrast, import of longer nucleic acids with a length of over 30nt into the nucleus was suppressed. PMID:27597250

  7. Allosterically Regulated Phosphatase Activity from Peptide-PNA Conjugates Folded Through Hybridization.

    Science.gov (United States)

    Machida, Takuya; Dutt, Som; Winssinger, Nicolas

    2016-07-18

    The importance of spatial organization in short peptide catalysts is well recognized. We synthesized and screened a library of peptides flanked by peptide nucleic acids (PNAs) such that the peptide would be constrained in a hairpin loop upon hybridization. A screen for phosphatase activity led to the discovery of a catalyst with >25-fold rate acceleration over the linear peptide. We demonstrated that the hybridization-enforced folding of the peptide is necessary for activity, and designed a catalyst that is allosterically controlled using a complementary PNA sequence. PMID:27320214

  8. Identification of antisense long noncoding RNAs that function as SINEUPs in human cells

    DEFF Research Database (Denmark)

    Schein, Aleks; Zucchelli, Silvia; Kauppinen, Sakari;

    2016-01-01

    , increasing PPP1R12A protein translation in human cells. The SINEUP activity depends on the aforementioned sense-antisense interaction and a free right Alu monomer repeat element at the 3' end of R12A-AS1. In addition, we identify another human antisense lncRNA with SINEUP activity. Our results demonstrate...

  9. Intra-Amygdala Injections of CREB Antisense Impair Inhibitory Avoidance Memory: Role of Norepinephrine and Acetylcholine

    Science.gov (United States)

    Canal, Clinton E.; Chang, Qing; Gold, Paul E.

    2008-01-01

    Infusions of CREB antisense into the amygdala prior to training impair memory for aversive tasks, suggesting that the antisense may interfere with CRE-mediated gene transcription and protein synthesis important for the formation of new memories within the amygdala. However, the amygdala also appears to modulate memory formation in distributed…

  10. Construction of antisense Bmi-1 expression plasmid and its inhibitory effect on K562 cells proliferation

    Institute of Scientific and Technical Information of China (English)

    MENG Xiu-xiang; LIU Wei-hong; LIU Dan-dan; ZHAO Xin-yu; SU Ben-li

    2005-01-01

    Background Bmi-1 gene determines the proliferative capacity of normal and leukemia stem cells. Expression of Bmi-1 has been found in all types of myeloid leukemia cells in both humans and mice. This study aimed at assessing the effect of antisense Bmi-1 expression on K562 cells proliferation and p16 protein (p16) expression.Results K562 cells transfected with antisense Bmi-1 plasmid grew significantly slower than that of controls (the parental K562 and cells transfected with empty plasmid). The colony forming ability of antisense Bmi-1 plasmid transfected cells decreased significantly (P<0.01) compared with controls. The p16 expression of cells transfected with antisense Bmi-1 was upgraded more apparently than that of controls.Conclusion The antisense Bmi-1 gene can inhibit the growth of K562 cell and upgrade expression of p16 in K562 cells.

  11. Dissecting and Exploiting Nonribosomal Peptide Synthetases

    Institute of Scientific and Technical Information of China (English)

    Qing-Tao SHEN; Xiu-Lan CHEN; Cai-Yun SUN; Yu-Zhong ZHANG

    2004-01-01

    A large number of therapeutically useful cyclic and linear peptides of bacteria or fungal origin are synthesized via a template-directed, nucleic-acid-independent nonribosomal mechanism. This process is carried out by mega-enzymes called nonribosomal peptide synthetases (NRPSs). NRPSs contain repeated coordinated groups of active sites called modules, and each module is composed of several domains with different catalytic activities. The familiarity to these domains lays base for the future genetic engineering of NRPSs to generate entirely "unnature" Products. The details about NRPSs domain structures and the exploitation of NRPSs are described in this review.

  12. Cis-Antisense Transcription Gives Rise to Tunable Genetic Switch Behavior: A Mathematical Modeling Approach.

    Science.gov (United States)

    Bordoy, Antoni E; Chatterjee, Anushree

    2015-01-01

    Antisense transcription has been extensively recognized as a regulatory mechanism for gene expression across all kingdoms of life. Despite the broad importance and extensive experimental determination of cis-antisense transcription, relatively little is known about its role in controlling cellular switching responses. Growing evidence suggests the presence of non-coding cis-antisense RNAs that regulate gene expression via antisense interaction. Recent studies also indicate the role of transcriptional interference in regulating expression of neighboring genes due to traffic of RNA polymerases from adjacent promoter regions. Previous models investigate these mechanisms independently, however, little is understood about how cells utilize coupling of these mechanisms in advantageous ways that could also be used to design novel synthetic genetic devices. Here, we present a mathematical modeling framework for antisense transcription that combines the effects of both transcriptional interference and cis-antisense regulation. We demonstrate the tunability of transcriptional interference through various parameters, and that coupling of transcriptional interference with cis-antisense RNA interaction gives rise to hypersensitive switches in expression of both antisense genes. When implementing additional positive and negative feed-back loops from proteins encoded by these genes, the system response acquires a bistable behavior. Our model shows that combining these multiple-levels of regulation allows fine-tuning of system parameters to give rise to a highly tunable output, ranging from a simple-first order response to biologically complex higher-order response such as tunable bistable switch. We identify important parameters affecting the cellular switch response in order to provide the design principles for tunable gene expression using antisense transcription. This presents an important insight into functional role of antisense transcription and its importance towards

  13. 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.

  14. Toward Peptide Nucleic Acid (PNA) Directed Peptide Translation Using Ester Based Aminoacyl Transfer

    DEFF Research Database (Denmark)

    Singhal, Abhishek; Bagnacani, Valentina; Corradini, Roberto;

    2014-01-01

    of phenylalanine occurs either between two ends of a PNA stem loop structure, between two PNAs in a duplex, or between two PNAs assembled on a PNA template, we show that bona fide template instructed phenylalanine transfer can take place. Thus, we have identified conditions which allow template assisted...

  15. Parallel Synthesis of Cell-Penetrating Peptide Conjugates of PMO Toward Exon Skipping Enhancement in Duchenne Muscular Dystrophy

    OpenAIRE

    O'Donovan, Liz; Okamoto, Itaru; Arzumanov, Andrey A; Williams, Donna L.; Deuss, Peter; Gait, Michael J.

    2015-01-01

    We describe two new methods of parallel chemical synthesis of libraries of peptide conjugates of phosphorodiamidate morpholino oligonucleotide (PMO) cargoes on a scale suitable for cell screening prior to in vivo analysis for therapeutic development. The methods represent an extension of the SELection of PEPtide CONjugates (SELPEPCON) approach previously developed for parallel peptide-peptide nucleic acid (PNA) synthesis. However, these new methods allow for the utilization of commercial PMO ...

  16. Highly stable triple helix formation by homopyrimidine (l)-acyclic threoninol nucleic acids with single stranded DNA and RNA

    DEFF Research Database (Denmark)

    Kumar, Vipin; Kesavan, Venkitasamy; Gothelf, Kurt Vesterager

    2015-01-01

    Acyclic (l)-threoninol nucleic acid (aTNA) containing thymine, cytosine and adenine nucleobases were synthesized and shown to form surprisingly stable triplexes with complementary single stranded homopurine DNA or RNA targets. The triplex structures consist of two (l)-aTNA strands and one DNA or...... RNA, and these triplexes are significantly stronger than the corresponding DNA or RNA duplexes as shown in competition experiments. As a unique property the (l)-aTNAs exclusively form triplex structures with DNA and RNA and no duplex structures are observed by gel electrophoresis. The results were...... compared to the known enantiomer (d)-aTNA, which forms much weaker triplexes depending upon temperature and time. It was demonstrated that (l)-aTNA triplexes are able to stop primer extension on a DNA template, showing the potential of (l)-aTNA for antisense applications....

  17. Detection of nucleic acid sequences by invader-directed cleavage

    Science.gov (United States)

    Brow, Mary Ann D.; Hall, Jeff Steven Grotelueschen; Lyamichev, Victor; Olive, David Michael; Prudent, James Robert

    1999-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The 5' nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based by charge.

  18. Novel Biochip Platform for Nucleic Acid Analysis

    Directory of Open Access Journals (Sweden)

    Juan J. Diaz-Mochon

    2012-06-01

    Full Text Available This manuscript describes the use of a novel biochip platform for the rapid analysis/identification of nucleic acids, including DNA and microRNAs, with very high specificity. This approach combines a unique dynamic chemistry approach for nucleic acid testing and analysis developed by DestiNA Genomics with the STMicroelectronics In-Check platform, which comprises two microfluidic optimized and independent PCR reaction chambers, and a sequential microarray area for nucleic acid capture and identification by fluorescence. With its compact bench-top “footprint” requiring only a single technician to operate, the biochip system promises to transform and expand routine clinical diagnostic testing and screening for genetic diseases, cancers, drug toxicology and heart disease, as well as employment in the emerging companion diagnostics market.

  19. JAWS: Just Add Water System - A device for detection of nucleic acids in Martian ice caps

    DEFF Research Database (Denmark)

    Hansen, Anders J.; Willerslev, Eske; Mørk, Søren;

    2002-01-01

    with a regulation of pH and salt concentrations e.g. the MOD systems and could be installed on a planetary probe melting its way down the Martian ice caps e.g. the NASA Cryobot. JAWS can be used for detection of remains of ancient life preserved in the Martian ice as well as for detection of contamination brought......The design of a device for nucleic acid detection in the Martian ice caps is presented; the Just Add Water System (JAWS). It is based on fiber-optic PNA (peptide nucleic acid) light up probe random microsphere universal array technology. JAWS is designed to be part of a larger system...

  20. Multifunctional Nucleic Acids for Tumor Cell Treatment

    DEFF Research Database (Denmark)

    Pofahl, Monika; Wengel, Jesper; Mayer, Günter

    2014-01-01

    We report on a multifunctional nucleic acid, termed AptamiR, composed of an aptamer domain and an antimiR domain. This composition mediates cell specific delivery of antimiR molecules for silencing of endogenous micro RNA. The introduced multifunctional molecule preserves cell targeting, anti......-proliferative and antimiR function in one 37-nucleotide nucleic acid molecule. It inhibits cancer cell growth and induces gene expression that is pathologically damped by an oncomir. These findings will have a strong impact on future developments regarding aptamer- and antimiR-related applications for tumor targeting...

  1. Macromolecular mimicry of nucleic acid and protein

    DEFF Research Database (Denmark)

    Nautrup Pedersen, Gitte; Nyborg, Jens; Clark, Brian F

    1999-01-01

    of the concept of macromolecular mimicry. Macromolecular mimicry has further been proposed among initiation and release factors, thereby adding a new element to the description of protein synthesis in bacteria. Such mimicry has also been observed in other biological processes such as autoimmunity, DNA repair......Although proteins and nucleic acids consist of different chemical components, proteins can mimic structures and possibly also functions of nucleic acids. Recently, structural mimicry was observed between two elongation factors in bacterial protein biosynthesis leading to the introduction...

  2. Functional Analysis of Polyphenol Oxidases by Antisense/Sense Technology

    Directory of Open Access Journals (Sweden)

    Jutharat Attajarusit

    2007-07-01

    Full Text Available Polyphenol oxidases (PPOs catalyze the oxidation of phenolics to quinones, the secondary reactions of which lead to oxidative browning and postharvest losses of many fruits and vegetables. PPOs are ubiquitous in angiosperms, are inducible by both biotic and abiotic stresses, and have been implicated in several physiological processes including plant defense against pathogens and insects, the Mehler reaction, photoreduction of molecular oxygen by PSI, regulation of plastidic oxygen levels, aurone biosynthesis and the phenylpropanoid pathway. Here we review experiments in which the roles of PPO in disease and insect resistance as well as in the Mehler reaction were investigated using transgenic tomato (Lycopersicon esculentum plants with modified PPO expression levels (suppressed PPO and overexpressing PPO. These transgenic plants showed normal growth, development and reproduction under laboratory, growth chamber and greenhouse conditions. Antisense PPO expression dramatically increased susceptibility while PPO overexpression increased resistance of tomato plants to Pseudomonas syringae. Similarly, PPO-overexpressing transgenic plants showed an increase in resistance to various insects, including common cutworm (Spodoptera litura (F., cotton bollworm (Helicoverpa armigera (Hübner and beet army worm (Spodoptera exigua (Hübner, whereas larvae feeding on plants with suppressed PPO activity had higher larval growth rates and consumed more foliage. Similar increases in weight gain, foliage consumption, and survival were also observed with Colorado potato beetles (Leptinotarsa decemlineata (Say feeding on antisense PPO transgenic tomatoes. The putative defensive mechanisms conferred by PPO and its interaction with other defense proteins are discussed. In addition, transgenic plants with suppressed PPO exhibited more favorable water relations and decreased photoinhibition compared to nontransformed controls and transgenic plants

  3. EFFECTS OF CYCLOOXYGENASE-2 ANTISENSE VECTOR ON PROLIFERATION OF HUMAN CHOLANGIOCARCINOMA CELLS

    Institute of Scientific and Technical Information of China (English)

    Gao-song Wu; Sheng-quan Zou; Xiao-yong Wu; Fa-zu Qiu

    2004-01-01

    Objective To transfect antisense vector of human cyclooxygenase-2 (COX-2) gene into COX-2 highly expressing cholangiocarcinoma cell line QBC939 and explore its biological activities and role in carcinogenesis.Methods QBC939 cells were transfected with antisense vector of human COX-2 gene using LipoVecTM transfecting technique. Transfected cells were selected with G418; COX-2 mRNA was examined using reverse transcription polymerase chain reaction (RT-PCR) and COX-2 protein expression was detected by immunocytochemistry using isozyme selective antibodies. The proliferative status of transfected cells was measured by using methabenzthiazuron (MTT) assay; Cell cycle and apoptosis were analyzed by using flow cytometry.Results RT-PCR showed a lower COX-2 mRNA level in antisense vector transfected cells and immunocytochemistry showed a weaker COX-2 protein expression in antisense vector transfected cells. The antisense vector transfected cells proliferative index decreased significantly (P< 0.01), the percentage of S phase decreased remarkably (P< 0.05) in antisense vector transfected cells (9.27% ± 1.91%) compared with that in QBC939 cells without transfection(16.35% ± 2.87%), and the percentage of G0/G 1 phase increased remarkably (P < 0.05) in antisense vector transfected cells (75.16%±4.13%) compared with that in QBC939 cells without transfection (57.31%± 10.16%). Transfection with antisense vector of human COX-2 gene had no significant influence on the apoptosis in QBC939 cells (P > 0.05).Conclusion Transfection with antisense vector of human COX-2 gene could inhibit the proliferation of human cholangiocarcinoma QBC939 cells.

  4. Locked and unlocked nucleosides in functional nucleic acids

    DEFF Research Database (Denmark)

    Doessing, Holger; Vester, Birte

    2011-01-01

    Nucleic acids are able to adopt a plethora of structures, many of which are of interest in therapeutics, bio- or nanotechnology. However, structural and biochemical stability is a major concern which has been addressed by incorporating a range of modifications and nucleoside derivatives. This rev...... review summarizes the use of locked nucleic acid (LNA) and un-locked nucleic acid (UNA) monomers in functional nucleic acids such as aptamers, ribozymes, and DNAzymes....

  5. Inhibitory effect of IGF-Ⅱ antisense RNA on malignant phenotype of hepatocellular carcinoma

    Institute of Scientific and Technical Information of China (English)

    Dong Hua Yang; Ming Qing Zhang; Han Rong Qin; Zi Rong Fan; Jiang Du; Chong Xu; Qiao Ming Liang; Ji Fang Mao

    2000-01-01

    @@INTRODUCTION According to the therapeutic effect and strategy of antisense RNA for hepatocellular carcinoma (HCC), we have specifically synthesized partial cDNA of human insulin-like growth factor Ⅱ (IGFⅡ ) and constructed IGF-Ⅱ cDNA antisense eukaryotic expression vector. The constructed vector was introduced into hepatoma cell line SMMC-7721 to block the intrinsic IGF- Ⅱexpression. The biological behavior changes of hepatoma cells were observed. All these would provide scientific basis for IGF- Ⅱ antisense RNA in the treatment of HCC.

  6. EFFECT OF TWO NEW BCL-2 ANTISENSES ON DRUG-SENSITIVITY OF CELLS FROMN LEUKEMIA PATIENTS

    Institute of Scientific and Technical Information of China (English)

    LEI Xiao-yong; ZHANG Huan

    2005-01-01

    Objective:To investigate the effect of two antisense oligonucleotides on cell surviving, bcl-2 expression and apoptosis of leukemia cells. Methods: The experimental assays were performed with cell culture, immunochemistry and flowcytometry. Results: The two antisense oligodeoxynucleotides, combined with Vp16 or Ara-c or DNR, were able to decline the survival rate of myeleukemic cells, downregulate bcl-2 gene expression and induce apoptosis of leukemic cells significantly, as compared with Vp16 or Ara-c or DNR alone. Conclusion: It is possible for the two new bcl-2 antisenses to be developed into clinical trials for leukemia and tumor with bcl-2 gene overexpression.

  7. An antisense RNA that governs the expression kinetics of a multifunctional virulence gene

    OpenAIRE

    Lee, Eun-Jin; Groisman, Eduardo A.

    2010-01-01

    Genome-wide transcriptome analyses of several bacterial species have recently uncovered a hitherto unappreciated amount of antisense transcription. However, the physiological role, regulation and significance of such antisense transcripts are presently unclear. We now report the identification of a cis-encoded 1.2 kb long antisense RNA – termed AmgR – that is complementary to the mgtC portion of the mgtCBR polycistronic message from Salmonella enterica. The mgtCBR mRNA specifies the MgtC prot...

  8. Inhibition of Leukemic Cell Telomerase Activity by Antisense Phosphorothioate Oligodeoxynucleotides

    Institute of Scientific and Technical Information of China (English)

    HEDongmei; ZHANGYuan

    2002-01-01

    Objective To evaluate the effect of human telomerase reverse transcriptase(hTERT) gene antisense oligodeoxynucleotide (ASON) on telomerase activity in K562 cells.Methods Telomerase activity was detemined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA) in K562 cells treated with ASODN and hTERTmRNA expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The hTERTmRNA level was decreased,and telomerase activity was significantly inhibited when the K562 cells were treated with ASODN for 48 h. Conclusion It is suggested that hTETR ASODN might specifically inhibit telomrase activity of K562 cells at translation level,and it is further proved that hTERT gene has significant correlation with telopmerase activity.

  9. Antisense oligonucleotide for tissue factor inhibits hepatic ischemic reperfusion injury.

    Science.gov (United States)

    Nakamura, Kenji; Kadotani, Yayoi; Ushigome, Hidetaka; Akioka, Kiyokazu; Okamoto, Masahiko; Ohmori, Yoshihiro; Yaoi, Takeshi; Fushiki, Shinji; Yoshimura, Rikio; Yoshimura, Norio

    2002-09-27

    Tissue factor (TF) is an initiation factor for blood coagulation and its expression is induced on endothelial cells during inflammatory or immune responses. We designed an antisense oligodeoxynucleotide (AS-1/TF) for rat TF and studied its effect on hepatic ischemic reperfusion injury. AS-1/TF was delivered intravenously to Lewis rats. After 10 h, hepatic artery and portal vein were partially clamped. Livers were reperfused after 180 min and harvested. TF expression was studied using immunohistochemical staining. One of 10 rats survived in a 5-day survival rate and TF was strongly stained on endothelial cells in non-treatment group. However, by treatment with AS-1/TF, six of seven survived and TF staining was significantly reduced. Furthermore, we observed that fluorescein-labeled AS-1/TF was absorbed into endothelial cells. These results suggest that AS-1/TF can strongly suppress the expression of TF and thereby inhibit ischemic reperfusion injury to the rat liver. PMID:12270110

  10. Chemical consequences of irradiating nucleic acids

    International Nuclear Information System (INIS)

    On the basis of literature data, a discussion is presented of the DNA damage which would be produced in a cellular environment and an attempt is made to place this damage in perspective as a potential hazard in food irradiation. The topics discussed are radiation damage mechanisms, OH reactions with DNA, base products, sugar products, and evaluation of damage from irradiated nucleic acids

  11. Non-instrumented nucleic acid amplification assay

    Science.gov (United States)

    Weigl, Bernhard H.; Domingo, Gonzalo; Gerlach, Jay; Tang, Dennis; Harvey, Darrel; Talwar, Nick; Fichtenholz, Alex; van Lew, Bill; LaBarre, Paul

    2008-02-01

    We have developed components of a diagnostic disposable platform that has the dual purpose of providing molecular diagnostics at the point of care (POC) as well as stabilizing specimens for further analysis via a centralized surveillance system. This diagnostic is targeted for use in low-resource settings by minimally trained health workers. The disposable device does not require any additional instrumentation and will be almost as rapid and simple to use as a lateral flow strip test - yet will offer the sensitivity and specificity of nucleic acid amplification tests (NAATs). The low-cost integrated device is composed of three functional components: (1) a sample-processing subunit that generates clean and stabilized DNA from raw samples containing nucleic acids, (2) a NA amplification subunit, and (3) visual amplicon detection sub-unit. The device integrates chemical exothermic heating, temperature stabilization using phase-change materials, and isothermal nucleic acid amplification. The aim of developing this system is to provide pathogen detection with NAAT-level sensitivity in low-resource settings where there is no access to instrumentation. If a disease occurs, patients would be tested with the disposable in the field. A nucleic acid sample would be preserved within the spent disposable which could be sent to a central laboratory facility for further analysis if needed.

  12. Antisense oligonucleotide induction of progerin in human myogenic cells.

    Directory of Open Access Journals (Sweden)

    Yue-Bei Luo

    Full Text Available We sought to use splice-switching antisense oligonucleotides to produce a model of accelerated ageing by enhancing expression of progerin, translated from a mis-spliced lamin A gene (LMNA transcript in human myogenic cells. The progerin transcript (LMNA Δ150 lacks the last 150 bases of exon 11, and is translated into a truncated protein associated with the severe premature ageing disease, Hutchinson-Gilford progeria syndrome (HGPS. HGPS arises from de novo mutations that activate a cryptic splice site in exon 11 of LMNA and result in progerin accumulation in tissues of mesodermal origin. Progerin has also been proposed to play a role in the 'natural' ageing process in tissues. We sought to test this hypothesis by producing a model of accelerated muscle ageing in human myogenic cells. A panel of splice-switching antisense oligonucleotides were designed to anneal across exon 11 of the LMNA pre-mRNA, and these compounds were transfected into primary human myogenic cells. RT-PCR showed that the majority of oligonucleotides were able to modify LMNA transcript processing. Oligonucleotides that annealed within the 150 base region of exon 11 that is missing in the progerin transcript, as well as those that targeted the normal exon 11 donor site induced the LMNA Δ150 transcript, but most oligonucleotides also generated variable levels of LMNA transcript missing the entire exon 11. Upon evaluation of different oligomer chemistries, the morpholino phosphorodiamidate oligonucleotides were found to be more efficient than the equivalent sequences prepared as oligonucleotides with 2'-O-methyl modified bases on a phosphorothioate backbone. The morpholino oligonucleotides induced nuclear localised progerin, demonstrated by immunostaining, and morphological nuclear changes typical of HGPS cells. We show that it is possible to induce progerin expression in myogenic cells using splice-switching oligonucleotides to redirect splicing of LMNA. This may offer a model

  13. Antisense angiopoietin-1 inhibits tumorigenesis and angiogenesis of gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Jun Wang; Kai-Chun Wu; De-Xin Zhang; Dai-Ming Fan

    2006-01-01

    AIM: To investigate the effect of angiopoietin-1 (Ang-1)on biological behaviors in vitro and tumorigenesis and angiogenesis in vitro of human gastric cancer cells.METHODS: Human full-length Ang-1 gene was cloned from human placental tissues by RT-PCR method.Recombinant human Ang-1 antisense eukaryotic expression vector was constructed by directional cloning,and transfected by lipofectin method into human gastric cancer line SGC7901 with high Ang-1 expression level.Inhibition efficiency was confirmed by semi- quantitive PCR and Western blot method. Cell growth curve and cell cycle were observed with MTT assays and flow cytometry, respectively. Nude mice tumorigenicity test was employed to compare in vitro tumorigenesis of cells with Ang-1 suppression. Microvessel density (MVD) of implanted tumor tissues was analyzed by immunohistochemistry for factor Ⅷ staining.RESULTS: Full-length Ang-1 gene was successfully cloned and stable transfectants were established,namely 7Ang1- for antisense, and 7901P for empty vector transfected. 7Ang1- cells showed down-regulated Ang-1 expression, while its in vitro proliferation and cell cycle distribution were not significantly changed.In contrast, xenograft of 7Ang1- cells in nude mice had lower volume and weight than those of 7901P after 30 days' implantation (P<0.01, 293.00±95.54 mg vs. 624.00±77.78 mg) accompanied with less vessel formation with MVD 6.00±1.73 compared to 7901P group 8.44±1.33 (P<0.01).CONCLUSION: Ang-1 may play an important role in tumorigenesis and angiogenesis of gastric cancer, and targeting its expression may be beneficial for the therapy of gastric cancer.

  14. Advances in magnetofection-magnetically guided nucleic acid delivery

    International Nuclear Information System (INIS)

    Magnetofection is nucleic acid delivery to cells supported and site-specifically guided by the attractive forces of magnetic fields acting on nucleic acid shuttles (vectors) which are associated with magnetic nanoparticles. Recent progress with the method confirms its general applicability with small and large nucleic acids and viruses. The method's therapeutic application as well as mechanistic studies will be discussed

  15. Sensitive detection of nucleic acids by PNA hybridization directed co-localization of fluorescent beads

    DEFF Research Database (Denmark)

    Shiraishi, Takehiko; Deborggraeve, Stijn; Büscher, Philippe;

    2011-01-01

    We have designed a pair of biotinylated peptide nucleic acid (PNA) probes targeting two sequences in 18S rRNA (from the parasite Trypanosoma brucei) at a distance of 191 nt (corresponding to maximum distance of ca. 60 nm) from each other. The PNA probes were individually bound to (strept....... The assay detected the parasite 18S rRNA down to 1.6 fmol while there was no such co-localization visible with human 18S rRNA not containing the PNA targets. Furthermore, the assay showed positive detection with 1.6 ng of total RNA (corresponding to RNA from ca. 300 parasites). Upon further optimization...

  16. Cis-encoded noncoding antisense RNAs in streptococci and other low GC Gram (+ bacterial pathogens.

    Directory of Open Access Journals (Sweden)

    Kyu Hong eCho

    2015-03-01

    Full Text Available Due to recent advances of bioinformatics and high throughput sequencing technology, discovery of regulatory noncoding RNAs in bacteria has been increased to a great extent. Based on this bandwagon, many studies searching for trans-acting small noncoding RNAs in streptococci have been performed intensively, especially in the important human pathogen, group A and B streptococci. However, studies for cis-encoded noncoding antisense RNAs in streptococci have been scarce. A recent study shows antisense RNAs are involved in virulence gene regulation in group B streptococcus, S. agalactiae. This suggests antisense RNAs could have important roles in the pathogenesis of streptococcal pathogens. In this review, we describe recent discoveries of chromosomal cis-encoded antisense RNAs in streptococcal pathogens and other low GC Gram (+ bacteria to provide a guide for future studies.

  17. STUDY ON THE INHIBITORY EFFECT OF ANTISENSE ETAR OLIGODEOXYNUCLEOTIDES ON THE PROLIFERATION OF VASCULAR SMOOTH CELLS

    Institute of Scientific and Technical Information of China (English)

    张岚; 张柏根; 张纪蔚; 钱济先; 张皓; 黄晓钟

    2002-01-01

    Objective To study the inhibitory effect of antisense endothelin receptor A (ETAR) on the proliferation of the vascular smooth muscle cells. Methods The sense, antisense and mismatched ODNs for ETAR were designed and synthetized. The study was carried out using MTT method and binding assays.Results ETAR-ODNs could move successfully across VSMC membranes. Photo-absorption in the MTT test was reduced significantly (P<0.05) in the antisense group at 5μmol/L; the reduction of CPM also occurred in the 125I-ET-1 specific binding assay; and the sense and mismatched ODNs groups did not show this reduction. Conclusion Our study suggested that the antisense oligomers inhibited the proliferation of VSMCs by hindering the translation of target mRNA and by reducing the production of related protein.

  18. The effects of antisense PTEN gene transfection on the growth and invasion of glioma cells

    Institute of Scientific and Technical Information of China (English)

    CHEN Hong-jie; ZHENG Zhao-cong; WANG Ru-mi; WANG Shou-sen; YANG Wei-zhong

    2006-01-01

    Objective:To study the effects of antisense PTEN gene on the growth and invasion of glioma cells. Methods:A pcDNA3. 1/Hygro (-) recombinant plasmid containing antisense PTEN gene fragment was constructed. Glioma cells of primary culture were transfected with antisense PTEN gene vector and stably transfected clones were selected. Then, the different growth and invasion abilities and the different MMP9 mRNA expressions of three kinds of cells were observed, including the transfected cells, untransfected cells and the cells transfected with empty vector. Results :The abilities of growth and invasion of the transfected cells and the expressions of MMP9 mRNA were obviously enhanced. Conclusion: Antisense PTEN gene could have a negative impact on the growth and invasion of primary culture glioma cells.

  19. Spt4 selectively regulates the expression of C9orf72 sense and antisense mutant transcripts.

    Science.gov (United States)

    Kramer, Nicholas J; Carlomagno, Yari; Zhang, Yong-Jie; Almeida, Sandra; Cook, Casey N; Gendron, Tania F; Prudencio, Mercedes; Van Blitterswijk, Marka; Belzil, Veronique; Couthouis, Julien; Paul, Joseph West; Goodman, Lindsey D; Daughrity, Lillian; Chew, Jeannie; Garrett, Aliesha; Pregent, Luc; Jansen-West, Karen; Tabassian, Lilia J; Rademakers, Rosa; Boylan, Kevin; Graff-Radford, Neill R; Josephs, Keith A; Parisi, Joseph E; Knopman, David S; Petersen, Ronald C; Boeve, Bradley F; Deng, Ning; Feng, Yanan; Cheng, Tzu-Hao; Dickson, Dennis W; Cohen, Stanley N; Bonini, Nancy M; Link, Christopher D; Gao, Fen-Biao; Petrucelli, Leonard; Gitler, Aaron D

    2016-08-12

    An expanded hexanucleotide repeat in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia (c9FTD/ALS). Therapeutics are being developed to target RNAs containing the expanded repeat sequence (GGGGCC); however, this approach is complicated by the presence of antisense strand transcription of expanded GGCCCC repeats. We found that targeting the transcription elongation factor Spt4 selectively decreased production of both sense and antisense expanded transcripts, as well as their translated dipeptide repeat (DPR) products, and also mitigated degeneration in animal models. Knockdown of SUPT4H1, the human Spt4 ortholog, similarly decreased production of sense and antisense RNA foci, as well as DPR proteins, in patient cells. Therapeutic targeting of a single factor to eliminate c9FTD/ALS pathological features offers advantages over approaches that require targeting sense and antisense repeats separately. PMID:27516603

  20. Construction and transfection of sense/antisense eukaryotic expression vectors for human cathepsin L gene

    Institute of Scientific and Technical Information of China (English)

    Maolin He; Anmin Chen

    2005-01-01

    Objective: To obtain sense/antisense eukaryotic expression vectors for human cathepsin L gene, and study the biological effects on human osteosarcoma cell line MG-63 after transfection. Methods: Cathepsin L gene sense/antisense eukaryotic expression vectors were constructed with recombinant technology and transfected into the human osteosarcoma cell line MG-63. The expression of cathepsin L gene mRNA was examined with RT-PCR and the expression of cathepsin L was examined with Western blot. Results: The sense/antisense recombinant eukaryotic expression vectors for cathepsin L were successfully constructed and transfected into MG-63 cell.Conclusion: Antisense cathepsin L gene can significantly inhibit the expression of cathepsin L mRNA and protein.

  1. Targeted Skipping of Human Dystrophin Exons in Transgenic Mouse Model Systemically for Antisense Drug Development

    OpenAIRE

    Bo Wu; Ehsan Benrashid; Peijuan Lu; Caryn Cloer; Allen Zillmer; Mona Shaban; Qi Long Lu

    2011-01-01

    Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD) patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs). However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD/...

  2. Bolaamphiphile-based nanocomplex delivery of phosphorothioate gapmer antisense oligonucleotides as a treatment for Clostridium difficile

    Science.gov (United States)

    Hegarty, John P; Krzeminski, Jacek; Sharma, Arun K; Guzman-Villanueva, Diana; Weissig, Volkmar; Stewart, David B

    2016-01-01

    Despite being a conceptually appealing alternative to conventional antibiotics, a major challenge toward the successful implementation of antisense treatments for bacterial infections is the development of efficient oligonucleotide delivery systems. Cationic vesicles (bolasomes) composed of dequalinium chloride (“DQAsomes”) have been used to deliver plasmid DNA across the cardiolipin-rich inner membrane of mitochondria. As cardiolipin is also a component of many bacterial membranes, we investigated the application of cationic bolasomes to bacteria as an oligonucleotide delivery system. Antisense sequences designed in silico to target the expression of essential genes of the bacterial pathogen, Clostridium difficile, were synthesized as 2′-O-methyl phosphorothioate gapmer antisense oligonucleotides (ASO). These antisense gapmers were quantitatively assessed for their ability to block mRNA translation using luciferase reporter and C. difficile protein expression plasmid constructs in a coupled transcription–translation system. Cationic bolaamphiphile compounds (dequalinium derivatives) of varying alkyl chain length were synthesized and bolasomes were prepared via probe sonication of an aqueous suspension. Bolasomes were characterized by particle size distribution, zeta potential, and binding capacities for anionic oligonucleotide. Bolasomes and antisense gapmers were combined to form antisense nanocomplexes. Anaerobic C. difficile log phase cultures were treated with serial doses of gapmer nanocomplexes or equivalent amounts of empty bolasomes for 24 hours. Antisense gapmers for four gene targets achieved nanomolar minimum inhibitory concentrations for C. difficile, with the lowest values observed for oligonucleotides targeting polymerase genes rpoB and dnaE. No inhibition of bacterial growth was observed from treatments at matched dosages of scrambled gapmer nanocomplexes or plain, oligonucleotide-free bolasomes compared to untreated control cultures. We

  3. Microinjection of antisense c-mos oligonucleotides prevents meiosis II in the maturing mouse egg.

    OpenAIRE

    O'Keefe, S J; Wolfes, H; Kiessling, A A; Cooper, G M

    1989-01-01

    Injection of antisense oligonucleotides was used to investigate the function of c-mos in murine oocytes. Oocytes injected with antisense c-mos oligonucleotides completed the first meiotic division but failed to initiate meiosis II. Instead, loss of c-mos function led to chromosome decondensation, reformation of a nucleus after meiosis I, and cleavage to two cells. Therefore, c-mos is required for meiosis II during murine oocyte maturation.

  4. Cathepsin B antisense oligodeoxynucleotide suppresses invasive potential of MG-63 cells

    Institute of Scientific and Technical Information of China (English)

    2008-01-01

    Objective To study the biological effects of cathepsin B phosporothioated antisense oligodeoxynucleotide on human osteosarcoma cell line MG-63 after transfection.Methods A 18-mer phosphorothioate antisense oligodeoxynucleotide(ASODN)targeted against the cathepsin B mRNA was transfected into the human osteosarcoma cell line MG-63 by lipofectamine 2000.The sense and nonsense oligodeoxynucleotides to cathepsin B and blank vector were used as controls.The expression of cathepsin B mRNA was examined by RT-PCR an...

  5. Detection of nucleic acids by multiple sequential invasive cleavages 02

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Jeff G. (Madison, WI); Lyamichev, Victor I. (Madison, WI); Mast, Andrea L. (Madison, WI); Brow, Mary Ann D. (Madison, WI)

    2002-01-01

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

  6. Detection of nucleic acids by multiple sequential invasive cleavages

    Energy Technology Data Exchange (ETDEWEB)

    Hall, Jeff G; Lyamichev, Victor I; Mast, Andrea L; Brow, Mary Ann D

    2012-10-16

    The present invention relates to means for the detection and characterization of nucleic acid sequences, as well as variations in nucleic acid sequences. The present invention also relates to methods for forming a nucleic acid cleavage structure on a target sequence and cleaving the nucleic acid cleavage structure in a site-specific manner. The structure-specific nuclease activity of a variety of enzymes is used to cleave the target-dependent cleavage structure, thereby indicating the presence of specific nucleic acid sequences or specific variations thereof. The present invention further relates to methods and devices for the separation of nucleic acid molecules based on charge. The present invention also provides methods for the detection of non-target cleavage products via the formation of a complete and activated protein binding region. The invention further provides sensitive and specific methods for the detection of human cytomegalovirus nucleic acid in a sample.

  7. Genome-wide analysis of antisense transcription with Affymetrix exon array

    Directory of Open Access Journals (Sweden)

    Jung Yong-chul

    2008-01-01

    Full Text Available Abstract Background A large number of natural antisense transcripts have been identified in human and mouse genomes. Study of their potential functions clearly requires cost-efficient method for expression analysis. Results Here we show that Affymetrix Exon arrays, which were designed to detect conventional transcripts in the sense orientation, can be used to monitor antisense expression across all exonic loci in mammalian genomes. Through modification of the cDNA synthesis protocol, we labeled single-strand cDNA in the reverse orientation as in the standard protocol, thus enabling the detection of antisense transcripts using the same array. Applying this technique to human Jurkat cells, we identified antisense transcription at 2,088 exonic loci of 1,516 UniGene clusters. Many of these antisense transcripts were not observed previously and some were validated by orientation-specific RT-PCR. Conclusion Our results suggest that with a modified protocol Affymetrix human, mouse and rat Exon arrays can be used as a routine method for genome-wide analysis of antisense transcription in these genomes.

  8. Bidirectional regulation between WDR83 and its natural antisense transcript DHPS in gastric cancer

    Institute of Scientific and Technical Information of China (English)

    Wen-Yu Su; Xuan Kong; Qin-Yan Gao; Li-Ping Wei; Jing-Yuan Fang; Jiong-Tang Li; Yun Cui; Jie Hong; Wan Du; Ying-Chao Wang; Yan-Wei Lin; Hua Xiong; Ji-Lin Wang

    2012-01-01

    Natural antisense transcripts (NATs) exist ubiquitously in mammalian genomes and play roles in the regulation of gene expression.However,both the existence of bidirectional antisense RNA regulation and the possibility of proteincoding genes that function as antisense RNAs remain speculative.Here,we found that the protein-coding gene,deoxyhypusine synthase (DHPS),as the NAT of WDR83,concordantly regulated the expression of WDR83 mRNA and protein.Conversely,WDR83 also regulated DHPS by antisense pairing in a concordant manner.WDR83 and DHPS were capable of forming an RNA duplex at overlapping 3′ untranslated regions and this duplex increased their mutual stability,which was required for the bidirectional regulation.As a pair of protein-coding cis-sense/antisense transcripts,WDR83 and DHPS were upregulated simultaneously and correlated positively in gastric cancer (GC),driving GC pathophysiology by promoting cell proliferation.Furthermore,the positive relationship between WDR83 and DHPS was also observed in other cancers.The bidirectional regulatory relationship between WDR83 and DHPS not only enriches our understanding of antisense regulation,but also provides a more complete understanding of their functions in tumor development.

  9. Studies of Liposomal bcl-2 Antisense Oligode-oxynucleofide Induction of Apoptosis in Raji Cells

    Institute of Scientific and Technical Information of China (English)

    DongmeiHe; HuanZhong

    2004-01-01

    OBJECTIVE To explore the effect of liposomal G3139 and transfected antisense phosphorothioate oligodeoxynucleotides directed against the coding region of the bcl-2 messenger RNA and the translation site on apoptosis in Raji cells.METHODS Cytotoxic effects were measured by use of the MTT method; The expression levels of Bcl-2 protein were assayed by immunofiuorescence using a fluoresce isothiocyanate label. Apoptosis was determined by morphological observation and flow cytometric analysis.RESULTS The 2 antisense oligonucleotides and G3139 can reduce Bcl-2 protein levels and Raji cell viability (IC50=4.54, 4.72 and 4.26 μmol/L, respectively), and induce apoptosis. A scrambled sequence control oligonucleotide and empty liposomes did not alter cell viability, Bcl-2 protein expression or apoptosis rates. There was no difference in reducing Bcl-2 protein levels and apoptosis rates found among the 3 antisense oligonucleotides.CONCLUSION The 2 antisense oligodeoxynucleotides of bcl-2 messenger RNA can effectively induce apoptosis of Raji cells. The 2 antisense sequences and G3139 have a similarity in their antisense effect.

  10. [Exon skipping therapy for Duchenne muscular dystrophy by using antisense Morpholino].

    Science.gov (United States)

    Takeda, Shin'ichi

    2009-11-01

    Duchenne muscular dystrophy (DMD) is caused by the lack of dystrophin protein at the sarcolemma. Exon skipping by antisense oligonucleotides is a novel method to restore the reading frame of the mutated DMD gene, and rescue dystrophin production. We recently reported that systemic delivery of Morpholino antisense oligonucleotides targeting exon 6 and 8 of the canine DMD gene, efficiently recovered functional dystrophin proteins at the sarcolamma of dystrophic dogs, and improved performance of affected dogs without serious side effects (Yokota et al., Ann Neurol. 65 (6): 667-676, 2009). To optimize therapeutic antisense Morpholinos for more frequent mutations of the DMD gene, we designed antisense Morpholinos targeting exon 51 of the mouse DMD gene, and injected them separately or in combination into the muscles of mdx52 mice, in which exon 52 has been deleted by a gene targeting technique (Araki et al., 1997). We also tried systemic delivery of antisense Morpholino to skip exon 51 in mdx52 mice. It is important to verify the effectiveness and side effects of antisense Morpholino in experimental animal models such as dystrophic dogs or mdx52 mice, before clinical trials in DMD patients. PMID:20030230

  11. Effective Antisense Gene Regulation via Noncationic, Polyethylene Glycol Brushes.

    Science.gov (United States)

    Lu, Xueguang; Jia, Fei; Tan, Xuyu; Wang, Dali; Cao, Xueyan; Zheng, Jiamin; Zhang, Ke

    2016-07-27

    Negatively charged nucleic acids are often complexed with polycationic transfection agents before delivery. Herein, we demonstrate that a noncationic, biocompatible polymer, polyethylene glycol, can be used as a transfection vector by forming a brush polymer-DNA conjugate. The brush architecture provides embedded DNA strands with enhanced nuclease stability and improved cell uptake. Because of the biologically benign nature of the polymer component, no cytotoxicity was observed. This approach has the potential to address several long-lasting challenges in oligonucleotide therapeutics. PMID:27420413

  12. [Development of Nucleic Acid-Based Adjuvant for Cancer Immunotherapy].

    Science.gov (United States)

    Kobiyama, Kouji; Ishii, Ken J

    2015-09-01

    Since the discovery of the human T cell-defined tumor antigen, the cancer immunotherapy field has rapidly progressed, with the research and development of cancer immunotherapy, including cancer vaccines, being conducted actively. However, the disadvantages of most cancer vaccines include relatively weak immunogenicity and immune escape or exhaustion. Adjuvants with innate immunostimulatory activities have been used to overcome these issues, and these agents have been shown to enhance the immunogenicity of cancer vaccines and to act as mono-therapeutic anti-tumor agents. CpG ODN, an agonist for TLR9, is one of the promising nucleic acid-based adjuvants, and it is a potent inducer of innate immune effector functions. CpG ODN suppresses tumor growth in the absence of tumor antigens and peptide administration. Therefore, CpG ODN is expected to be useful as a cancer vaccine adjuvant as well as a cancer immunotherapy agent. In this review, we discuss the potential therapeutic applications and mechanisms of CpG ODN for cancer immunotherapy. PMID:26469159

  13. [Development of Nucleic Acid-Based Adjuvant for Cancer Immunotherapy].

    Science.gov (United States)

    Kobiyama, Kouji; Ishii, Ken J

    2015-09-01

    Since the discovery of the human T cell-defined tumor antigen, the cancer immunotherapy field has rapidly progressed, with the research and development of cancer immunotherapy, including cancer vaccines, being conducted actively. However, the disadvantages of most cancer vaccines include relatively weak immunogenicity and immune escape or exhaustion. Adjuvants with innate immunostimulatory activities have been used to overcome these issues, and these agents have been shown to enhance the immunogenicity of cancer vaccines and to act as mono-therapeutic anti-tumor agents. CpG ODN, an agonist for TLR9, is one of the promising nucleic acid-based adjuvants, and it is a potent inducer of innate immune effector functions. CpG ODN suppresses tumor growth in the absence of tumor antigens and peptide administration. Therefore, CpG ODN is expected to be useful as a cancer vaccine adjuvant as well as a cancer immunotherapy agent. In this review, we discuss the potential therapeutic applications and mechanisms of CpG ODN for cancer immunotherapy.

  14. Revised genomic structure of the human ghrelin gene and identification of novel exons, alternative splice variants and natural antisense transcripts

    Directory of Open Access Journals (Sweden)

    Herington Adrian C

    2007-08-01

    Full Text Available Abstract Background Ghrelin is a multifunctional peptide hormone expressed in a range of normal tissues and pathologies. It has been reported that the human ghrelin gene consists of five exons which span 5 kb of genomic DNA on chromosome 3 and includes a 20 bp non-coding first exon (20 bp exon 0. The availability of bioinformatic tools enabling comparative analysis and the finalisation of the human genome prompted us to re-examine the genomic structure of the ghrelin locus. Results We have demonstrated the presence of an additional novel exon (exon -1 and 5' extensions to exon 0 and 1 using comparative in silico analysis and have demonstrated their existence experimentally using RT-PCR and 5' RACE. A revised exon-intron structure demonstrates that the human ghrelin gene spans 7.2 kb and consists of six rather than five exons. Several ghrelin gene-derived splice forms were detected in a range of human tissues and cell lines. We have demonstrated ghrelin gene-derived mRNA transcripts that do not code for ghrelin, but instead may encode the C-terminal region of full-length preproghrelin (C-ghrelin, which contains the coding region for obestatin and a transcript encoding obestatin-only. Splice variants that differed in their 5' untranslated regions were also found, suggesting a role of these regions in the post-transcriptional regulation of preproghrelin translation. Finally, several natural antisense transcripts, termed ghrelinOS (ghrelin opposite strand transcripts, were demonstrated via orientation-specific RT-PCR, 5' RACE and in silico analysis of ESTs and cloned amplicons. Conclusion The sense and antisense alternative transcripts demonstrated in this study may function as non-coding regulatory RNA, or code for novel protein isoforms. This is the first demonstration of putative obestatin and C-ghrelin specific transcripts and these findings suggest that these ghrelin gene-derived peptides may also be produced independently of preproghrelin

  15. Nucleic acid compositions and the encoding proteins

    Science.gov (United States)

    Preston, III, James F.; Chow, Virginia; Nong, Guang; Rice, John D.; St. John, Franz J.

    2014-09-02

    The subject invention provides at least one nucleic acid sequence encoding an aldouronate-utilization regulon isolated from Paenibacillus sp. strain JDR-2, a bacterium which efficiently utilizes xylan and metabolizes aldouronates (methylglucuronoxylosaccharides). The subject invention also provides a means for providing a coordinately regulated process in which xylan depolymerization and product assimilation are coupled in Paenibacillus sp. strain JDR-2 to provide a favorable system for the conversion of lignocellulosic biomass to biobased products. Additionally, the nucleic acid sequences encoding the aldouronate-utilization regulon can be used to transform other bacteria to form organisms capable of producing a desired product (e.g., ethanol, 1-butanol, acetoin, 2,3-butanediol, 1,3-propanediol, succinate, lactate, acetate, malate or alanine) from lignocellulosic biomass.

  16. Total Nucleic Acid Extraction from Soil

    OpenAIRE

    sprotocols

    2015-01-01

    Authors: Roey Angel ### Abstract The following protocol is intended for the simultaneous extraction of DNA and RNA from various soil samples along with suggestions on how to tweak the protocol for soil with higher humic content. The protocol has been used by many and results in very high yields of nucleic acids, typically much more than commercial kits. For buffers and solutions used in this protocol, please see accompanying document Buffers and Solutions for TNA Extractions.pdf. ...

  17. Novel applications of locked nucleic acids.

    Science.gov (United States)

    Veedu, Rakesh N; Vester, Birte; Wengel, Jesper

    2007-01-01

    Locked Nucleic Acid (LNA) nucleoside triphosphates were prepared and their substrate properties for different polymerases during primer extension and PCR experiments investigated. Phusion High Fidelity DNA polymerase and 9( degrees )Nm(TM) DNA polymerase readily accept LNA nucleoside 5'-triphosphates as substrates in primer extension assays. However, in PCR assays, However, in PCR assays, DNA 9oN(m) polymerase proved to be the best for amplification employing the LNA-A nucleotide. PMID:18029570

  18. Nanoparticle Delivery of Antisense Oligonucleotides and Their Application in the Exon Skipping Strategy for Duchenne Muscular Dystrophy

    OpenAIRE

    Falzarano, Maria Sofia; Passarelli, Chiara; Ferlini, Alessandra

    2014-01-01

    Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA,...

  19. Sequence Selective Recognition of Double-Stranded RNA at Physiologically Relevant Conditions Using PNA-Peptide Conjugates

    OpenAIRE

    Muse, Oluwatoyosi; Zengeya, Thomas; Mwaura, Juddy; Hnedzko, Dziyana; McGee, Dennis W.; Grewer, Christof T.; Rozners, Eriks

    2013-01-01

    Conjugation of short peptide nucleic acids (PNA) with tetralysine peptides strongly enhanced triple helical binding to RNA at physiologically relevant conditions. The PNA hexamers and heptamers carrying cationic nucleobase and tetralysine modifications displayed high binding affinity for complementary double-stranded RNA without compromising sequence selectivity. The PNA-peptide conjugates had unique preference for binding double-stranded RNA, while having little, if any, affinity for double-...

  20. Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum

    Directory of Open Access Journals (Sweden)

    López-Barragán María J

    2011-11-01

    Full Text Available Abstract Background It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent and pattern of antisense transcripts in different developmental stages remain largely unknown. Results We have sequenced seven bidirectional libraries from ring, early and late trophozoite, schizont, gametocyte II, gametocyte V, and ookinete, and four strand-specific libraries from late trophozoite, schizont, gametocyte II, and gametocyte V of the 3D7 parasites. Alignment of the cDNA sequences to the 3D7 reference genome revealed stage-specific antisense transcripts and novel intron-exon splicing junctions. Sequencing of strand-specific cDNA libraries suggested that more genes are expressed in one direction in gametocyte than in schizont. Alternatively spliced genes, antisense transcripts, and stage-specific expressed genes were also characterized. Conclusions It is necessary to continue to sequence cDNA from different developmental stages, particularly those of non-erythrocytic stages. The presence of antisense transcripts in some gametocyte and ookinete genes suggests that these antisense RNA may play an important role in gene expression regulation and parasite development. Future gene expression studies should make use of directional cDNA libraries. Antisense transcripts may partly explain the observed discrepancy between levels of mRNA and protein expression.

  1. A specific scenario for the origin of life and the genetic code based on peptide/oligonucleotide interdependence.

    Science.gov (United States)

    Griffith, Robert W

    2009-12-01

    Among various scenarios that attempt to explain how life arose, the RNA world is currently the most widely accepted scientific hypothesis among biologists. However, the RNA world is logistically implausible and doesn't explain how translation arose and DNA became incorporated into living systems. Here I propose an alternative hypothesis for life's origin based on cooperation between simple nucleic acids, peptides and lipids. Organic matter that accumulated on the prebiotic Earth segregated into phases in the ocean based on density and solubility. Synthesis of complex organic monomers and polymerization reactions occurred within a surface hydrophilic layer and at its aqueous and atmospheric interfaces. Replication of nucleic acids and translation of peptides began at the emulsified interface between hydrophobic and aqueous layers. At the core of the protobiont was a family of short nucleic acids bearing arginine's codon and anticodon that added this amino acid to pre-formed peptides. In turn, the survival and replication of nucleic acid was aided by the peptides. The arginine-enriched peptides served to sequester and transfer phosphate bond energy and acted as cohesive agents, aggregating nucleic acids and keeping them at the interface.

  2. Conformational analysis of Infectious bursal disease virus (IBDV derived cell penetrating peptide (CPP analogs

    Directory of Open Access Journals (Sweden)

    Vinay G. Joshi

    2013-12-01

    Full Text Available Aim: This study was designed to develop peptide analogs of Infectious Bursal Disease (IBD virus VP5 protein segment having cell penetrating ability to improve their interaction with cargo molecule (Nucleic acid without affecting the backbone conformation. Materials and Methods: IBDV VP5 protein segment designated as RATH peptide were synthesized using solid phase peptide synthesis and their solution conformation was elucidated using CD spectroscopy in polar (water and apolar (TFE solvents. Cell penetrating ability of RATH-CONH2 was observed using FITC labeled peptide internalization in to HeLa cells under fluorescent microscopy. The efficacy of RATH analog interactions with nucleic acids was evaluated using FITC labeled oligonucleotides by fluorescence spectroscopy and plasmid constructs in gel retardation assay. Results: CD spectra of RATH analogs in water and apolar trifluroethanol (TFE helped to compare their secondary structures which were almost similar with dominant beta conformations suggesting successful induction of positive charge in the analogs without affecting back bone conformation of CPP designed. Cell penetrating ability of RATH CONH2 in HeLa cell was more than 90%. The fluorescence spectroscopy and plasmid constructs in gel retardation assay demonstrated successful interaction of amide analogs with nucleic acid. Conclusion: Intentional changes made in IBDV derived peptide RATH COOH to RATH CONH2 did not showed major changes in backbone conformation and such modifications may help to improve the cationic charge in most CPPs to interact with nucleic acid. [Vet World 2013; 6(6.000: 307-312

  3. A homogeneous nucleic acid hybridization assay based on strand displacement.

    OpenAIRE

    Vary, C P

    1987-01-01

    A homogeneous nucleic acid hybridization assay which is conducted in solution and requires no separation steps is described. The assay is based on the concept of strand displacement. In the strand displacement assay, an RNA "signal strand" is hybridized within a larger DNA strand termed the "probe strand", which is, in turn, complementary to the target nucleic acid of interest. Hybridization of the target nucleic acid with the probe strand ultimately results in displacement of the RNA signal ...

  4. Microspectrophotometric quantitation of nucleic acid and protein in irradiated epidermis.

    Science.gov (United States)

    Conti, C J; Giménez, I B; Cabrini, R L

    1976-03-01

    Nucleic acid and proteins of newborn rat tail subjected to local X-irradiation were microspectrophotometrically studied. Feulgen, gallocyanine chrom-alum and naphthol yellow S methods were performed for demonstration of DNA, total nucleic acid and proteins respectively. The amount of proteins and total nucleic acid increases concomitantly with reactional acanthosis. However, the proteins and nucleic acid decrease as from day 3 post-irradiation. A tentative interpretation of the results would point to a giantization of the epidermic cells not only caused by aqueous imbition but also by an actual increase of the cellular protoplasm. PMID:1258094

  5. Flexibility of nucleic acids: from DNA to RNA

    CERN Document Server

    Bao, Lei; Jin, Lei; Tan, Zhi-Jie

    2015-01-01

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids.

  6. Microspectrophotometric quantitation of nucleic acid and protein in irradiated epidermis

    International Nuclear Information System (INIS)

    Nucleic acid and proteins of newborn rat tail epidermis subjected to local X-irradiation were microspectrophotometrically studied. Feulgen, gallocyanine chrom-alum and naphthol yellow S methods were performed for demonstration of DNA, total nucleic acid and proteins respectively. The amount of proteins and total nucleic acid increases concomitantly with reactional acanthosis. However, the proteins and nucleic acid decrease as from day 3 post-irradiation. A tentative interpretation of the results would point to a giantization of the epidermic cells not only caused by aqueous imbition but also by an actual increase of the cellular protoplasm. (orig.)

  7. BTEB2 antisense RNA inhibits intimal hyperplasia in a rat carotid balloon injury model

    Institute of Scientific and Technical Information of China (English)

    LI De; HE Guo-xiang; TANG Bing; TANG Bo

    2006-01-01

    Objective: To investigate the effects of basic transcriptional element binding protein-2(BTEB2) antisense RNA on vascular smooth muscle cells (VSMCs) proliferation and the neointimal formation after carotid balloon injury in rats. Methods: The cultured VSMCs were transfected with an adenoviral vector containing BTEB2 antisense gene, Ad ASBTEB2. Effects of BTEB2 antisense RNA on the expression of BTEB2 were investigated by Western blot analysis. The cell cycle was analyzed using flow cytometry. Ad ASBTEB2, control adenoviral vector Ad. LacZ or PBS was transduced into the rat carotid artery after balloon injury. The expression of BTEB2 at 7, 14, and 21 d after gene transfer was detected by immunohistochemistry and neointima-to-media (I/M) area ratio at these time points was calculated. Results: The cell cycle was arrested in G0/G1 phase and the expression of BETB2 was downregulated after transfection with Ad ASBTEB2. Ad ASBTEB2 treatment reduced I/M area ratios on day 7, 14, and 21 after injury by 45%, 50% and 53% respectively, whereas the Ad LacZ treatment did not significantly alter these ratios compared with control group. Conclusion: BTEB2 antisense RNA mediated by adenoviral vector inhibits proliferation of VSMCs and significantly reduces neointimal hyperplasia in the rat carotid balloon injury model. BTEB2 antisense RNA is a potential therapeutic approach to preventing neointimal formation after balloon injury.

  8. Induction of apoptosis and inhibition of proliferation in Hep-2 by antisense survivin RNA in vitro

    Institute of Scientific and Technical Information of China (English)

    2006-01-01

    Objective: To study induction of apoptosis and inhibition of proliferation in Hep-2 by antisense survivin RNA. Methods: Antisense survivin RNA expression vector was constructed and then was transfected to human laryngeal carcinoma cell line Hep-2 by lipofectamine. HpEGFP/survivin cells (transfected with the combinant of antisense survivin RNA) were obstained by using G418. The levels of survivin protein before and after transfection were determined by Western-blot. Proliferation activity was measured by MTT assay. The experiment of colony formation in soft agar was carried out for assessing ability of proliferation of Hep-2 cell. Apoptosis was assessed by flow cytometry and acrdine orange(AO).Results:After antisense survivin RNA plasmids were transfected, the level of survivin protein was inhibited in Hep-2. ComPared with control, proliferation of HpEGFP/survivin cells were suppressed significantly. The experiment of colony formation in soft agar showed the ability of colony formation decreased in HpEGFP/survivin cells compared to control (P<0.05). Apoptosis rate increased about 1.81 folds compared with control. Conclusion: The antisense survivin RNA can partly inhibit the level of survivin protein expression in Hep-2 and can induce apoptosis and inhibit the proliferation of Hep-2 by down-regulating the expression of endogenous survivin in vitro.

  9. Expression of an Antisense BcMF3 Affects Microsporogenesis and Pollen Tube Growth in Arabidopsis

    Institute of Scientific and Technical Information of China (English)

    LIU Le-cheng; CAO Jia-shu; YU Xiao-lin; XIANG Xun; FEI Yong-jun

    2006-01-01

    In an effort to provide some information relevant to the molecular mechanism of genic male sterility in plants, BcMF3 gene that encodes a pectin methylesterase was isolated from the fertile B line of Chinese cabbage-pak-choi (Brassica rapa ssp.chinensis, syn. B. campestris ssp. chinensis). In the present paper, a 455-bp antisense cDNA fragment of BcMF3 was introduced to binary vector pBI121, and then was mobilized into Agrobacterium tumefaciens strain LBA4404. The A.tumefaciens harboring the BcMF3 antisense fragment was transformed to Arabidopsis thaliana by floral dip. Scanning electronic microscopy examination demonstrated that 47.8% of BcMF3 antisense pollen grains exhibited abnormal shape,which might lead to decreased germination of pollens, suggesting that the product of BcMF3 gene plays an important role during microsporogenesis. The evidence on burst of 45.7% of BcMF3 antisense pollen tubes in vitro and a majority of BcMF3 antisense pollens restricted within the stigmatic tissue revealed that BcMF3 is involved in aiding the growth of pollen tubes. The results suggest that BcMF3 acts at both stages of microsporogensis and pollen tube growth.

  10. Intracerebroventricular Administration of Mineralocorticoid Receptor Antisense Oligonucleotides Attenuates Salt Appetite in the Rat.

    Science.gov (United States)

    Ma; Itharat; Fluharty; Sakai

    1997-10-01

    The anterior ventral third ventricle (AV3V) region of the brain contains high concentrations of mineralocorticoid receptors (MR) and glucocorticoid receptors (GR) that are important in the maintenance of body fluid and electrolyte balance as well as other physiological processes. Daily intracerebroventricular pulse injections of MR antisense oligonucleotides significantly suppressed deoxycorticosterone acetate (DOCA) induced salt appetite in a dose-related manner. Similar administration of GR antisense or scrambled/sense oligonucleotide into the third ventricle failed to inhibit salt appetite. Salt appetite aroused after adrenalectomy was not suppressed by MR antisense oligonucleotide treatments but was suppressed by an antisense oligonucleotide directed against the angiotensin II AT1 receptor subtype. Receptor binding analysis demonstrated that MR and GR oligonucleotide treatments each reduced their respective receptor subtypes. Finally, although GR antisense oligonucleotide treatment was ineffective in suppressing DOCA-induced salt appetite, this treatment did increase stress induced corticosterone release as well as delayed the recovery of corticosterone to basal levels after stress. PMID:9787254

  11. Obstructive Effects of Ultrasonic Microbubble Intensifier on CHG-5 Cell with Survivin Antisense Oligonucleotides Transfection

    Institute of Scientific and Technical Information of China (English)

    CAO Hong-ying; CAO You-de; WANG Zhi-gang; LI Pan

    2008-01-01

    Objective:To study the effects on human glioma cell line CHG-5 by ultrasonic microbubble intensifier with survivin antisense oligonucleotides (ASODN)transfection. Methods: Antisense oligonucleotides targeting survivin mRNA was designed and synthesized.Four regimen groups were designed,group A:survivin antisense oligonucleotides transfected with ultrasonic microbubble intensifier combined with ultrasound irradiation,group B: survivin antisense oligonucleotides transfected with lipofectamine combined with ultrasound irradiation,group C:survivin antisense oligonucelotides with lipofectamine transfection.group D:blank control.The expression changes of surviving protein were measured by immunohistochemical staining and Western blotting,and MTr assay was used to measure the changes of proliferation.Results:Survivin protein expression in group A was decreased significantly in human glioma cell line CHG-5 than other groups(P<0.05),and the proliferating rate of CHG-5 in group A was also significantly inhibited(P<0.05).Conclusion:Ultrasonic microbubble intensifier transfection combined with ultrasound irradiation is a promising method in gene transfection effectively and noninvasively.

  12. A locked nucleic Acid-based nanocrawler

    DEFF Research Database (Denmark)

    Astakhova, I Kira; Pasternak, Karol; Campbell, Meghan A;

    2013-01-01

    excimer formation and pyrene-perylene interstrand Förster resonance energy transfer. We furthermore demonstrate that the nanocrawler selectively and reversibly moves along the road, followed by a bright and consistent fluorescence response for up to 10 cycles without any loss of signal.......Herein we introduce a novel fluorescent LNA/DNA machine, a nanocrawler, which reversibly moves along a directionally polar complementary road controlled by affinity-enhancing locked nucleic acid (LNA) monomers and additional regulatory strands. Polyaromatic hydrocarbon (PAH) dyes attached to 2...

  13. Correction of a Cystic Fibrosis Splicing Mutation by Antisense Oligonucleotides.

    Science.gov (United States)

    Igreja, Susana; Clarke, Luka A; Botelho, Hugo M; Marques, Luís; Amaral, Margarida D

    2016-02-01

    Cystic fibrosis (CF), the most common life-threatening genetic disease in Caucasians, is caused by ∼2,000 different mutations in the CF transmembrane conductance regulator (CFTR) gene. A significant fraction of these (∼13%) affect pre-mRNA splicing for which novel therapies have been somewhat neglected. We have previously described the effect of the CFTR splicing mutation c.2657+5G>A in IVS16, showing that it originates transcripts lacking exon 16 as well as wild-type transcripts. Here, we tested an RNA-based antisense oligonucleotide (AON) strategy to correct the aberrant splicing caused by this mutation. Two AONs (AON1/2) complementary to the pre-mRNA IVS16 mutant region were designed and their effect on splicing was assessed at the RNA and protein levels, on intracellular protein localization and function. To this end, we used the 2657+5G>A mutant CFTR minigene stably expressed in HEK293 Flp-In cells that express a single copy of the transgene. RNA data from AON1-treated mutant cells show that exon 16 inclusion was almost completely restored (to 95%), also resulting in increased levels of correctly localized CFTR protein at the plasma membrane (PM) and with increased function. A novel two-color CFTR splicing reporter minigene developed here allowed the quantitative monitoring of splicing by automated microscopy localization of CFTR at the PM. The AON strategy is thus a promising therapeutic approach for the specific correction of alternative splicing.

  14. Rational genomics I: antisense open reading frames and codon bias in short-chain oxido reductase enzymes and the evolution of the genetic code.

    Science.gov (United States)

    Duax, William L; Huether, Robert; Pletnev, Vladimir Z; Langs, David; Addlagatta, Anthony; Connare, Sonjay; Habegger, Lukas; Gill, Jay

    2005-12-01

    The short-chain oxidoreductase (SCOR) family of enzymes includes over 6000 members, extending from bacteria and archaea to humans. Nucleic acid sequence analysis reveals that significant numbers of these genes are remarkably free of stopcodons in reading frames other than the coding frame, including those on the antisense strand. The genes from this subset also use almost entirely the GC-rich half of the 64 codons. Analysis of a million hypothetical genes having random nucleotide composition shows that the percentage of SCOR genes having multiple open reading frames exceeds random by a factor of as much as 1 x 10(6). Nevertheless, screening the content of the SWISS-PROT TrEMBL database reveals that 15% of all genes contain multiple open reading frames. The SCOR genes having multiple open reading frames and a GC-rich coding bias exhibit a similar GC bias in the nucleotide triple composition of their DNA. This bias is not correlated with the GC content of the species in which the SCOR genes are found. One possible explanation for the conservation of multiple open reading frames and extreme bias in nucleic acid composition in the family of Rossman folds is that the primordial member of this family was encoded early using only very stable GC-rich DNA and that evolution proceeded with extremely limited introduction of any codons having two or more adenine or thymine nucleotides. These and other data suggest that the SCOR family of enzymes may even have diverged from a common ancestor before most of the AT-rich half of the genetic code was fully defined.

  15. NALDB: nucleic acid ligand database for small molecules targeting nucleic acid.

    Science.gov (United States)

    Kumar Mishra, Subodh; Kumar, Amit

    2016-01-01

    Nucleic acid ligand database (NALDB) is a unique database that provides detailed information about the experimental data of small molecules that were reported to target several types of nucleic acid structures. NALDB is the first ligand database that contains ligand information for all type of nucleic acid. NALDB contains more than 3500 ligand entries with detailed pharmacokinetic and pharmacodynamic information such as target name, target sequence, ligand 2D/3D structure, SMILES, molecular formula, molecular weight, net-formal charge, AlogP, number of rings, number of hydrogen bond donor and acceptor, potential energy along with their Ki, Kd, IC50 values. All these details at single platform would be helpful for the development and betterment of novel ligands targeting nucleic acids that could serve as a potential target in different diseases including cancers and neurological disorders. With maximum 255 conformers for each ligand entry, our database is a multi-conformer database and can facilitate the virtual screening process. NALDB provides powerful web-based search tools that make database searching efficient and simplified using option for text as well as for structure query. NALDB also provides multi-dimensional advanced search tool which can screen the database molecules on the basis of molecular properties of ligand provided by database users. A 3D structure visualization tool has also been included for 3D structure representation of ligands. NALDB offers an inclusive pharmacological information and the structurally flexible set of small molecules with their three-dimensional conformers that can accelerate the virtual screening and other modeling processes and eventually complement the nucleic acid-based drug discovery research. NALDB can be routinely updated and freely available on bsbe.iiti.ac.in/bsbe/naldb/HOME.php. Database URL: http://bsbe.iiti.ac.in/bsbe/naldb/HOME.php. PMID:26896846

  16. Repair of Thalassemic Human β -globin mRNA in Mammalian Cells by Antisense Oligonucleotides

    Science.gov (United States)

    Sierakowska, Halina; Sambade, Maria J.; Agrawal, Sudhir; Kole, Ryszard

    1996-11-01

    In one form of β -thalassemia, a genetic blood disorder, a mutation in intron 2 of the β -globin gene (IVS2-654) causes aberrant splicing of β -globin pre-mRNA and, consequently, β -globin deficiency. Treatment of mammalian cells stably expressing the IVS2-654 human β -globin gene with antisense oligonucleotides targeted at the aberrant splice sites restored correct splicing in a dose-dependent fashion, generating correct human β -globin mRNA and polypeptide. Both products persisted for up to 72 hr posttreatment. The oligonucleotides modified splicing by a true antisense mechanism without overt unspecific effects on cell growth and splicing of other pre-mRNAs. This novel approach in which antisense oligonucleotides are used to restore rather than to down-regulate the activity of the target gene is applicable to other splicing mutants and is of potential clinical interest.

  17. Effects of CIITA antisense RNA on the expression of HLA class Ⅱ molecules

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To study the effect of the major histocompatibility complex class Ⅱ (MHCⅡ) transactivator (CIITA) antisense RNA on the expression of the human leukemia (HLA) class Ⅱ molecules, 5′ end cDNA sequence of CIITA gene was cloned, and antisense RNA expression vector pcDNA-Ⅱ was constructed. HeLa cells transfected with pcDNA-Ⅱ and pcDNA3 were induced by IFN-g for 3 d. The expression of HLA class Ⅱ molecules on HeLa/pcDNA-Ⅱ cells was significantly decreased, while it has no effect on the expression of HLA class Ⅰ molecules. This result suggests that the CIITA antisense RNA can inhibit the expression of HLA class Ⅱ molecules in HeLa cells. It also implies a promising approach to generate immune tolerance in graft transplantation.

  18. Antimicrobial peptides.

    Science.gov (United States)

    Zhang, Ling-Juan; Gallo, Richard L

    2016-01-11

    Antimicrobial peptides and proteins (AMPs) are a diverse class of naturally occurring molecules that are produced as a first line of defense by all multicellular organisms. These proteins can have broad activity to directly kill bacteria, yeasts, fungi, viruses and even cancer cells. Insects and plants primarily deploy AMPs as an antibiotic to protect against potential pathogenic microbes, but microbes also produce AMPs to defend their environmental niche. In higher eukaryotic organisms, AMPs can also be referred to as 'host defense peptides', emphasizing their additional immunomodulatory activities. These activities are diverse, specific to the type of AMP, and include a variety of cytokine and growth factor-like effects that are relevant to normal immune homeostasis. In some instances, the inappropriate expression of AMPs can also induce autoimmune diseases, thus further highlighting the importance of understanding these molecules and their complex activities. This Primer will provide an update of our current understanding of AMPs. PMID:26766224

  19. Antimicrobial Peptides

    Directory of Open Access Journals (Sweden)

    Ali Adem Bahar

    2013-11-01

    Full Text Available The rapid increase in drug-resistant infections has presented a serious challenge to antimicrobial therapies. The failure of the most potent antibiotics to kill “superbugs” emphasizes the urgent need to develop other control agents. Here we review the history and new development of antimicrobial peptides (AMPs, a growing class of natural and synthetic peptides with a wide spectrum of targets including viruses, bacteria, fungi, and parasites. We summarize the major types of AMPs, their modes of action, and the common mechanisms of AMP resistance. In addition, we discuss the principles for designing effective AMPs and the potential of using AMPs to control biofilms (multicellular structures of bacteria embedded in extracellular matrixes and persister cells (dormant phenotypic variants of bacterial cells that are highly tolerant to antibiotics.

  20. Functional comparison of antisense proteins of HTLV-1 and HTLV-2 in viral pathogenesis

    Directory of Open Access Journals (Sweden)

    Benoit eBarbeau

    2013-08-01

    Full Text Available The production of antisense transcripts from the 3’ long terminal repeat (LTR in human T-lymphotropic retroviruses has now been clearly demonstrated. After the identification of the antisense strand-encoded HTLV-1 bZIP (HBZ factor, we reported that HBZ could interact with CREB transcription factors and consequently turn off the important activating potential of the viral Tax protein on HTLV-1 5’ LTR promoter activity. We have recently accumulated new results demonstrating that antisense transcripts also exist in HTLV-2, -3 and -4. Furthermore, our data have confirmed the existence of encoded proteins from these antisense transcripts (termed antisense proteins of HTLVs or APHs. APHs are also involved in the down-regulation of Tax-dependent viral transcription. In this review, we will focus on the different molecular mechanisms used by HBZ and APH-2 to control viral expression. While HBZ interacts with CREB through its basic zipper domain, APH-2 binds to this cellular factor through a five amino acid motif localized in its carboxyl terminus. Moreover, unlike APH-2, HBZ possesses an N-terminal activation domain that also contributes to the inhibition of the viral transcription by interacting with the KIX domain of p300/CBP. On the other hand, HBZ was found to induce T-cell proliferation while APH-2 was unable to promote such proliferation. Interestingly, HTLV-2 has not been causally linked to human T-cell leukemia, while HTLV-1 is responsible for the development of the Adult T-cell Leukemia/Lymphoma (ATLL. We will further discuss the possible role played by antisense proteins in the establishment of pathologies induced by viral infection.

  1. Large-scale analysis of antisense transcription in wheat using the Affymetrix GeneChip Wheat Genome Array

    Directory of Open Access Journals (Sweden)

    Settles Matthew L

    2009-05-01

    Full Text Available Abstract Background Natural antisense transcripts (NATs are transcripts of the opposite DNA strand to the sense-strand either at the same locus (cis-encoded or a different locus (trans-encoded. They can affect gene expression at multiple stages including transcription, RNA processing and transport, and translation. NATs give rise to sense-antisense transcript pairs and the number of these identified has escalated greatly with the availability of DNA sequencing resources and public databases. Traditionally, NATs were identified by the alignment of full-length cDNAs or expressed sequence tags to genome sequences, but an alternative method for large-scale detection of sense-antisense transcript pairs involves the use of microarrays. In this study we developed a novel protocol to assay sense- and antisense-strand transcription on the 55 K Affymetrix GeneChip Wheat Genome Array, which is a 3' in vitro transcription (3'IVT expression array. We selected five different tissue types for assay to enable maximum discovery, and used the 'Chinese Spring' wheat genotype because most of the wheat GeneChip probe sequences were based on its genomic sequence. This study is the first report of using a 3'IVT expression array to discover the expression of natural sense-antisense transcript pairs, and may be considered as proof-of-concept. Results By using alternative target preparation schemes, both the sense- and antisense-strand derived transcripts were labeled and hybridized to the Wheat GeneChip. Quality assurance verified that successful hybridization did occur in the antisense-strand assay. A stringent threshold for positive hybridization was applied, which resulted in the identification of 110 sense-antisense transcript pairs, as well as 80 potentially antisense-specific transcripts. Strand-specific RT-PCR validated the microarray observations, and showed that antisense transcription is likely to be tissue specific. For the annotated sense-antisense

  2. Inhibiting effect of antisense hTRT on telomerase activity of human liver cancer cell line SMMC-7721

    Institute of Scientific and Technical Information of China (English)

    牟娇; 李晓冬; 杨庆; 贾凤岐; 卫立辛; 郭亚军; 吴孟超

    2003-01-01

    Objective: To induce changes in biological character of human liver cancer cell line SMMC-7721 by blocking the expression of telomerase genes hTRT and to explore its value in cancer gene therapy. Methods: The vehicle for eukaryotic expression of antisense hTRT was constructed and then transfected into SMMC-7721 cells. The effects of antisense hTRT gene on telomerase activity, cancer cell growth and malignant phenotypes were analyzed. Results: The obtained transfectants that could express antisense hTRT gene stably showed marked decrease in telomerase activity; the shortening of telomere was obvious; cells presented contact growth inhibition; in nude mice transplantation, the rate of tumor induction dramatically decreased. Conclusion: Antisense hTRT gene expression can significantly inhibit telomerase activity of cancer cells and decrease malignant phenotypes in vitro and in vivo. Therefore, as a telomerase inhibitor, antisense hTRT gene may be a new pathway for cancer therapy.

  3. Combination Adenovirus-Mediated HSV-tk/GCV and Antisense IGF-1 Gene Therapy for Rat Glioma

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    Objective To investigate the effects of combination adenovirus-mediated HSV-tk/GCV system and antisense IGF-1 gene therapy for rat glioma and analyze the mechanism.Methods Using the recombinant adenovirus vector,GCV killing effeciency after combined gene transfer of HSV-tk and antisense IGF-1 was observed in vitro.Rat glioma was treated with HSV-tk/GCV and antisense IGF-1 and the survival rate of rats was observed.Results C6 cells transfected with tk and antisense IGF-1 gene were more sensitive to GCV than that transfected with tk gene alone.The survival of the combination gene therapy group was prolonged significantly and large amounts of CD+4,CD+8 lymphocytes were detected in the tumor tissues.Conclusion Antisense IGF-1 gene may enhance the tumor-killing effects of HSV-tk/GCV.

  4. Intragenic pausing and anti-sense transcription within the murine c-myc locus.

    OpenAIRE

    Nepveu, A; Marcu, K B

    1986-01-01

    We present a detailed analysis of strand-specific transcription in different regions of the murine c-myc locus. In normal and transformed cell lines, RNA polymerase II directed transcription occurs in the sense and anti-sense direction. Three noncontiguous regions show a high level of transcription in the anti-sense orientation: upstream of the first exon, within the first intron and in the 3' part of the gene (intron 2 and exon 3). In a cell line carrying a c-myc amplification (54c12), anti-...

  5. Inhibition of human immunodeficiency virus type 1 multiplication by antisense and sense RNA expression.

    OpenAIRE

    Joshi, S; Van Brunschot, A; Asad, S.; van der Elst, I; Read, S. E.; Bernstein, A

    1991-01-01

    Human immunodeficiency virus type 1 (HIV-1) primarily infects CD4+ lymphocytes and macrophages and causes AIDS in humans. Retroviral vectors allowing neomycin phosphotransferase (npt) gene expression were engineered to express 5' sequences of HIV-1 RNA in the antisense or sense orientation and used to transform the human CD4+ lymphocyte-derived MT4 cell line. Cells expressing antisense or sense RNA to the HIV-1 tat mRNA leader sequence, as part of the 3' untranslated region of the npt mRNA, r...

  6. Survivin antisense compound inhibits proliferation and promotes apoptosis in liver cancer cells

    Institute of Scientific and Technical Information of China (English)

    De-Jian Dai; Cai-De Lu; Ri-Yong Lai; Jun-Ming Guo; Hua Meng; Wei-Sheng Chen; Jun Gu

    2005-01-01

    AIM: To evaluate the effects of survivin on cell proliferation and apoptosis in liver cancer.METHODS: MTT assay was used to generate and optimize phosphorothioate antisense oligonucleotides (ODNs)LipofectamineTM2000 (LiP) compound by varying ODNs (μg):LiP (μL) ratios from 1:0.5 to 1:5. Then, liver cancer cells (HepG2) were transfected with the compound. By using RT-PCR and Western blot, the expression levels of survivin mRNA and proteins were detected in HepG2 cells treated with antisense compounds (ODNs:LiP = 1:4), and compared with those treated with sense compounds (1:4) as control.MTT assay was applied to the determination of cell proliferation in HepG2 cells. Active caspase-3 was evaluated by flow cytometric analysis. The morphological changes were assessed by electron microscopy. Laser scanning confocal microscopy was performed to detect the subcellular localization of survivin proteins in treated and untreated cells.RESULTS: Antisense compounds (1:4) down-regulated survivin expression (mRNA and protein) in a dose-dependent manner with an IC50 of 250 nmol/L. Its maximum effect was achieved at a concentration of 500 nmol/L, at whichmRNA and protein levels were down-regulated by 80%.The similar results were found in MTT assay. Antisense compound (1:4)-treated cells revealed increased caspase3-like protease activity compared with untreated cells.Untreated cells as control were primarily negative for the presence of active-caspase-3. As shown by transmission electron microscopy, treated cells with antisense compounds (1:4) resulted in morphological changes such as blebbing and loss of microvilli, vacuolization in the cytoplasm,condensation of the cytoplasm and nuclei, and fragmented chromatin. Immunofluorescence analysis confirmed the presence of survivin protein pool inside the cytoplasm in untreated cells. Labeled-FITC immunofluorescence staining of survivin clearly showed that survivin was distributed mainly in a spotted form inside the cytoplasm. Whereas

  7. Use of electrophoretic mobility to determine the secondary structure of a small antisense RNA.

    OpenAIRE

    Jacques, J P; Susskind, M M

    1991-01-01

    Natural antisense RNAs have stem-loop (hairpin) secondary structures that are important for their function. The sar antisense RNA of phage P22 is unusual: the 3' half of the molecule forms an extensive stem-loop, but potential structures for the 5' half are not predicted to be thermodynamically stable. We devised a novel method to determine the secondary structure of sar RNA by examining the electrophoretic mobility on non-denaturing gels of numerous sar mutants. The results show that the wil...

  8. Antisense silencing of the creA gene in Aspergillus nidulans

    DEFF Research Database (Denmark)

    Bautista, L. F.; Aleksenko, Alexei Y.; Hentzer, Morten;

    2000-01-01

    Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect was appr......Antisense expression of a portion of the gene encoding the major carbon catabolite repressor CREA in Aspergillus nidulans resulted in a substantial increase in the levels of glucose-repressible enzymes, both endogenous and heterologous, in the presence of glucose. The derepression effect...

  9. Construction of neuron specific vector of human antisense noggin gene expression

    Institute of Scientific and Technical Information of China (English)

    Shengnian Zhou; Chengshan Li; Xiansen Wei; Liqing Liu; Zhengda Zhang

    2010-01-01

    The noggin gene is present in the central nervous system in embryonic and postnatal mammals,and plays an important role in maintaining nervous system development and physiological function.A 0.76-kb sequence of human noggin gene was cloned by polymerase chain reaction with the digestion site of Hind Ⅲ and Xba l on the 5' end.The cloned fragment was reversely inserted into pCS2+[Tα1]-GFP plasmid,an neural cell-specific antisense eukaryotic expression vector.The plasmid expresses antisense for human noggin specifically in neurons,which may facilitate understanding of the physiological function of noggin.

  10. Effect of antisense oligonucleotides targeting telomerase catalytic subunit on tumor cell proliferationin vitro

    Institute of Scientific and Technical Information of China (English)

    2002-01-01

    To screen specific antitumor drugs targeting telomerase catalytic subunit (hEST2), 12 different hEST2 antisense oligonucleotides were designed based on hEST2 mRNA second structure and transfected into tumor cell lines by the lipofectin-mediated method. Cell growth activity was evaluated by MTT assay. hEST212 was picked out and its specificity, antitumor tree and continuous effect were analyzed. The results showed that hEST212 had promising antitumor activity in vitro, hEST2 can be used as a pratical target and an antisense drug candidate for cancer.

  11. Hepatotoxicity of high affinity gapmer antisense oligonucleotides is mediated by RNase H1 dependent promiscuous reduction of very long pre-mRNA transcripts.

    Science.gov (United States)

    Burel, Sebastien A; Hart, Christopher E; Cauntay, Patrick; Hsiao, Jill; Machemer, Todd; Katz, Melanie; Watt, Andy; Bui, Huynh-Hoa; Younis, Husam; Sabripour, Mahyar; Freier, Susan M; Hung, Gene; Dan, Amy; Prakash, T P; Seth, Punit P; Swayze, Eric E; Bennett, C Frank; Crooke, Stanley T; Henry, Scott P

    2016-03-18

    High affinity antisense oligonucleotides (ASOs) containing bicylic modifications (BNA) such as locked nucleic acid (LNA) designed to induce target RNA cleavage have been shown to have enhanced potency along with a higher propensity to cause hepatotoxicity. In order to understand the mechanism of this hepatotoxicity, transcriptional profiles were collected from the livers of mice treated with a panel of highly efficacious hepatotoxic or non-hepatotoxic LNA ASOs. We observed highly selective transcript knockdown in mice treated with non-hepatotoxic LNA ASOs, while the levels of many unintended transcripts were reduced in mice treated with hepatotoxic LNA ASOs. This transcriptional signature was concurrent with on-target RNA reduction and preceded transaminitis. Remarkably, the mRNA transcripts commonly reduced by toxic LNA ASOs were generally not strongly associated with any particular biological process, cellular component or functional group. However, they tended to have much longer pre-mRNA transcripts. We also demonstrate that the off-target RNA knockdown and hepatotoxicity is attenuated by RNase H1 knockdown, and that this effect can be generalized to high affinity modifications beyond LNA. This suggests that for a certain set of ASOs containing high affinity modifications such as LNA, hepatotoxicity can occur as a result of unintended off-target RNase H1 dependent RNA degradation. PMID:26553810

  12. Biological activity and biotechnological aspects of locked nucleic acids

    DEFF Research Database (Denmark)

    Lundin, Karin E; Højland, Torben; Hansen, Bo;

    2013-01-01

    Locked nucleic acid (LNA) is one of the most promising new nucleic acid analogues that has been produced under the past two decades. In this chapter, we have tried to cover many of the different areas, where this molecule has been used to improve the function of synthetic oligonucleotides (ONs). ...

  13. MEANS AND METHODS FOR CLONING NUCLEIC ACID SEQUENCES

    NARCIS (Netherlands)

    Geertsma, Eric Robin; Poolman, Berend

    2008-01-01

    The invention provides means and methods for efficiently cloning nucleic acid sequences of interest in micro-organisms that are less amenable to conventional nucleic acid manipulations, as compared to, for instance, E.coli. The present invention enables high-throughput cloning (and, preferably, expr

  14. Nucleic acid based fluorescent sensor for mercury detection

    Science.gov (United States)

    Lu, Yi; Liu, Juewen

    2013-02-05

    A nucleic acid enzyme comprises an oligonucleotide containing thymine bases. The nucleic acid enzyme is dependent on both Hg.sup.2+and a second ion as cofactors, to produce a product from a substrate. The substrate comprises a ribonucleotide, a deoxyribonucleotide, or both.

  15. EGVII endoglucanase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel (Los Gatos, CA); Goedegebuur, Frits (Vlaardingen, NL); Ward, Michael (San Francisco, CA); Yao, Jian (Sunnyvale, CA)

    2009-05-05

    The present invention provides an endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  16. EGVI endoglucanase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel (Los Gatos, CA); Goedegebuur, Frits (Vlaardingen, NL); Ward, Michael (San Francisco, CA); Yao, Jian (Sunnyvale, CA)

    2010-10-05

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  17. EGVI endoglucanase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel (Los Gatos, CA); Goedegebuur, Frits (Vlaardingen, NL); Ward, Michael (San Francisco, CA); Yao, Jian (Sunnyvale, CA)

    2010-10-12

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl6, and the corresponding EGVI amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVI, recombinant EGVI proteins and methods for producing the same.

  18. EGVII endoglucanase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel (Los Gatos, CA); Goedegebuur, Frits (Vlaardingen, NL); Ward, Michael (San Francisco, CA); Yao, Jian (Sunnyvale, CA)

    2008-11-11

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  19. EGVII endoglucanase and nucleic acids encoding the same

    Energy Technology Data Exchange (ETDEWEB)

    Dunn-Coleman, Nigel (Los Gatos, CA); Goedegebuur, Frits (Vlaardingen, NL); Ward, Michael (San Francisco, CA); Yao, Jian (Sunnyvale, CA)

    2012-02-14

    The present invention provides a novel endoglucanase nucleic acid sequence, designated egl7, and the corresponding EGVII amino acid sequence. The invention also provides expression vectors and host cells comprising a nucleic acid sequence encoding EGVII, recombinant EGVII proteins and methods for producing the same.

  20. Transient receptor potential channel A1 involved in calcitonin gene-related peptide release in neurons

    Institute of Scientific and Technical Information of China (English)

    Nobumasa Ushio; Yi Dai; Shenglan Wang; Tetsuo Fukuoka; Koichi Noguchi

    2013-01-01

    Transient receptor potential channel A1 is one of the important transducers of noxious stimuli in the primary afferents, which may contribute to generation of neurogenic inflammation and hyperalgesia. The present study was designed to investigate if activation of transient receptor potential channel A1 may induce calcitonin gene-related peptide release from the primary afferent neurons. We found that application of al yl isothiocyanate, a transient receptor potential channel A1 activator, caused calcitonin gene-related peptide release from the cultured rat dorsal root ganglion neurons. Knock-down of transient receptor potential channel A1 with an antisense oligodeoxynucleotide prevented calcitonin gene-related peptide release by al yl isothiocyanate application in cultured dorsal root ganglion neurons. Thus, we concluded that transient receptor potential channel A1 activation caused calcitonin gene-related peptide release in sensory neurons.

  1. Delivery of antisense oligonucleotide to the cornea by iontophoresis.

    Science.gov (United States)

    Berdugo, M; Valamanesh, F; Andrieu, C; Klein, C; Benezra, D; Courtois, Y; Behar-Cohen, F

    2003-04-01

    We wished to evaluate the potential of iontophoresis to promote the delivery of antisense oligonucleotides (ODN) directed at the vascular endothelial growth factor (VEGF)-R2 receptor (KDR/Flk) to the cornea of the rat eye. Fluorescence (CY5)-labeled ODNs in phosphate-buffered saline (PBS) (20 microM) were locally administered to rat eyes, and their fate within the anterior segment was studied. Thirty-four male, 5-week-old Wistar rats were used for all experiments. The rats were divided in four groups. In group I (12 rats, 12 eyes), the ODNs (20 microM) were delivered by iontophoresis (300 microA for 5 minutes) using a specially designed corneal applicator. In group II (12 rats, 12 eyes), the ODNs (20 microM) were delivered using the same applicator, but no electrical current was applied. In group III (6 rats, 6 eyes), a corneal neovascular reaction was induced prior to the application of ODNs (20 microM), and iontophoresis electrical current was delivered as for group I rats. Group IV (4 rats, 4 eyes) received ODN (60 microM) iontophoresis application (300 microA for 5 minutes) and were used for ODN integrity studies. The animals were killed 5 minutes, 90 minutes, and 24 hours after a single ODN application and studied. Topically applied ODNs using the same iontophoresis applicator but without current do not penetrate the cornea and remain confined to the superficial epithelial layer. ODNs delivered with transcorneoscleral iontophoresis penetrate into all corneal layers and are also detected in the iris. In corneas with neovascularization, ODNs were particularly localized within the vascular endothelial cells of the stroma. ODNs extracted from eye tissues 24 hours after iontophoresis remained unaltered. The iontophoresis current did not cause any detectable ocular damage under these conditions. Iontophoresis promotes the delivery of ODNs to the anterior segment of the eye, including all corneal layers. Iontophoresis of ODNs directed at VEGF-R2 may be used for the

  2. Click chemistry generated model DNA-peptide heteroconjugates as tools for mass spectrometry.

    Science.gov (United States)

    Flett, Fiona J; Walton, Jeffrey G A; Mackay, C Logan; Interthal, Heidrun

    2015-10-01

    UV cross-linking of nucleic acids to proteins in combination with mass spectrometry is a powerful technique to identify proteins, peptides, and the amino acids involved in intermolecular interactions within nucleic acid-protein complexes. However, the mass spectrometric identification of cross-linked nucleic acid-protein heteroconjugates in complex mixtures and MS/MS characterization of the specific sites of cross-linking is extremely challenging. As a tool for the optimization of sample preparation, ionization, fragmentation, and detection by mass spectrometry, novel synthetic DNA-peptide heteroconjugates were generated to act as mimics of UV cross-linked heteroconjugates. Click chemistry was employed to cross-link peptides to DNA oligonucleotides. These heteroconjugates were fully characterized by high resolution FTICR mass spectrometry and by collision-induced dissociation (CID) following nuclease P1 digestion of the DNA moiety to a single nucleotide monophosphate. This allowed the exact site of the cross-linking within the peptide to be unambiguously assigned. These synthetic DNA-peptide heteroconjugates have the potential to be of use for a variety of applications that involve DNA-peptide heteroconjugates.

  3. Nucleic Acid-Based Nanodevices in Biological Imaging.

    Science.gov (United States)

    Chakraborty, Kasturi; Veetil, Aneesh T; Jaffrey, Samie R; Krishnan, Yamuna

    2016-06-01

    The nanoscale engineering of nucleic acids has led to exciting molecular technologies for high-end biological imaging. The predictable base pairing, high programmability, and superior new chemical and biological methods used to access nucleic acids with diverse lengths and in high purity, coupled with computational tools for their design, have allowed the creation of a stunning diversity of nucleic acid-based nanodevices. Given their biological origin, such synthetic devices have a tremendous capacity to interface with the biological world, and this capacity lies at the heart of several nucleic acid-based technologies that are finding applications in biological systems. We discuss these diverse applications and emphasize the advantage, in terms of physicochemical properties, that the nucleic acid scaffold brings to these contexts. As our ability to engineer this versatile scaffold increases, its applications in structural, cellular, and organismal biology are clearly poised to massively expand. PMID:27294440

  4. Therapeutic Antisense Oligonucleotides against Cancer: Hurdling to the Clinic

    Science.gov (United States)

    Moreno, Pedro; Pêgo, Ana

    2014-10-01

    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.

  5. The seeds of Lotus japonicus lines transformed with sense, antisense, and sense/antisense galactomannan galactosyltransferase constructs have structurally altered galactomannans in their endosperm cell walls.

    Science.gov (United States)

    Edwards, Mary E; Choo, Tze-Siang; Dickson, Cathryn A; Scott, Catherine; Gidley, Michael J; Reid, J S Grant

    2004-03-01

    Galactomannan biosynthesis in legume seed endosperms involves two Golgi membrane-bound glycosyltransferases, mannan synthase and galactomannan galactosyltransferase (GMGT). GMGT specificity is an important factor regulating the distribution and amount of (1-->6)-alpha-galactose (Gal) substitution of the (1-->4)-beta-linked mannan backbone. The model legume Lotus japonicus is shown now to have endospermic seeds with endosperm cell walls that contain a high-Gal galactomannan (mannose [Man]/Gal = 1.2-1.3). Galactomannan biosynthesis in developing L. japonicus endosperms has been mapped, and a cDNA encoding a functional GMGT has been obtained from L. japonicus endosperms during galactomannan deposition. L. japonicus has been transformed with sense, antisense, and sense/antisense ("hairpin loop") constructs of the GMGT cDNA. Some of the sense, antisense, and sense/antisense transgenic lines exhibited galactomannans with altered (higher) Man/Gal values in their (T(1) generation) seeds, at frequencies that were consistent with posttranscriptional silencing of GMGT. For T(1) generation individuals, transgene inheritance was correlated with galactomannan composition and amount in the endosperm. All the azygous individuals had unchanged galactomannans, whereas those that had inherited a GMGT transgene exhibited a range of Man/Gal values, up to about 6 in some lines. For Man/Gal values up to 4, the results were consistent with lowered Gal substitution of a constant amount of mannan backbone. Further lowering of Gal substitution was accompanied by a slight decrease in the amount of mannan backbone. Microsomal membranes prepared from the developing T(2) generation endosperms of transgenic lines showed reduced GMGT activity relative to mannan synthase. The results demonstrate structural modification of a plant cell wall polysaccharide by designed regulation of a Golgi-bound glycosyltransferase.

  6. Cell penetrating peptide delivery of splice directing oligonucleotides as a treatment for Duchenne muscular dystrophy.

    Science.gov (United States)

    Betts, Corinne A; Wood, Matthew J A

    2013-01-01

    Duchenne muscular dystrophy is a severe, X-linked muscle wasting disorder caused by the absence of an integral structural protein called dystrophin. This is caused by mutations or deletions in the dystrophin gene which disrupt the reading frame, thereby halting the production of a functional protein. A number of potential therapies have been investigated for the treatment of this disease including utrophin upregulation, 'stop-codon read through' aminoglycosides and adeno-associated virus gene replacement as well as stem cell therapy. However, the most promising treatment to date is the use of antisense oligonucleotides which cause exon skipping by binding to a specific mRNA sequence, skipping the desired exon, thereby restoring the reading frame and producing a truncated yet functional protein. The results from recent 2'OMePS and morpholino clinical trials have renewed hope for Duchenne patients; however in vivo studies in a mouse model, mdx, have revealed low systemic distribution and poor delivery of oligonucleotides to affected tissues such as the brain and heart. However a variety of cell penetrating peptides directly conjugated to antisense oligonucleotides have been shown to enhance delivery in Duchenne model systems with improved systemic distribution and greater efficacy compared to 'naked' antisense oligonucleotides. These cell penetrating peptides, combined with an optimised dose and dosing regimen, as well as thorough toxicity profile have the potential to be developed into a promising treatment which may be progressed to clinical trial. PMID:23140454

  7. Quantitative thermodynamic predication of interactions between nucleic acid and non-nucleic acid species using Microsoft excel.

    Science.gov (United States)

    Zou, Jiaqi; Li, Na

    2013-09-01

    Proper design of nucleic acid sequences is crucial for many applications. We have previously established a thermodynamics-based quantitative model to help design aptamer-based nucleic acid probes by predicting equilibrium concentrations of all interacting species. To facilitate customization of this thermodynamic model for different applications, here we present a generic and easy-to-use platform to implement the algorithm of the model with Microsoft(®) Excel formulas and VBA (Visual Basic for Applications) macros. Two Excel spreadsheets have been developed: one for the applications involving only nucleic acid species, the other for the applications involving both nucleic acid and non-nucleic acid species. The spreadsheets take the nucleic acid sequences and the initial concentrations of all species as input, guide the user to retrieve the necessary thermodynamic constants, and finally calculate equilibrium concentrations for all species in various bound and unbound conformations. The validity of both spreadsheets has been verified by comparing the modeling results with the experimental results on nucleic acid sequences reported in the literature. This Excel-based platform described here will allow biomedical researchers to rationalize the sequence design of nucleic acid probes using the thermodynamics-based modeling even without relevant theoretical and computational skills. PMID:23849929

  8. Quantitative thermodynamic predication of interactions between nucleic acid and non-nucleic acid species using Microsoft excel.

    Science.gov (United States)

    Zou, Jiaqi; Li, Na

    2013-09-01

    Proper design of nucleic acid sequences is crucial for many applications. We have previously established a thermodynamics-based quantitative model to help design aptamer-based nucleic acid probes by predicting equilibrium concentrations of all interacting species. To facilitate customization of this thermodynamic model for different applications, here we present a generic and easy-to-use platform to implement the algorithm of the model with Microsoft(®) Excel formulas and VBA (Visual Basic for Applications) macros. Two Excel spreadsheets have been developed: one for the applications involving only nucleic acid species, the other for the applications involving both nucleic acid and non-nucleic acid species. The spreadsheets take the nucleic acid sequences and the initial concentrations of all species as input, guide the user to retrieve the necessary thermodynamic constants, and finally calculate equilibrium concentrations for all species in various bound and unbound conformations. The validity of both spreadsheets has been verified by comparing the modeling results with the experimental results on nucleic acid sequences reported in the literature. This Excel-based platform described here will allow biomedical researchers to rationalize the sequence design of nucleic acid probes using the thermodynamics-based modeling even without relevant theoretical and computational skills.

  9. Nucleic Acid Aptamers for Living Cell Analysis

    Science.gov (United States)

    Xiong, Xiangling; Lv, Yifan; Chen, Tao; Zhang, Xiaobing; Wang, Kemin; Tan, Weihong

    2014-06-01

    Cells as the building blocks of life determine the basic functions and properties of a living organism. Understanding the structure and components of a cell aids in the elucidation of its biological functions. Moreover, knowledge of the similarities and differences between diseased and healthy cells is essential to understanding pathological mechanisms, identifying diagnostic markers, and designing therapeutic molecules. However, monitoring the structures and activities of a living cell remains a challenging task in bioanalytical and life science research. To meet the requirements of this task, aptamers, as “chemical antibodies,” have become increasingly powerful tools for cellular analysis. This article reviews recent advances in the development of nucleic acid aptamers in the areas of cell membrane analysis, cell detection and isolation, real-time monitoring of cell secretion, and intracellular delivery and analysis with living cell models. Limitations of aptamers and possible solutions are also discussed.

  10. Structural studies of nucleic acids and proteins involved in nucleic acid recognition

    OpenAIRE

    Russo Krauss, Irene

    2010-01-01

    This PhD thesis focuses on the structural analysis of the protein-nucleic acid recognition. In particular the research work has been focalized on two different kinds of proteins and their nucleotide ligands. The first part concerns the structural characterization of complexes between human α-thrombin, a protein of physiological and pathological relevance, and two oligonucleotide aptamers (the so called thrombin binding aptamer and a modified version of it), which adopt a G-quadruplex fold. Th...

  11. Intumescent features of nucleic acids and proteins

    Energy Technology Data Exchange (ETDEWEB)

    Alongi, Jenny, E-mail: jenny.alongi@polito.it; Cuttica, Fabio; Blasio, Alessandro Di; Carosio, Federico; Malucelli, Giulio

    2014-09-10

    Highlights: • The combustion resistance of DNA and caseins to different heat fluxes was studied. • Upon heating, DNA and caseins exhibited an intumescent behaviour. • The char derived from DNA was more stable and coherent than that from caseins. - Abstract: Are nucleic acids and proteins intumescent molecules? In order to get an answer, in the present manuscript, powders of deoxyribose nucleic acids (DNA) and caseins have been exposed to different heat fluxes under a cone calorimeter source and to the direct application of a propane flame. Under these conditions, DNA and caseins exhibited a typical intumescent behaviour, generating a coherent expanded cellular carbonaceous residue (char), extremely resistant to heat exposure. The resulting volumetric expansion as well as the resistance of the formed char turned out to be dependent on (i) the chemical structure of the chosen biomacromolecule, (ii) the evolution of ammonia and (iii) the adopted heat flux in cone calorimetry tests (namely, 25, 35, 50 and 75 kW/m{sup 2}). The presence of ribose units within the DNA backbone determined the formation of highly expanded and coherent residues as compared to those obtained from caseins. Indeed, under a heat flux of 35 kW/m{sup 2}, when a carbon source (i.e. common cane sugar) was added to caseins, the resulting char was similar to that formed by DNA. Furthermore, the char expansion was ascribed to the evolution of ammonia released by these biomacromolecules upon heating, as detected by thermogravimetry coupled to infrared spectroscopy, and confirmed by scanning electron microscopy experiments performed on the bubbles present in the residues of flammability tests.

  12. Recognition of pathogen-associated nucleic acids by endosomal nucleic acid-sensing toll-like receptors

    Institute of Scientific and Technical Information of China (English)

    Xiaobing He; Huaijie Jia; Zhizhong Jing; Dingxiang Liu

    2013-01-01

    Foreign nucleic acids,the essential signature molecules of invading pathogens that act as danger signals for host cells,are detected by endosomal nucleic acid-sensing tolllike receptors (TLRs) 3,7,8,9,and 13.These TLRs have evolved to recognize ‘non-self' nucleic acids within endosomal compartments and rapidly initiate innate immune responses to ensure host protection through induction of type Ⅰ interferons,inflammatory cytokines,chemokines,and co-stimulatory molecules and maturation of immune cells.In this review,we highlight our understanding of the recognition of pathogen-associated nucleic acids and activation of corresponding signaling pathways through endosomal nucleic acid-sensing TLRs 3,7,8,9,and 13 for an enormous diversity of pathogens,with particular emphasis on their compartmentalization,intracellular trafficking,proteolytic cleavage,autophagy,and regulatory programs.

  13. THE EFFECT OF ANTISENSE OLIGONUCLEOTIDE ON THE INTERLEUKIN-5 IN THE SUPERNATANTS OF SPLEEN CELL CULTURES OF ASTHMATIC MICE

    Institute of Scientific and Technical Information of China (English)

    王美琴; 白春学; 钮善福; 方晓惠; 陈常庆; 陈波

    2001-01-01

    To explore the effect of antisense oligonucleotide on the production of IL-5 by mouse spleen T lymphocytes.Methods Based on the IL-5 cDNA sequence of mouse, a segment of antisense oligonucleotide was designed and synthesized. 5’-labeling of antisense oligonucleotide was signed by T4 PNK in order that the efficiency of stearylamine liposome in transfecting antisense oligonucleotide can be evaluated. Asthma model was duplicated with ovalbumin(OVA) absorbed to aluminum hydroxide. T lymphocytes of mice were separated by nylon fiber method, then T lymphocytes transfected with different concentration of antisense oligonucleotide with cation stearylamine liposme were incubated respectively in order to observe the effect of antisense oligonucleotide on Il-5 production by T lymphocytes. IL-5 levels in the supernatants of T lymphocyte cultures were determined by ELISA.Results Stearylamine liposome could markedly increase the efficiency of antisense oligonucleotide transfection. The transfection efficiency of antisense oligouncleotide increased approximately 12 times at a ratio of 1: 15m/m (antisense oligonucleotide to SA liposome). In healthy and asthma Balb/c mice, IL-5 was not detectable in the supernatants of T lymphocyte cultures without stimulated with OVA; however, IL-5 was increased markedly in the supernatants of T lymphocyte cultures stimulated with OVA. After transfection with different concentrations of antisense oligonucleotide, IL-5 levels in the supernatants of T lymphocyte cultures were significantly lower than those in control cultured without antisense oligonucleotide transfection. IL-5 levels decreased from 44.60±6.23 pg/ml to 30.70±7.362 pg/ml, 17.20±6.181 pg/ml and 8.16±2.34 pg/ml respectively. And IL-5 synthesis was inhibited by 31.17%, 61.43% and 81.7% respectively.Conclusion IL-5 synthesis could be obviously inhibited by antisense oligonucleotide and showed a markedly correlation between dose and effectiveness. It suggests the production

  14. An in vivo transcriptome data set of natural antisense transcripts from Plasmodium falciparum clinical isolates

    Directory of Open Access Journals (Sweden)

    Amit Kumar Subudhi

    2014-12-01

    Full Text Available Antisense transcription is pervasive among biological systems and one of the products of antisense transcription is natural antisense transcripts (NATs. Emerging evidences suggest that they are key regulators of gene expression. With the discovery of NATs in Plasmodium falciparum, it has been suggested that these might also be playing regulatory roles in this parasite. However, all the reports describing the diversity of NATs have come from parasites in culture condition except for a recent study published by us. In order to explore the in vivo diversity of NATs in P. falciparum clinical isolates, we performed a whole genome expression profiling using a strand-specific 244 K microarray that contains probes for both sense and antisense transcripts. In this report, we describe the experimental procedure and analysis thereof of the microarray data published recently in Gene Expression Omnibus (GEO under accession number GSE44921. This published data provide a wealth of information about the prevalence of NATs in P. falciparum clinical isolates from patients with diverse malaria related disease conditions. Supplementary information about the description and interpretation of the data can be found in a recent publication by Subudhi et al. in Experimental Parasitology (2014.

  15. Refinement of antisense oligonucleotide mediated exon skipping as therapy for Duchenne muscular dystrophy

    NARCIS (Netherlands)

    Heemskerk, Johannes Antonius

    2011-01-01

    In recent years, modulation of mRNA has emerged as a promising therapeutic tool. For instance, in the field of neuromuscular disorders therapeutic strategies are being developed for several diseases, including antisense oligonucleotide (AON) mediated exon skipping for Duchenne Muscular Dystrophy (DM

  16. Improving the nutritional quality of the barley and wheat grain storage proteins by antisense technology

    DEFF Research Database (Denmark)

    Sikdar, Md. Shafiqul Islam; Lange, Mette; Aaslo, Per;

    2011-01-01

    gliadins) are also available from Germany and UK. We have grown them under different N regimes (high, medium and low N) in semi-field conditions. Previously five different antisense C-hordein lines of barley have been characterized in our laboratory. The analyses revealed that the lysine, threonine...

  17. Effect of C-myc Antisense Oligodeoxynucleotides on Hypoxia-induced Proliferation of Pulmonary Vascular Pericytes

    Institute of Scientific and Technical Information of China (English)

    2000-01-01

    To study the effect of c-myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin-11-dUTP-labeled cDNA,3H-thymidine incorporation, immunocytochemical technique and image analysis methods were used to observe the effect of c-myc antisense ODNs on expression of c-myc gene and proliferating cell nuclear antigen (PCNA), and 3H-thymidine incorporation of PC induced by hypoxia. The results showed that hypoxia could significantly enhance the expression of c-myc and PCNA (P<0.01), and elevate 3H-thymidine incorporation of PC (P<0.01), but antisense ODNs could significantly inhibit the expression of c-myc and PCNA (P<0.05), and 3H-thymidine incorporation of PC (P<0.01). It was suggested that hypoxia could promote the proliferation of PC by up-regulating the expression of c-myc gene, but c-myc antisense ODNs could inhibit hypoxia-induced proliferation of PC by downregulating the expression of c-myc gene.

  18. Molecular characterization of a stable antisense chalcone synthase phenotype in strawberry (Fragaria ananassa)

    NARCIS (Netherlands)

    Lunkenbein, S.; Coiner, H.; Vos, de C.H.; Schaart, J.G.; Boone, M.J.; Krens, F.A.; Schwab, W.

    2006-01-01

    An octaploid (Fragaria × ananassa cv. Calypso) genotype of strawberry was transformed with an antisense chalcone synthase (CHS) gene construct using a ripening related CHS cDNA from Fragaria × ananassa cv. Elsanta under the control of the constitutive CaMV 35S promoter via Agrobacterium tumefaciens.

  19. TSUNAMI: an antisense method to phenocopy splicing-associated diseases in animals

    OpenAIRE

    Sahashi, Kentaro; Hua, Yimin; Ling, Karen K Y; Hung, Gene; Rigo, Frank; Horev, Guy; Katsuno, Masahisa; Sobue, Gen; Ko, Chien-Ping; Bennett, C. Frank; Krainer, Adrian R.

    2012-01-01

    This study presents an antisense oligonucleotide methodology to phenocopy a disease—in this case, the motor neuron disease spinal muscular atrophy in mice. Sahashi et al. show that it is possible to fine-tune disease severity through dose-dependent effects on RNA splicing, making this a novel animal model for monitoring disease onset and progression as well as testing candidate therapeutics.

  20. Inhibition of lipoxygenase in lentil protoplasts by expression of antisense RNA

    NARCIS (Netherlands)

    Vliegenthart, J.F.G.; Maccarrone, M.; Hilbers, M.P.; Finazzi Agrò, A.

    1995-01-01

    A number of plasmids were constructed containing chimeric genes consisting of fragments of antisense-oriented lentil lipoxygenase cDNA. The different constructs were tested for their ability to lower lipoxygenase activity in lentil protoplasts. Plasmids containing a full length lentil lipoxygenase c

  1. Mismatched single stranded antisense oligonucleotides can induce efficient dystrophin splice switching

    Directory of Open Access Journals (Sweden)

    Kole Ryszard

    2011-10-01

    Full Text Available Abstract Background Antisense oligomer induced exon skipping aims to reduce the severity of Duchenne muscular dystrophy by redirecting splicing during pre-RNA processing such that the causative mutation is by-passed and a shorter but partially functional Becker muscular dystrophy-like dystrophin isoform is produced. Normal exons are generally targeted to restore the dystrophin reading frame however, an appreciable subset of dystrophin mutations are intra-exonic and therefore have the potential to compromise oligomer efficiency, necessitating personalised oligomer design for some patients. Although antisense oligomers are easily personalised, it remains unclear whether all patient polymorphisms within antisense oligomer target sequences will require the costly process of producing and validating patient specific compounds. Methods Here we report preclinical testing of a panel of splice switching antisense oligomers, designed to excise exon 25 from the dystrophin transcript, in normal and dystrophic patient cells. These patient cells harbour a single base insertion in exon 25 that lies within the target sequence of an oligomer shown to be effective at removing exon 25. Results It was anticipated that such a mutation would compromise oligomer binding and efficiency. However, we show that, despite the mismatch an oligomer, designed and optimised to excise exon 25 from the normal dystrophin mRNA, removes the mutated exon 25 more efficiently than the mutation-specific oligomer. Conclusion This raises the possibility that mismatched AOs could still be therapeutically applicable in some cases, negating the necessity to produce patient-specific compounds.

  2. Antisense to the glucocorticoid receptor in hippocampal dentate gyrus reduces immobility in forced swim test

    NARCIS (Netherlands)

    Korte, S.M.; de Kloet, E.R.; Buwalda, B; Bouman, S.D.; Bohus, B

    1996-01-01

    Immobility time of rats in the forced swim test was reduced after bilateral infusion of an 18-mer antisense phosphorothioate oligodeoxynucleotide targeted to the glucocorticoid receptor mRNA into the dentate gyrus of the hippocampus. Vehicle-, sense- and scrambled sequence-treated animals spent sign

  3. Antisense oligonucleotides as innovative therapeutic strategy in the treatment of high-grade gliomas.

    Science.gov (United States)

    Caruso, Gerardo; Caffo, Mariella; Raudino, Giuseppe; Alafaci, Concetta; Salpietro, Francesco M; Tomasello, Francesco

    2010-01-01

    Despite the intensive recent research in cancer therapy, the prognosis in patients affected by high-grade gliomas is still very unfavorable. The efficacy of classical anti-cancer strategies is seriously limited by lack of specific therapies against malignant cells. The extracellular matrix plays a pivotal role in processes such as differentiation, apoptosis, and migration in both the normal and the pathologic nervous system. Glial tumors seem to be able to create a favorable environment for the invasion of glioma cells in cerebral parenchyma when they combine with the extracellular matrix via cell surface receptors. Glioma cells synthesize matrix proteins, such as tenascin, laminin, fibronectin that facilitate the tumor cell's motility. New treatments have shown to hit the acting molecules in the tumor growth and to increase the efficacy and minimize the toxicity. Antisense oligonucleotides are synthetic stretches of DNA which hybridize with specific mRNA strands. The specificity of hybridization makes antisense method an interesting strategy to selectively modulate the expression of genes involved in tumorigenesis. In this review we will focus on the mechanisms of action of antisense oligonucleotides and report clinical and experimental studies on the treatment of high-grade gliomas. We will also report the patents of preclinical and/or clinical studies that adopt the antisense oligonucleotide therapy list in cerebral gliomas.

  4. Effects of antisense oligonucleotides on the expression of macrophage migration inhibitory factor on macrophages

    Institute of Scientific and Technical Information of China (English)

    WEIYINGCHEN; GUANGRANLI; XUEQINGYU; XIAOYANLI; XIAOYANG

    2005-01-01

    To investigate the effects of antisense oligonucleotides on the expression of macrophage migration inhibitory factor (MIF) on macrophages, the mouse phosphorothioate oligonucleotides were designed and synthesized with the sequences of antisense, 5'-TACGGATACAAGTAGCAC-3';Sense, 5'-ATGC-CTATGTTCATCGTG-3';Missense, 5'-CTCTCAGACTCGATCTGT-3'. These phosphorothioate oligonucleotides were then transfected into cultured macrophages (RAW264.7) by luciferase vector, and the transfected macrophages were incubated with Lipopolysaccharide (LPS) (1 ng/ml) for various periods of times and collected afterwards. The content of MIF protein in the cultural supernatants was determined by ELISA, cellular RNA extracted and the expression of MIF mRNA was examined by RT-PCR analysis.The experimental results showed that LPS could induce a time-dependent specific expression of MIF on macrophages, in which the MIF mRNA in cells and the MIF protein in cultural supernatants appeared after 3 h and reached their highest concentration at 9-12 h after LPS stimulation. The levels of mRNA and proteins in the macrophages treated with antisense olignucleotides were decreased significantly after stimulation with LPS in comparison with that of stimulation with LPS alone or with that with LPS plus sense or missense oligonucleotides.There were no differences among those without LPS stimulation. It is concluded that macrophages stimulated with LPS express MIF, and the antisense olignucleotides of MIF inhibitthe expression of MIF mRNA as well as the secretion of MIF proteins in macrophages.

  5. Regulation of Polyphosphate Kinase Production by Antisense RNA in Pseudomonas fluorescens Pf0-1

    OpenAIRE

    Silby, Mark W.; Julie S Nicoll; Levy, Stuart B.

    2012-01-01

    Pseudomonas spp. adapt rapidly to environmental fluctuations. Loss or overproduction of polyphosphate reduces the fitness of Pseudomonas fluorescens Pf0-1, indicating the importance of the fine-tuning of polyphosphate production. An antisense RNA was investigated and shown to regulate the polyphosphate kinase gene (ppk) by a posttranscriptional mechanism reducing ppk transcript abundance.

  6. Effects of recombinant epidermal growth factor receptor antisense adenovirus combined with irradiation on breast cancer cells

    International Nuclear Information System (INIS)

    Objective: To investigate the effects of a recombinant antisense adenovirus for epidermal growth factor receptor (EGFR) combined with irradiation on breast cancer cells. Methods: Human EGFR cDNA fragment was subcloned in the opposite orientation to the cytomegaloviral promoter and inserted into a E1/E3-deleted type 5 adenoviral vector to obtain AdE5 construct which expresses EGFR antisense RNA. Combined with γ-ray irradiation, its effects on clonogenicity and cell cycle phase distribution were studied in a human breast cancer line MDA-MB-23. Results: EGFR protein expression was dramatically inhibited in MDA-MB-231 cells after AdE5 infection. The post-irradiation clonogenicity was reduced by AdE5 in a viral and irradiation dose-dependent manner. Further cytometric analysis showed that AdE5 infection at a MOI of 300 pfu/cell induced a cell cycle progression from radio-resistant G0 + G1 phases to radiosensitive G2 + M phases, resulting in a synergistic effect after combination of these two treatments. Conclusions: The transduction of EGFR antisense RNA by adenoviral vector is effective for antisense strategy targeting EGFR, and increases the cell-killing effect of ionizing radiation on breast cancer cells.(authors)

  7. Sustained Release of Cx43 Antisense Oligodeoxynucleotides from Coated Collagen Scaffolds Promotes Wound Healing.

    Science.gov (United States)

    Gilmartin, Daniel J; Soon, Allyson; Thrasivoulou, Christopher; Phillips, Anthony R J; Jayasinghe, Suwan N; Becker, David L

    2016-07-01

    Antisense oligodeoxynucleotides targeting the mRNA of the gap junction protein Cx43 promote tissue repair in a variety of different wounds. Delivery of the antisense drug has most often been achieved by a thermoreversible hydrogel, Pluronic F-127, which is very effective in the short term but does not allow for sustained delivery over several days. For chronic wounds that take a long time to heal, repeated dosing with the drug may be desirable but is not always compatible with conventional treatments such as the weekly changing of compression bandages on venous leg ulcers. Here the coating of collagen scaffolds with antisense oligonucleotides is investigated and a way to provide protection of the oligodeoxynucleotide drug is found in conjunction with sustained release over a 7 d period. This approach significantly reduces the normal foreign body reaction to the scaffold, which induces an increase of Cx43 protein and an inhibition of healing. As a result of the antisense integration into the scaffold, inflammation is reduced with the rate of wound healing and contracture is significantly improved. This coated scaffold approach may be very useful for treating venous leg ulcers and also for providing a sustained release of any other types of oligonucleotide drugs that are being developed. PMID:27253638

  8. Effect of TGF-β1 antisense oligodeoxynucleotide on renal function in chronic renal failure rats

    Institute of Scientific and Technical Information of China (English)

    Law Chung HIONG; Kiew Lik VOON; Nor Azizan ABDULLAH; Munavvar A SATTAR; Nazarina AbduRAHMAN; Abdul Hye KHAN; Edward James JOHNS

    2008-01-01

    Aim:The aim of the present study was to investigate the effectiveness of trans-forming growth factor (TGF)-β1 antisense oligodeoxynucleotides (ODN) in ame-liorating deteriorated kidney function in rats with puromycin-induced chronic renal failure (CRF). Methods:Saline, puromycin, puromycin+TGF-β1 antisense ODN or puromycin+scrambled ODN were administered to unilaterally nephrecto-mized rats. Renal hemodynamic and excretory measurements were taken in the anaesthetized rats that had undergone surgical procedure. Results:It was ob-served that in the CRF rats, there was a marked reduction in the renal blood flow (RBF), glomerular filtration rate (GFR), severe proteinuria, and almost 6-fold in-creased fractional excretion of sodium (FE Na+) as compared to that in the control rats (all P<0.05). It was further observed that in the CRF rats, the treatment with TGF-β1 antisense, but not scrambled ODN, markedly attenuated the reduction of RBF, GFR, and proteinuria and markedly prevented the increase of the FE Na+ (all P<0.05). In addition, the renal hypertrophy in the CRF group (P<0.05 vs non-renal failure control) was markedly attenuated after treatment with TGF-1 antisense ODN (P<0.05). Focal segmental glomerulosclerosis was evident only in the un-treated and scrambled ODN-treated CRF groups. An interesting observation of this study was that in the CRF rats, although there was marked attenuating and preventive effects of the TGF-β1 antisense ODN on the deteriorated renal functions, the antisense treatment did not cause any marked change in the renal expression of TGF-β1 at the protein level. Conclusion:Collectively, the data obtained sug-gests that TGF-β1 antisense ODN possesses beneficial effects in puromycin-induced chronic renal failure and that the deterioration in morphology and im-paired renal function in this pathological state is in part dependent upon the action of TGF-β1 within the kidney.

  9. Oligonucleotide delivery with cell surface binding and cell penetrating Peptide amphiphile nanospheres.

    Science.gov (United States)

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

    2015-05-01

    A drug delivery system designed specifically for oligonucleotide therapeutics can ameliorate the problems associated with the in vivo delivery of these molecules. The internalization of free oligonucleotides is challenging, and cytotoxicity is the main obstacle for current transfection vehicles. To develop nontoxic delivery vehicles for efficient transfection of oligonucleotides, we designed a self-assembling peptide amphiphile (PA) nanosphere delivery system decorated with cell penetrating peptides (CPPs) containing multiple arginine residues (R4 and R8), and a cell surface binding peptide (KRSR), and report the efficiency of this system in delivering G-3129, a Bcl-2 antisense oligonucleotide (AON). PA/AON (peptide amphiphile/antisense oligonucleotide) complexes were characterized with regards to their size and secondary structure, and their cellular internalization efficiencies were evaluated. The effect of the number of arginine residues on the cellular internalization was investigated by both flow cytometry and confocal imaging, and the results revealed that uptake efficiency improved as the number of arginines in the sequence increased. The combined effect of cell penetration and surface binding property on the cellular internalization and its uptake mechanism was also evaluated by mixing R8-PA and KRSR-PA. R8 and R8/KRSR decorated PAs were found to drastically increase the internalization of AONs compared to nonbioactive PA control. Overall, the KRSR-decorated self-assembled PA nanospheres were demonstrated to be noncytotoxic delivery vectors with high transfection rates and may serve as a promising delivery system for AONs. PMID:25828697

  10. Effect of CD44 Suppression by Antisense Oligonucleotide on Attachment of Human Trabecular Meshwork Cells to HA

    Institute of Scientific and Technical Information of China (English)

    李中国; 张虹

    2004-01-01

    The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and primary open-angle glaucoma (POAG) investigated. CD44-specific antisense oligonucleotide was delivered with cationic lipid to cultured human trabecular meshwork cells. The expression of CD44 suppressed by CD44-specific antisense oligonucleotide was detected by RT-PCR and Western blotting. The effect of CD44 suppression by specific antisense oligonucleotide on attachment of trabecular meshwork cells to HA was measured by MTT assay. Results showed that expression of CD44 was suppressed by CD4, specific antisense oligonucleotide. Antisense oligonucleotide also suppressed the adhesion of human trabecular meshwork cells to HA in a concentration dependent manner. It was concluded that attachment of human trabecular meshwork cells to HA was decreased when CD44 was suppressed by specific antisense oligonucleotide. CD44might play a role in pathogenesis of POAG by affecting the adhesion of trabecular meshwork cells to HA.

  11. Antisense oligonucleotide to insulin—like growth factor Ⅱ induces apotosis in human ovarian cancer AO cell line

    Institute of Scientific and Technical Information of China (English)

    YINDELING; LUPU; 等

    1998-01-01

    The effects of antisense oligonucleotide to insulin0like growth factor -Ⅱ(IGFⅡ)to induce apotosis in human ovarian cancer cells were evaluated.Antiproliferation effects of antisense to IGFⅡin ovarian cancer AO cells were determined by 3H-thymidine incorporation.Apoptosis of the IGFⅡ antisense-treated cells was quantitated by both nuclear condensation and flow cytometry after cells were stained with propidium iodide,IGFⅡ antisense(4.5μM) treatment of 48h maximally inhibited proliferation of AO cells,More than 25% of IGFⅡantisense-treated cells(4.5μM for 24h) had undergone apoptosis,whereas less than 3% of the cells were apoptotic in either IGFⅡ sense-treated cells or untreated cells.Antisense oligonucleotide to IGFⅡ significantly inhibited cell proliferation and induced apoptosis in human ovarian cancer AO cell.These data suggest that IGFII may be a potential target in treatment of ovarian cancer and antisense oligonucleotide to IGFⅡ may serve as a therapeutic approach.

  12. Suppression of intracranial glioma tumorigenesis with vascular endothelial growth factor antisense oligonucleotide in rats

    Institute of Scientific and Technical Information of China (English)

    李维方; 张光霁; 朱诚; 金由辛; 卢亦成

    2003-01-01

    Objective: To observe the inhibition of intracranial glioma tumorigenesis by vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotide (ODN) in rats. Methods: Totally 20 μl Hank's liquid containing 1×106 C6 glioma cells was seeded into rat right caudate putamen in high-flow microinfusion with stereotactic technique. VEGF antisense ODN was simultaneously used with glioma cell. Each rat of the treated groupⅠ and the treated group Ⅱ was treated with 1 000 μmol/L VEGF antisense ODN. Each rat of the treated group Ⅲ and the treated group Ⅳ was treated with 2 000 μmol/L VEGF antisense ODN. The experimental periods of the treated group Ⅰ, the treated group Ⅲ and the control group Ⅰ were 2 weeks, those of the treated group Ⅱ, the treated group Ⅳ and the control group Ⅱ were 3 weeks. Before sacrifice, MRI was performed on each rat. Tumor magnitude and pathologic examination were detected after samples were dissected. Results: The survival state of all treated rats was better, and that of the control rats was in severe danger. The tumor volumes of the treated group Ⅰ and the treated group Ⅱ were remarkably lessened. Tumor tissue could not be found macroscopically in the brain samples of the treated group Ⅲ and the treated group Ⅳ, but tumor nest could be found with microscopy. Tumors of the treated groupⅠand the treated group Ⅱ had weak expressions of VEGF mRNA and VEGF, while normal brains and the samples of the treated group Ⅲ and the treated group Ⅳ had negative expressions, but tumors of the control groups had strong expressions. Conclusion: VEGF antisense ODN used early in situ can suppress angiogenesis and growth of rat intracranial glioma to retard tumorigenesis.

  13. Effects of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 on liver fibrosis in rats

    Institute of Scientific and Technical Information of China (English)

    JIANG Wei; WANG Ji-yao; YANG Chang-qing; LIU Wen-bin; WANG Yi-qing; HE Bo-ming

    2005-01-01

    Background No efficient therapy for liver fibrosis has been available. This study was aimed to provide evidence that the introduction of a plasmid expressing antisense tissue inhibitor of metalloproteinase-1 (TIMP-1) into a rat model of immunologically induced liver fibrosis can result in the increased activity of interstitial collagenase, thus enhancing the degradation of collagen.Methods Real-time nested polymerase chain reaction (RT-Nested-PCR) and gene recombination techniques were used to construct a rat antisense TIMP-1 recombinant plasmid that can be expressed in eukaryotic cells. Both the recombinant plasmid and an empty vector (pcDNA3) were encapsulated with glycosyl-poly-L-lysine and injected into rats suffering from pig serum-induced liver fibrosis. The expression of exogenous transfected plasmid was assessed by Northern blot, RT-PCR, and Western blot. Hepatic interstitial collagenase activity was detected using fluorescinisothiocyanate (FITC)-labeled type Ⅰ collagen. In addition to hepatic hydroxyproline content, hepatic collagen types Ⅰ and Ⅲ were detected by immunohistochemical staining, and the stages of liver fibrosis by Van Gieson staining.Results Exogenous antisense TIMP-1 was successfully expressed in vivo and could block the gene and protein expression of TIMP-1. Active and latent hepatic interstitial collagenase activities were elevated (P<0.01), hepatic hydroxyproline content and the accumulation of collagen types Ⅰ and Ⅲ were lowered, and liver fibrosis was alleviated in the antisense TIMP-1 group (P<0.01) as compared with the model group. Conclusion The results demonstrate that antisense TIMP-1 recombinant plasmids have some inhibitory effect on liver fibrosis.

  14. Antisense EGFR sequence enhances apoptosis in a human hepatoma cell line BEL—7404

    Institute of Scientific and Technical Information of China (English)

    FUTAO; HELIU; 等

    1996-01-01

    Effects of antisense epidermal growth factor receptor (EGFR) sequence on apoptotic cell death were examined in a human hepatoma cell line BEL-7404 cells.In the cells of JX-1,a sub clone of BEL-7404 stably transfected with antisense EGFR vector (Cell Research,3:75,1993),an enhanced rate(9.5%) of spontaneous apoptosis was detected by flow cytometry,whereas the rates of spontaneous apoptosis in JX-0 cells,a sub-clone of BEL-7404 transfected by control vector,and the parent BEL-7404 transfected by control vector,and the parent BEL-7404 transfected by control vector,and the parent BEL-7404 cells were almost equal and about 1.7%.Serum-starvation for 72h increased the rate of apoptosis of JX-lcells up to 33.7%,while JX-0 and BEL-7404 cells,under the same condition,produced less than 5% of apoptotic cells.Observation with electron microscope demonstrated that condensation and fragmentation of chromatin and formation of apoptotic bodies often occurred in JX-1 cells,especially during serumstarvation.These results,combined with the data of DNA fragmentation Elisa test,suggested that antisense EGFR sequence enhances apoptosis in the human hepatoma cells.Comparison of intracellular Ca2+ level and the responsiveness of JX-1 cells to the induced action of EGF and tharpsigargin (TG) treatment with that of control JX-0 cells indicated that antisense egfr might interrupt the EGF/EGFR sigaling pathway resulting in the decreass of intracellular Ca2+ pool content as well as the responsiveness of these cells to the extracellular signals.These findings suggest that antisense EGFR either directly or indirectly regulates Ca2+ storage in endoplasmic reticulum,thereby enhances apoptosis in the human hepatoma cells.

  15. Tailoring Conformation-Induced Chromism of Polythiophene Copolymers for Nucleic Acid Assay at Resource Limited Settings.

    Science.gov (United States)

    Rajwar, Deepa; Ammanath, Gopal; Cheema, Jamal Ahmed; Palaniappan, Alagappan; Yildiz, Umit Hakan; Liedberg, Bo

    2016-04-01

    Here we report on the design and synthesis of cationic water-soluble thiophene copolymers as reporters for colorimetric detection of microRNA (miRNA) in human plasma. Poly(3-alkoxythiophene) (PT) polyelectrolytes with controlled ratios of pendant groups such as triethylamine/1-methyl imidazole were synthesized for optimizing interaction with target miRNA sequence (Tseq). Incorporation of specific peptide nucleic acid (PNA) sequences with the cationic polythiophenes yielded distinguishable responses upon formation of fluorescent PT-PNA-Tseq triplex and weakly fluorescent PT-Tseq duplex, thereby enabling selective detection of target miRNA. Unlike homopolymers of PT (hPT), experimental results indicate the possibility of utilizing copolymers of PT (cPT) with appropriate ratios of pendant groups for miRNA assay in complex matrices such as plasma. As an illustration, colorimetric responses were obtained for lung cancer associated miRNA sequence (mir21) in human plasma, with a detection limit of 10 nM, illustrating the feasibility of proposed methodology for clinical applications without involving sophisticated instrumentation. The described methodology therefore possesses high potential for low-cost nucleic acid assays in resource-limited settings. PMID:26956217

  16. Nucleic acid X-ray crystallography via direct selenium derivatization.

    Science.gov (United States)

    Lin, Lina; Sheng, Jia; Huang, Zhen

    2011-09-01

    X-ray crystallography has proven to be an essential tool for structural studies of bio-macromolecules at the atomic level. There are two major bottle-neck problems in the macromolecular crystal structure determination: phasing and crystallization. Although the selenium derivatization is routinely used for solving novel protein structures through the MAD phasing technique, the phase problem is still a critical issue in nucleic acid crystallography. The background and current progress of using direct selenium-derivatization of nucleic acids (SeNA) to solve the phase problem and to facilitate nucleic acid crystallization for X-ray crystallography are summarized in this tutorial review. PMID:21666919

  17. Nucleic acid detection system and method for detecting influenza

    Energy Technology Data Exchange (ETDEWEB)

    Cai, Hong; Song, Jian

    2015-03-17

    The invention provides a rapid, sensitive and specific nucleic acid detection system which utilizes isothermal nucleic acid amplification in combination with a lateral flow chromatographic device, or DNA dipstick, for DNA-hybridization detection. The system of the invention requires no complex instrumentation or electronic hardware, and provides a low cost nucleic acid detection system suitable for highly sensitive pathogen detection. Hybridization to single-stranded DNA amplification products using the system of the invention provides a sensitive and specific means by which assays can be multiplexed for the detection of multiple target sequences.

  18. Assays for urinary biomarkers of oxidatively damaged nucleic acids

    DEFF Research Database (Denmark)

    Weimann, Allan; Broedbaek, Kasper; Henriksen, Trine;

    2012-01-01

    Abstract The analysis of oxidized nucleic acid metabolites can be performed by a variety of methodologies: liquid chromatography coupled with electrochemical or mass-spectrometry detection, gas chromatography coupled with mass spectrometry, capillary electrophoresis and ELISA (Enzyme...... and skills requirement. The available ELISA methods present considerable specificity problems and cannot be recommended at present. The oxidized nucleic acid metabolites in urine are assumed to originate from the DNA and RNA. However, direct evidence is not available. A possible contribution from...... can easily be expanded to analyze the oxidized ribonucleosides. The urinary measurement of oxidized nucleic acid metabolites provides a non-invasive measurement of oxidative stress to DNA and RNA....

  19. Measuring protein-protein and protein-nucleic Acid interactions by biolayer interferometry.

    Science.gov (United States)

    Sultana, Azmiri; Lee, Jeffrey E

    2015-01-01

    Biolayer interferometry (BLI) is a simple, optical dip-and-read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber-optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin-based BLI to analyze DNA-protein and protein-protein interactions. A quantitative set of equilibrium binding affinities (K(d)) and rates of association and dissociation (k(a)/k(d)) can be measured in minutes using nanomole quantities of sample.

  20. Antisense oligodeoxynucleotide inhibition as a potent diagnostic tool for gene function in plant biology

    Energy Technology Data Exchange (ETDEWEB)

    Jansson, Christer; Sun, Chuanxin; Ghebramedhin, Haile; Hoglund, Anna-Stina; Jansson, Christer

    2008-01-15

    Antisense oligodeoxynucleotide (ODN) inhibition emerges as an effective means for probing gene function in plant cells. Employing this method we have established the importance of the SUSIBA2 transcription factor for regulation of starch synthesis in barley endosperm, and arrived at a model for the role of the SUSIBAs in sugar signaling and source-sink commutation during cereal endosperm development. In this addendum we provide additional data demonstrating the suitability of the antisense ODN technology in studies on starch branching enzyme activities in barley leaves. We also comment on the mechanism for ODN uptake in plant cells. Antisense ODNs are short (12-25 nt-long) stretches of single-stranded ODNs that hybridize to the cognate mRNA in a sequence-specific manner, thereby inhibiting gene expression. They are naturally occurring in both prokaryotes and eukaryotes where they partake in gene regulation and defense against viral infection. The mechanisms for antisense ODN inhibition are not fully understood but it is generally considered that the ODN either sterically interferes with translation or promotes transcript degradation by RNase H activation. The earliest indication of the usefulness of antisense ODN technology for the purposes of molecular biology and medical therapy was the demonstration in 1978 that synthetic ODNs complementary to Raos sarcoma virus could inhibit virus replication in tissue cultures of chick embryo fibroblasts. Since then the antisense ODN technology has been widely used in animal sciences and as an important emerging therapeutic approach in clinical medicine. However, antisense ODN inhibition has been an under-exploited strategy for plant tissues, although the prospects for plant cells in suspension cultures to take up single-stranded ODNs was reported over a decade ago. In 2001, two reports from Malho and coworker demonstrated the use of cationic-complexed antisense ODNs to suppress expression of genes encoding pollen

  1. Aerosolized Medications for Gene and Peptide Therapy.

    Science.gov (United States)

    Laube, Beth L

    2015-06-01

    Inhalation therapy has matured to include drugs that: (1) deliver nucleic acids that either lead to the restoration of a gene construct or protein coding sequence in a population of cells or suppress or disrupt production of an abnormal gene product (gene therapy); (2) deliver peptides that target lung diseases such as asthma, sarcoidosis, pulmonary hypertension, and cystic fibrosis; and (3) deliver peptides to treat diseases outside the lung whose target is the systemic circulation (systemic drug delivery). These newer applications for aerosol therapy are the focus of this paper, and I discuss the status of each and the challenges that remain to their successful development. Drugs that are highlighted include: small interfering ribonucleic acid to treat lung cancer and Mycobacterium tuberculosis; vectors carrying the normal alpha-1 antitrypsin gene to treat alpha-1 antitrypsin deficiency; vectors carrying the normal cystic fibrosis transmembrane conductance regulator gene to treat cystic fibrosis; vasoactive intestinal peptide to treat asthma, pulmonary hypertension, and sarcoidosis; glutathione to treat cystic fibrosis; granulocyte-macrophage colony-stimulating factor to treat pulmonary alveolar proteinosis; calcitonin for postmenopausal osteoporosis; and insulin to treat diabetes. The success of these new aerosol applications will depend on many factors, such as: (1) developing gene therapy formulations that are safe for acute and chronic administrations to the lung, (2) improving the delivery of the genetic material beyond the airway mucus barrier and cell membrane and transferring the material to the cell cytoplasm or the cell nucleus, (3) developing aerosol devices that efficiently deliver genetic material and peptides to their lung targets over a short period of time, (4) developing devices that increase aerosol delivery to the lungs of infants, (5) optimizing the bioavailability of systemically delivered peptides, and (6) developing peptide formulations for

  2. Nanopore biosensors for detection of proteins and nucleic acids

    NARCIS (Netherlands)

    Maglia, Giovanni; Soskine, Mikhael

    2014-01-01

    Described herein are nanopore biosensors based on a modified cytolysin protein. The nanopore biosensors accommodate macromoiecules including proteins and nucleic acids, and may additionally comprise ligands with selective binding properties.

  3. Flexibility of nucleic acids: From DNA to RNA

    Science.gov (United States)

    Lei, Bao; Xi, Zhang; Lei, Jin; Zhi-Jie, Tan

    2016-01-01

    The structural flexibility of nucleic acids plays a key role in many fundamental life processes, such as gene replication and expression, DNA-protein recognition, and gene regulation. To obtain a thorough understanding of nucleic acid flexibility, extensive studies have been performed using various experimental methods and theoretical models. In this review, we will introduce the progress that has been made in understanding the flexibility of nucleic acids including DNAs and RNAs, and will emphasize the experimental findings and the effects of salt, temperature, and sequence. Finally, we will discuss the major unanswered questions in understanding the flexibility of nucleic acids. Project supported by the National Basic Research Program of China (Grant No. 2011CB933600), the National Natural Science Foundation of China (Grant Nos. 11175132, 11575128, and 11374234), and the Program for New Century Excellent Talents, China (Grant No. NCET 08-0408).

  4. Molecular Modeling of Nucleic Acid Structure: Energy and Sampling

    OpenAIRE

    Bergonzo, Christina; Galindo-Murillo, Rodrigo; Cheatham, Thomas E.

    2001-01-01

    An overview of computer simulation techniques as applied to nucleic acid systems is presented. This unit discusses methods used to treat the energy and to sample representative configurations. Emphasis is placed on molecular mechanics and empirical force fields.

  5. Computational Approaches to Nucleic Acid Origami.

    Science.gov (United States)

    Jabbari, Hosna; Aminpour, Maral; Montemagno, Carlo

    2015-10-12

    Recent advances in experimental DNA origami have dramatically expanded the horizon of DNA nanotechnology. Complex 3D suprastructures have been designed and developed using DNA origami with applications in biomaterial science, nanomedicine, nanorobotics, and molecular computation. Ribonucleic acid (RNA) origami has recently been realized as a new approach. Similar to DNA, RNA molecules can be designed to form complex 3D structures through complementary base pairings. RNA origami structures are, however, more compact and more thermodynamically stable due to RNA's non-canonical base pairing and tertiary interactions. With all these advantages, the development of RNA origami lags behind DNA origami by a large gap. Furthermore, although computational methods have proven to be effective in designing DNA and RNA origami structures and in their evaluation, advances in computational nucleic acid origami is even more limited. In this paper, we review major milestones in experimental and computational DNA and RNA origami and present current challenges in these fields. We believe collaboration between experimental nanotechnologists and computer scientists are critical for advancing these new research paradigms. PMID:26348196

  6. Hydrophilic magnetic latex for nucleic acid extraction, purification and concentration

    International Nuclear Information System (INIS)

    Core-shell magnetic latex particles bearing poly(N-isopropylacrylamide) in the shell were prepared by encapsulation of magnetic core using a precipitation polymerization process. The cationic character of the particles' surface is favorable for nucleic acid adsorption-desorption by controlling the pH and salinity of the medium. The concentration process of nucleic acids was presented and proven using DNA as a model

  7. Thermodynamics of RNA duplexes modified with unlocked nucleic acid nucleotides

    OpenAIRE

    Pasternak, Anna; Wengel, Jesper

    2010-01-01

    Thermodynamics provides insights into the influence of modified nucleotide residues on stability of nucleic acids and is crucial for designing duplexes with given properties. In this article, we introduce detailed thermodynamic analysis of RNA duplexes modified with unlocked nucleic acid (UNA) nucleotide residues. We investigate UNA single substitutions as well as model mismatch and dangling end effects. UNA residues placed in a central position makes RNA duplex structure less favourable by 4...

  8. Inhibition of Proliferation of Human Osteosarcoma Cells Transfected with PIN1 Antisense Gene

    Institute of Scientific and Technical Information of China (English)

    XIONG Wenhua; CHEN Anmin; GUO Fengjin

    2006-01-01

    Objective: To evaluate the inhibition of proliferation of human osteosarcoma cells transfected with Pin1 anti-sense gene. Methods: Different doses of antisense Pin1 gene (0, 20, 50, 100, 200, 250μL) were transfected into osteosarcoma MG-63 cells. The cells and culture supernatant before and after transfection were collected. The curve of cell growth was made by MTT method. The cell growth cycle and apoptosis were detected by FCM. The expression of Pin1 was detected by Western-blot and that of Pin1 mRNA by polymerase chain reaction (RT-PCR) respectively. Results: MTT and FCM assays indicated that the transfection by antisense Pin1 gene could inhibit MG-63 proliferation and induce apoptosis. Western-blot assays revealed that the antisense Pin1 gene-transfected MG-63 cells had weaker staining than those without transfected with antisense Pin1 gene, and staining intensity was negatively related with doses. The cells transfected by different doses of gene (0, 20, 50, 100, 200, 250μL) had different absorbance rate: 0.854±0.136, 0.866±0.138, 0.732±0.154, 0.611±0.121, 0.547±0.109, 0.398±0.113,0.320±0.151 respectively, with the difference being significant by F and q test (P<0.05). The expression of Pin1 mRNA had the similar results and its absorbance rate was 0.983±0.125, 0.988±0.127, 0.915±0.157,0.786±0.125, 0.608±0.124, 0.433±0.130, 0.410±0.158 respectively (P<0.05). Conclusion: The expression of Pin1 mRNA in MG-63 cells could be inhibited by antisense Pin1 gene, so to reduce the expression of Pin1 and depress the proliferation of human osteosarcoma cells MG-63.

  9. Effect of antisense human telomerase RNA on malignant behaviors of gastric carcinoma cell line SGC-7901

    Institute of Scientific and Technical Information of China (English)

    YANG Jin-liang; FANG Dian-chun; YANG Shi-ming; LUO Yuan-hui; LUO Kun-lun; LU Rong; LIU Wei-wen

    2001-01-01

    Objective: To study the effects of antisense human telomerase RNA (ahTR) transfection on the malignant behaviors of gastric carcinoma cell line SGC-7901 and its potential role in gene therapy for tumor. Methods: An antisense hTR eukaryotic expression vector containing the sequence of template region of telomere repeats was transfected into gastric carcinoma cell line SGC-7901 with liposome DOTAP. The expressions of hTR RNA and antisense hTR RNA were observed with RT-PCR, telomerase activity with PCR-ELISA. Telomere length was measured with Southern blot. Cell morphology and cellular proliferation capacity were studied with MTT assay. Cell cycle distribution and apoptotic state were observed with flow cytometry. Efficiency of clone formation in soft agar and tumorigencity in nude mice were examined and evaluated in ahTR-transfected 7901 cells, and plasmid pCL-neo transfected 7901 cells and parental 7901 cells served as control. Results: An antisense hTR eukaryotic expression vector was transfected into 7901 cells successfully. The telomerase activity in ahTR-transfected 7901 cells was decreased from 100% to about 25%, and telomere length in the cells shortened from 4.08 kb to 3.35 kb at 60 population doublings (PDs). Compared with parental 7901 and pCL-neo transfected 7901 cells, ahTR-transfected 7901 cells displayed some morphological changes, including decreased cell atypia and nucleus/cytoplasm ratio under light microscope. Furthermore, ahTR-transfected 7901 cells displayed growth inhibition, decreased invasive capacity in Borden's chamber invasive model, increased G0/G1 phase rate and apoptotic rate, and restored contact inhibition and density inhibition. Surprisingly, ahTR-transfected 7901 cells lost their capacity of clone formation in soft agar and carcinogensis in nude mice. Conclusion: Antisense hTR transfection can induce 7901 cell differentiation and reverse its malignant phenotype. This study provides an exciting approach for cancer therapy through the

  10. Sensors of Infection: Viral Nucleic Acid PRRs in Fish

    Directory of Open Access Journals (Sweden)

    Sarah Poynter

    2015-07-01

    Full Text Available Viruses produce nucleic acids during their replication, either during genomic replication or transcription. These nucleic acids are present in the cytoplasm or endosome of an infected cell, or in the extracellular space to be sensed by neighboring cells during lytic infections. Cells have mechanisms of sensing virus-generated nucleic acids; these nucleic acids act as flags to the cell, indicating an infection requiring defense mechanisms. The viral nucleic acids are called pathogen-associated molecular patterns (PAMPs and the sensors that bind them are called pattern recognition receptors (PRRs. This review article focuses on the most recent findings regarding nucleic acids PRRs in fish, including: Toll-like receptors (TLRs, RIG-I-like receptors (RLRs, cytoplasmic DNA sensors (CDSs and class A scavenger receptors (SR-As. It also discusses what is currently known of the downstream signaling molecules for each PRR family and the resulting antiviral response, either type I interferons (IFNs or pro-inflammatory cytokine production. The review highlights what is known but also defines what still requires elucidation in this economically important animal. Understanding innate immune systems to virus infections will aid in the development of better antiviral therapies and vaccines for the future.

  11. Human peptide transporters

    DEFF Research Database (Denmark)

    Nielsen, Carsten Uhd; Brodin, Birger; Jørgensen, Flemming Steen;

    2002-01-01

    Peptide transporters are epithelial solute carriers. Their functional role has been characterised in the small intestine and proximal tubules, where they are involved in absorption of dietary peptides and peptide reabsorption, respectively. Currently, two peptide transporters, PepT1 and PepT2...

  12. Effect of c- erbB2 Antisense Oligodeoxynucleotides on Radiosensitivity of Human Ovarian Cancer Cell Line

    Institute of Scientific and Technical Information of China (English)

    RENQing-Lan

    2003-01-01

    Object To explore tile effect of lipofectin - c - erbB2 antisense oligodeoxynucleotides on radiosensitivity of human ovarian cancer cell llne. Methods The expression of c - erbB2 was detected by means of RT - PCR, cellular response to irradiation was evaluated by tile colony forming assay. Results Lipofectin- c - erbB2 antisense oligodeoxynucleotides(AS- ODN) could suppress the expression of c - erbB2 , and significantly decreased the colony forming rate of human ovarian cancer cells after ionizing irradiation (P 0.05 ). Condusion c - erbB2 antisense oligodeoxynueleotides sensitized the SKOV3 to ionizing irradiation through decreasing the expression of e - erbB2 , which might be the result of the fact that c - erbB2 antisense oligodeoxynueleotides inhibit the eelluar signal transductionpathway relating to the radiation- resistant phenotype.

  13. Lipolysis and apoptosis of adipocytes induced by neuropeptide Y—Y5 receptor antisense oligodeoxynucleotides in obese rats

    Institute of Scientific and Technical Information of China (English)

    GONGHai-Xia; GUOXi-Rong; FEILi; GUOMei; LIUQian-Qi; CHENRong-Hua

    2003-01-01

    AIM:To investigate the influence of central administration of neuropeptide Y-Y5 receptor antisense oligodeoxynucleotides(ODN) on the body weight and fat pads of high-energy diet-induced obese rats, and the effects on white adipocyte lipolysis and apoptosis. METHODS: Y5 receptor antisense, sense, mismatched oligodeoxynucleotides (ODN) or vehicle were intracerebroventricularly injected, and average adipocyte area was calculated. DNA ladders were measured to evaluate adipocyte apoptosis, and RT-PCR was used to analyze the expression of bcl-2 and bax gene. RESULTS: (1) Central administration of Y5 receptor antisense ODN significantly decreased body weight, fat pads, and average adipocyte area. (2) DNA fragmentation was presented after electrophoresis at both epididymal and retroperitoneal adipose tissue. (3) The expression of bcl-2 gene was downregulated, while the expression of bax was upregulated. CONCLUSION:Lipolysis and adipocyte apoptosis may be important reasons for Y5 receptor antisense therapy.

  14. Analysis of the mechanism of protection in transgenic plants expressing the potato virus X coat protein or its antisense RNA

    OpenAIRE

    Hemenway, Cynthia; Fang, Rong-Xiang; Kaniewski, Wojciech K.; Chua, Nam-Hai; Tumer, Nilgun E.

    1988-01-01

    Transgenic tobacco plants engineered to express either the potato virus X (PVX) coat protein (CP+) or the antisense coat protein transcript (CP-antisense) were protected from infection by PVX, as indicated by reduced lesion numbers on inoculated leaves, delay or absence of systemic symptom development and reduction in virus accumulation in both inoculated and systemic leaves. The extent of protection observed in CP+ plants primarily depended upon the level of expression of the coat protein. P...

  15. Antisense oligonucleotides-induced local blockade of T-bet expression leads to airway inflammation in rats

    Institute of Scientific and Technical Information of China (English)

    Gang WANG; Chun-tao LIU; Zeng-li WANG; Li-li JIANG; Cuniang YAN; Feng-min LUO

    2006-01-01

    Aim: To explore whether local blockade of T-box expressed in T cells (T-bet) expression in the 1ungs could lead to airway inflammation. Methods: Twenty-four rats were randomly divided into 4 groups: saline group, ovalbumin (OVA)-sensitized group, nonsense group, and the antisense group. The OVA-sensitized rats were sensitized and challenged with OVA, and the rats in the nonsense and antisense groups were subjected to an aerosol delivery of the nonsense and antisense oligonucleotides (AS-ODN)of T-bet(0.1%, w/v). The levels of interferon-γ(IFN-γ), interleukin-4(IL-4), and IL-5 in the bronchoalveolar lavage fluid (BALF) were detected by ELISA, and the mRNA and the protein expression of T-bet and GATA-3 genes were examined by in situ hybridization and Western blot analysis, respectively. Results: The expression of T-bet mRNA and protein in the lungs of the rats in the antisense group were inhibited effectively. The lungs of the rats in the antisense and OVA-sensitized groups showed eosinophil and lymphocyte inflammatory infiltration, and eosinophilia located predominantly around the airways. The number of GATA-3 mRNA-positive cells and the level of GAllA-3 protein in the 1ungs of the rats in the antisense and the OVA-sensitized groups significantly increased. The level of IL-4 and IL-5 in the BALF in the antisense and OVA-sensitized groups were elevated, but the level of IFN-γ decreased markedly. Conclusion: Antisense ODN-induced local blockade of T-bet expression leads to airway inflammation with a selective alteration in patterns of cytokine expression and recruitment of eosinophil cells similar to that in the OVA-sensitized

  16. Enzyme-free translation of DNA into sequence-defined synthetic polymers structurally unrelated to nucleic acids

    Science.gov (United States)

    Niu, Jia; Hili, Ryan; Liu, David R.

    2013-04-01

    The translation of DNA sequences into corresponding biopolymers enables the production, function and evolution of the macromolecules of life. In contrast, methods to generate sequence-defined synthetic polymers with similar levels of control have remained elusive. Here, we report the development of a DNA-templated translation system that enables the enzyme-free translation of DNA templates into sequence-defined synthetic polymers that have no necessary structural relationship with nucleic acids. We demonstrate the efficiency, sequence-specificity and generality of this translation system by oligomerizing building blocks including polyethylene glycol, α-(D)-peptides, and β-peptides in a DNA-programmed manner. Sequence-defined synthetic polymers with molecular weights of 26 kDa containing 16 consecutively coupled building blocks and 90 densely functionalized β-amino acid residues were translated from DNA templates using this strategy. We integrated the DNA-templated translation system developed here into a complete cycle of translation, coding sequence replication, template regeneration and re-translation suitable for the iterated in vitro selection of functional sequence-defined synthetic polymers unrelated in structure to nucleic acids.

  17. Interactions of Night Blue with Nucleic Acids and Determination of Nucleic Acids Using Resonance Light Scattering Technique

    Institute of Scientific and Technical Information of China (English)

    吴会灵; 梁宏; 等

    2003-01-01

    The noncovalent interactions of night blue (NB) with several nucleic acids in buffer medium of Britton-Robinson at pH 4.1 have been studied by spectroscopic methods.It is shown that the binding of NB with nucleic acids involves the J-aggregation of NB molecules on the surface of nucleic acids.The aggregation was encouraged by polyanions nucleic acids,in which nucleic acids served for acting templates,In this connection,a new method of nucleic acids with sensitivity at nanogram level is proposed based on the measurement of enhanced resonance light scattering (RLS).The linear range of ctDNA,fsDNA and yRNA is 0.01-2.5,0.03-2.5 and 0.04-1.0 μg/mL,respectively,and the corresponding detection limits(3σ)are 9.4,7.3 and 5.7ng/mL at 2.5×1005mol/L of NB.Synthetic and real samples were analyzed with satisfactory results.

  18. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress.

    Science.gov (United States)

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-07-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  19. Tye7 regulates yeast Ty1 retrotransposon sense and antisense transcription in response to adenylic nucleotides stress

    Science.gov (United States)

    Servant, Géraldine; Pinson, Benoit; Tchalikian-Cosson, Aurélie; Coulpier, Fanny; Lemoine, Sophie; Pennetier, Carole; Bridier-Nahmias, Antoine; Todeschini, Anne Laure; Fayol, Hélène; Daignan-Fornier, Bertrand; Lesage, Pascale

    2012-01-01

    Transposable elements play a fundamental role in genome evolution. It is proposed that their mobility, activated under stress, induces mutations that could confer advantages to the host organism. Transcription of the Ty1 LTR-retrotransposon of Saccharomyces cerevisiae is activated in response to a severe deficiency in adenylic nucleotides. Here, we show that Ty2 and Ty3 are also stimulated under these stress conditions, revealing the simultaneous activation of three active Ty retrotransposon families. We demonstrate that Ty1 activation in response to adenylic nucleotide depletion requires the DNA-binding transcription factor Tye7. Ty1 is transcribed in both sense and antisense directions. We identify three Tye7 potential binding sites in the region of Ty1 DNA sequence where antisense transcription starts. We show that Tye7 binds to Ty1 DNA and regulates Ty1 antisense transcription. Altogether, our data suggest that, in response to adenylic nucleotide reduction, TYE7 is induced and activates Ty1 mRNA transcription, possibly by controlling Ty1 antisense transcription. We also provide the first evidence that Ty1 antisense transcription can be regulated by environmental stress conditions, pointing to a new level of control of Ty1 activity by stress, as Ty1 antisense RNAs play an important role in regulating Ty1 mobility at both the transcriptional and post-transcriptional stages. PMID:22379133

  20. Lignin reduction in transgenic poplars by expressing antisense CCoAOMT gene

    Institute of Scientific and Technical Information of China (English)

    LU Jing; ZHAO Huayan; WEI Jianhua; HE Yikun; SHI Chao; WANG Hongzhi; SONG Yanru

    2004-01-01

    The antisense Caffeoyl CoA O-methyltransferase (CCoAOMT) cDNA was transformed into Chinese white poplar (Populus tomentosa) mediated by Agrobacterium tumefaciens. Many factors affecting the transformation efficiency were studied and a stable transformation system was established. PCR-Southern blot analysis indicated that antisense CCoAOMT cDNA had been integrated into the genome of the transgenic poplars. RT-PCR and Western blot analyses demonstrated that the endogenous CCoAOMT gene was suppressed at both transcriptional and translational levels. Klason lignin content assay exhibited the lignin reduction to different degrees in transgenic poplars. The stems of partial transgenic poplars with the remarkable lignin reduction turned red, and the color distribution was stripped or spotted. Taken together, these results suggested that CCoAOMT gene would be a potential useful gene in altering lignin biosynthesis by biotechnology for improving wood properties.

  1. The Effects of Aerosolized STAT1 Antisense Oligodeoxynucleotides on Rat Pulmonary Fibrosis

    OpenAIRE

    Wang, Wenjun; Liao, Bin; Zeng, Ming; Zhu, Chen; Fan, Xianming

    2009-01-01

    Previous study showed that aerosolized signal transducer and activator of transcription-1 (STAT1) antisense oligodeoxynucleotide (ASON) inhibited the expression of STAT1 and ICAM-1 mRNA and protein in alveolar macrophages (AMs) and decreased the concentrations of TGF-β, PDGF and TNF-α in bronchioalveolar lavage fluid (BALF) in bleomycin (BLM)-induced rat pulmonary fibrosis. Administration of STAT1 ASON ameliorated alveolitis in rat pulmonary fibrosis. However, further investigations are neede...

  2. Over 20% of human transcripts might form sense–antisense pairs

    OpenAIRE

    Chen, Jianjun; Sun, Miao; Kent, W. James; Huang, Xiaoqiu; Xie, Hanqing; Wang, Wenquan; Zhou, Guolin; Shi, Run Zhang; Rowley, Janet D.

    2004-01-01

    The major challenge to identifying natural sense– antisense (SA) transcripts from public databases is how to determine the correct orientation for an expressed sequence, especially an expressed sequence tag sequence. In this study, we established a set of very stringent criteria to identify the correct orientation of each human transcript. We used these orientation-reliable transcripts to create 26 741 transcription clusters in the human genome. Our analysis shows that 22% (5880) of the human...

  3. Antisense Oligonucleotide-Mediated Exon Skipping for Duchenne Muscular Dystrophy: Progress and Challenges.

    OpenAIRE

    Arechavala-Gomeza, V.; Anthony, K.; Morgan, J; Muntoni, F.

    2012-01-01

    Duchenne muscular dystrophy (DMD) is the most common childhood neuromuscular disorder. It is caused by mutations in the DMD gene that disrupt the open reading frame (ORF) preventing the production of functional dystrophin protein. The loss of dystrophin ultimately leads to the degeneration of muscle fibres, progressive weakness and premature death. Antisense oligonucleotides (AOs) targeted to splicing elements within DMD pre-mRNA can induce the skipping of targeted exons, restoring the ORF an...

  4. Antisense Mediated Splicing Modulation For Inherited Metabolic Diseases: Challenges for Delivery

    OpenAIRE

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel; Desviat, Lourdes R.

    2014-01-01

    In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with suc...

  5. Directional gene expression and antisense transcripts in sexual and asexual stages of Plasmodium falciparum

    OpenAIRE

    López-Barragán María J; Lemieux Jacob; Quiñones Mariam; Williamson Kim C; Molina-Cruz Alvaro; Cui Kairong; Barillas-Mury Carolina; Zhao Keji; Su Xin-zhuan

    2011-01-01

    Abstract Background It has been shown that nearly a quarter of the initial predicted gene models in the Plasmodium falciparum genome contain errors. Although there have been efforts to obtain complete cDNA sequences to correct the errors, the coverage of cDNA sequences on the predicted genes is still incomplete, and many gene models for those expressed in sexual or mosquito stages have not been validated. Antisense transcripts have widely been reported in P. falciparum; however, the extent an...

  6. Antisense MMP-9 RNA inhibits malignant glioma cell growth in vitro and in vivo

    Institute of Scientific and Technical Information of China (English)

    Cuiyun Sun; Qian Wang; Hongxu Zhou; Shizhu Yu; Alain R.Simard; Chunsheng Kang; Yanyan Li

    2013-01-01

    The matrix-degrading metalloproteinases (MMPs),particularly MMP-9,play important roles in the pathogenesis and development of malignant gliomas.In the present study,the oncogenic role of MMP-9 in malignant glioma cells was investigated via antisense RNA blockade in vitro and in vivo.TJ905 malignant glioma cells were transfected with pcDNA3.0 vector expressing antisense MMP-9 RNA (pcDNA-ASMMP9),which significantly decreased MMP-9 expression,and cell proliferation was assessed.For in vivo studies,U251 cells,a human malignant glioma cell line,were implanted subcutaneously into 4-to 6-week-old BALB/c nude mice.The mice bearing well-established U251 gliomas were treated with intratumoral pcDNA-AS-MMP9-Lipofectamine complex (AS-MMP-9-treated group),subcutaneous injection of endostatin (endostatin-treated group),or both (combined therapy group).Mice treated with pcDNA (empty vector)-Lipofectamine served as the control group.Four or eight weeks later,the volume and weight of tumor,MMP-9 expression,microvessel density and proliferative activity were assayed.We demonstrate that pcDNA-AS-MMP9 significantly decreased MMP-9 expression and inhibited glioma cell proliferation.Volume and weight of tumor,MMP-9 expression,microvessel density and proliferative activity in the antisense-MMP-9-treated and therapeutic alliance groups were significantly lower than those in the control group.The results suggest that MMP-9 not only promotes malignant glioma cell invasiveness,but also affects tumor cell proliferation.Blocking the expression of MMP-9 with antisense RNA substantially suppresses the malignant phenotype of glioma cells,and thus can be used as an effective therapeutic strategy for malignant gliomas.

  7. Delivering Antisense Morpholino Oligonucleotides to Target Telomerase Splice Variants in Human Embryonic Stem Cells.

    Science.gov (United States)

    Radan, Lida; Hughes, Chris S; Teichroeb, Jonathan H; Postovit, Lynne-Marie; Betts, Dean H

    2016-01-01

    Morpholino oligonucleotides (MO) are an innovative tool that provides a means for examining and modifying gene expression outcomes by antisense interaction with targeted RNA transcripts. The site-specific nature of their binding facilitates focused modulation to alter splice variant expression patterns. Here we describe the steric-blocking of human telomerase reverse transcriptase (hTERT) Δα and Δβ splice variants using MO to examine cellular outcomes related to pluripotency and differentiation in human embryonic stem cells.

  8. Microbial Nucleic Acid Sensing in Oral and Systemic Diseases.

    Science.gov (United States)

    Crump, K E; Sahingur, S E

    2016-01-01

    One challenge in studying chronic infectious and inflammatory disorders is understanding how host pattern recognition receptors (PRRs), specifically toll-like receptors (TLRs), sense and respond to pathogen- or damage-associated molecular patterns, their communication with each other and different components of the immune system, and their role in propagating inflammatory stages of disease. The discovery of innate immune activation through nucleic acid recognition by intracellular PRRs such as endosomal TLRs (TLR3, TLR7, TLR8, and TLR9) and cytoplasmic proteins (absent in melanoma 2 and DNA-dependent activator of interferon regulatory factor) opened a new paradigm: Nucleic acid sensing is now implicated in multiple immune and inflammatory conditions (e.g., atherosclerosis, cancer), viral (e.g., human papillomavirus, herpes virus) and bacterial (e.g., Helicobacter pylori, pneumonia) diseases, and autoimmune disorders (e.g., systemic lupus erythematosus, rheumatoid arthritis). Clinical investigations reveal the overexpression of specific nucleic acid sensors in diseased tissues. In vivo animal models show enhanced disease progression associated with receptor activation. The involvement of nucleic acid sensors in various systemic conditions is further supported by studies reporting receptor knockout mice being either protected from or prone to disease. TLR9-mediated inflammation is also implicated in periodontal diseases. Considering that persistent inflammation in the oral cavity is associated with systemic diseases and that oral microbial DNA is isolated at distal sites, nucleic acid sensing may potentially be a link between oral and systemic diseases. In this review, we discuss recent advances in how intracellular PRRs respond to microbial nucleic acids and emerging views on the role of nucleic acid sensors in various systemic diseases. We also highlight new information on the role of intracellular PRRs in the pathogenesis of oral diseases including periodontitis

  9. Antisense-Mediated Depletion of Tomato Chloroplast Omega-3 Fatty Acid Desaturase Enhances Thermal Tolerance

    Institute of Scientific and Technical Information of China (English)

    Xun-Yan Liu; Jing-Hua Yang; Bin Li; Xiu-Mei Yang; Qing-Wei Meng

    2006-01-01

    A chloroplast-localized tomato (Lycopersicon esculentum Mill.) ω-3 fatty acid desaturase gene (LeFAD7) was isolated and characterized with regard to its sequence, response to various temperatures, and function in antisense transgenic tomato plants. The deduced amino acid sequence had four histidine-rich regions, of which three regions were highly conserved throughout the whole ω-3 fatty acid desaturase gene family.Southern blotting analysis showed that LeFAD7was encoded by a single copy gene and had two homologous genes in the tomato genome. Northern blot showed that LeFAD7was expressed in all organs and was especially abundant in leaf tissue. Meanwhile, expression of LeFAD7was induced by chilling stress (4 ℃),but was inhibited by high temperature (45 ℃), in leaves. Transgenic tomato plants were produced by integration of the antisense LeFAD7 DNA under the control of a CaMV35S promoter into the genome. Antisense transgenic plants with lower 18: 3 content could maintain a higher maximal photochemical efficiency (Fv/Fm)and O2 evolution rate than wild-type plants. These results suggested that silence of the LeFAD7 gene alleviated high-temperature stress. There was also a correlation between the low content of 18: 3 resulting from silence of the LeFAD7 gene and tolerance to high-temperature stress.

  10. Murine neurofibroma reversion by antisense RNA for HTLV-I tax

    Institute of Scientific and Technical Information of China (English)

    李昌本; Mark; C.Horowitz; Nancy; H.Ruddle

    1999-01-01

    Neurofibroma cell lines derived from mice transgenic for HTLV-I LTR tax express high levels of HTLV-I tax mRNA and protein and exhibit a transformed phenotype. A retrovirus vector carrying HTLV-I tax cDNA in reversed transcriptional orientation was stably transfected into the neurofibroma cells. Antisense RNA inhibited expression of the tax gene with a decrease of more than 40 % in both tax mRNA and protein. Tax antisense RNA reversed the transformed phenotype as exhibited by dramatic changes in cell morphology and growth characteristics. Expression of several cellular genes which are activated by Tax protein including GM-CSF, IL-6, LT/TNF, c-myc and LIF was down-regulated, while M-CSF and c-src proto-oncogene expressions were up-regulated. Accumulation of β-actin mRNA was not affected. The changes that occurred in the tax antisense expressing neurofibroma cells could be the consequence of the decreased concentration of Tax protein. These results also indicate that HTLV-I Tax protein is crucial for main

  11. Pseudomonas exotoxin antisense RNA selectively kills hepatitis B virus infected cells

    Institute of Scientific and Technical Information of China (English)

    Peter Hafkemeyer; Ulrich Brinkmann; Elizabeth Brinkmann; Ira Pastan; Hubert E Blum; Thomas F Baumert

    2008-01-01

    AIM: To present an approach for selectively killing retrovirus-infected cells that combines the toxicity of Pseudomonas exotoxin (PE) and the presence of reverse transcriptase (RT) in infected cells. METHODS: PE antisense toxin RNA has palindromic stem loops at its 5' and 3' ends enabling self-primed generation of cDNA in the presence of RT. The RT activity expressed in retrovirus-infected cells converts "antisense-toxin-RNA" into a lethal toxin gene exclusively in these cells. RESULTS: Using cotransfection studies with Peexpressing RNAs and β-gal expressing reporter plasmids, we show that, in HepG2 and HepG2. 2. 15 hepatomacells as well as in duck hepatitis B virus (DHBV) infected cells, HBV or DHBV-polymerase reverse transcribe a lethal cDNA copy of an antisense toxin RNA, which is composed of sequences complementary to a PE gene and eukaryotic transcription and translation signals. CONCLUSION: This finding may have important implications as a novel therapeutic strategy aimed at the elimination of HBV infection.

  12. HTLV-I antisense transcripts initiating in the 3'LTR are alternatively spliced and polyadenylated

    Directory of Open Access Journals (Sweden)

    Marriott Susan J

    2006-03-01

    Full Text Available Abstract Background Antisense transcription in retroviruses has been suggested for both HIV-1 and HTLV-I, although the existence and coding potential of these transcripts remain controversial. Thorough characterization is required to demonstrate the existence of these transcripts and gain insight into their role in retrovirus biology. Results This report provides the first complete characterization of an antisense retroviral transcript that encodes the previously described HTLV-I HBZ protein. In this study, we show that HBZ-encoding transcripts initiate in the 3' long terminal repeat (LTR at several positions and consist of two alternatively spliced variants (SP1 and SP2. Expression of the most abundant HBZ spliced variant (SP1 could be detected in different HTLV-I-infected cell lines and importantly in cellular clones isolated from HTLV-I-infected patients. Polyadenylation of HBZ RNA occurred at a distance of 1450 nucleotides downstream of the HBZ stop codon in close proximity of a typical polyA signal. We have also determined that translation mostly initiates from the first exon located in the 3' LTR and that the HBZ isoform produced from the SP1 spliced variant demonstrated inhibition of Tax and c-Jun-dependent transcriptional activation. Conclusion These results conclusively demonstrate the existence of antisense transcription in retroviruses, which likely plays a role in HTLV-I-associated pathogenesis through HBZ protein synthesis.

  13. Targeted skipping of human dystrophin exons in transgenic mouse model systemically for antisense drug development.

    Directory of Open Access Journals (Sweden)

    Bo Wu

    Full Text Available Antisense therapy has recently been demonstrated with great potential for targeted exon skipping and restoration of dystrophin production in cultured muscle cells and in muscles of Duchenne Muscular Dystrophy (DMD patients. Therapeutic values of exon skipping critically depend on efficacy of the drugs, antisense oligomers (AOs. However, no animal model has been established to test AO targeting human dystrophin exon in vivo systemically. In this study, we applied Vivo-Morpholino to the hDMD mouse, a transgenic model carrying the full-length human dystrophin gene, and achieved for the first time more than 70% efficiency of targeted human dystrophin exon skipping in vivo systemically. We also established a GFP-reporter myoblast culture to screen AOs targeting human dystrophin exon 50. Antisense efficiency for most AOs is consistent between the reporter cells, human myoblasts and in the hDMD mice in vivo. However, variation in efficiency was also clearly observed. A combination of in vitro cell culture and a Vivo-Morpholino based evaluation in vivo systemically in the hDMD mice therefore may represent a prudent approach for selecting AO drug and to meet the regulatory requirement.

  14. Cotton transgenics with Antisense AC1 gene for resistance against cotton leaf curl virus

    Directory of Open Access Journals (Sweden)

    J.Amudha, G.Balasubramani, V.G.Malathi, D.Monga, K.C.Bansal and K.R.Kranthi

    2010-07-01

    Full Text Available Cotton leaf curl virus is a devastating pest in the North India and in small pockets of Southern states. Cotton leaf curldisease (CLCuD is caused by a Geminivirus, transmitted by whitefly Bemisia tabaci vector. This is a serious problem inthe northern region and leads to yield losses up to 58% and 69% (ICAC recorder, 1999. Genetic engineering for cottontransgenics resistant to leaf curl disease (CLCuD through antisense RNA approach is potential to tackle the disease incotton. Cotton transgenics resistant to leaf curl disease (CLCuD using Antisense (rep (Replicase protein gene wasdeveloped via Agrobacterium mediated transformation. A binary vector carrying the Antisense rep gene along with thenpt II (neomycin phospho transferase gene driven by CaMV-35S promoter and NOS (nopaline synthase terminator wasused for transformation. The confirmation of the rep and npt II genes in the transgenic plants were verified by PCR andintegration of T-DNA into the plant genome was confirmed by Southern analysis. The individual transgenics were raisedin the green house and screened for the virus resistance. T2 progeny analysis showed classical Mendelian pattern ofinheritance.

  15. Intrathecal PLC(β3) oligodeoxynucleotides antisense potentiates acute morphine efficacy and attenuates chronic morphine tolerance.

    Science.gov (United States)

    Quanhong, Zhou; Ying, Xue; Moxi, Chen; Tao, Xu; Jing, Wang; Xin, Zhang; Li, Wang; Derong, Cui; Xiaoli, Zhang; Wei, Jiang

    2012-09-01

    Morphine is a mainstay for chronic pain treatment, but its efficacy has been hampered by physical tolerance. The underlying mechanism for chronic morphine induced tolerance is complicated and not well understood. PLC(β3) is regarded as an important factor in the morphine tolerance signal pathway. In this study, we determined intrathecal (i.t.) administration of an antisense oligodeoxynucleotide (ODN) of PLC(β3) could quicken the on-set antinociceptive efficacy of acute morphine treatment and prolong the maximum effect up to 4h. The antisense could also attenuate the development of morphine-induced tolerance and left shift the ED50 after 7 day of coadministration with morphine. These results probably were contributed by the PLC(β3) antisense ODN as they successfully knocked down protein expression levels and reduced activity of PLC(β3) in spinal cord in rats. The mismatch group had no such effects. The results confirmed the important involvement of PLC(β3) in both acute morphine efficacy and chronic morphine tolerance at spinal level in rats. This study may provide an idea for producing a novel adjuvant for morphine treatment.

  16. 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.

  17. A vector library for silencing central carbon metabolism genes with antisense RNAs in Escherichia coli.

    Science.gov (United States)

    Nakashima, Nobutaka; Ohno, Satoshi; Yoshikawa, Katsunori; Shimizu, Hiroshi; Tamura, Tomohiro

    2014-01-01

    We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination.

  18. Antisense repression of sucrose phosphate synthase in transgenic muskmelon alters plant growth and fruit development

    International Nuclear Information System (INIS)

    To unravel the roles of sucrose phosphate synthase (SPS) in muskmelon (Cucumis melo L.), we reduced its activity in transgenic muskmelon plants by an antisense approach. For this purpose, an 830 bp cDNA fragment of muskmelon sucrose phosphate synthase was expressed in antisense orientation behind the 35S promoter of the cauliflower mosaic virus. The phenotype of the antisense plants clearly differed from that of control plants. The transgenic plant leaves were markedly smaller, and the plant height and stem diameter were obviously shorter and thinner. Transmission electron microscope observation revealed that the membrane degradation of chloroplast happened in transgenic leaves and the numbers of grana and grana lamella in the chloroplast were significantly less, suggesting that the slow growth and weaker phenotype of transgenic plants may be due to the damage of the chloroplast ultrastructure, which in turn results in the decrease of the net photosynthetic rate. The sucrose concentration and levels of sucrose phosphate synthase decreased in transgenic mature fruit, and the fruit size was smaller than the control fruit. Together, our results suggest that sucrose phosphate synthase may play an important role in regulating the muskmelon plant growth and fruit development.

  19. Specificity of protein — Nucleic acid interaction and the biochemical evolution

    Science.gov (United States)

    Podder, S. K.; Basu, H. S.

    1984-12-01

    The water soluble carbodiimide mediated condensation of dipeptides of the general form Gly-X was carried out in the presence of mono- and poly-nucleotides. The observed yield of the tetrapeptide was found to be higher for peptide-nucleotide system of higher interaction specificity following mainly the anticodon-amino acid relationship (Basu, H.S. & Podder, S.K., 1981, Ind. J. Biochem. Biophys., 19, 251 253). The yield of the condensation product of L-peptide was more because of its higher interaction specificity. The extent of the racemization during the condensation of Gly-L-Phe, Gly-L-Tyr and Gly-D-Phe was found to be dependent on the specificity of the interaction —the higher the specificity, the lesser the racemization. The product formed was shown to have a catalytic effect on the condensation reaction. These data thus provide a mechanism showing how the specific interaction between amino acids/dipeptides and nucleic acids could lead to the formation of the ‘primitive’ translation machinery.

  20. Structural requirements for the procoagulant activity of nucleic acids.

    Directory of Open Access Journals (Sweden)

    Julia Gansler

    Full Text Available Nucleic acids, especially extracellular RNA, are exposed following tissue- or vessel damage and have previously been shown to activate the intrinsic blood coagulation pathway in vitro and in vivo. Yet, no information on structural requirements for the procoagulant activity of nucleic acids is available. A comparison of linear and hairpin-forming RNA- and DNA-oligomers revealed that all tested oligomers forming a stable hairpin structure were protected from degradation in human plasma. In contrast to linear nucleic acids, hairpin forming compounds demonstrated highest procoagulant activities based on the analysis of clotting time in human plasma and in a prekallikrein activation assay. Moreover, the procoagulant activities of the DNA-oligomers correlated well with their binding affinity to high molecular weight kininogen, whereas the binding affinity of all tested oligomers to prekallikrein was low. Furthermore, four DNA-aptamers directed against thrombin, activated protein C, vascular endothelial growth factor and nucleolin as well as the naturally occurring small nucleolar RNA U6snRNA were identified as effective cofactors for prekallikrein auto-activation. Together, we conclude that hairpin-forming nucleic acids are most effective in promoting procoagulant activities, largely mediated by their specific binding to kininogen. Thus, in vivo application of therapeutic nucleic acids like aptamers might have undesired prothrombotic or proinflammatory side effects.

  1. Nucleic acids in circulation: Are they harmful to the host?

    Indian Academy of Sciences (India)

    Indraneel Mittra; Naveen Kumar Nair; Pradyumna Kumar Mishra

    2012-06-01

    It has been estimated that 1011–1012 cells, primarily of haematogenous origin, die in the adult human body daily, and a similar number is regenerated to maintain homeostasis. Despite the presence of an efficient scavenging system for dead cells, considerable amounts of fragmented genetic material enter the circulation in healthy individuals. Elevated blood levels of extracellular nucleic acids have been reported in various disease conditions; such as ageing and age-related degenerative disorders, cancer; acute and chronic inflammatory conditions, severe trauma and autoimmune disorders. In addition to genomic DNA and nucleosomes, mitochondrial DNA is also found in circulation, as are RNA and microRNA. There is extensive literature that suggests that extraneously added nucleic acids have biological actions. They can enter into cells in vitro and in vivo and induce genetic transformation and cellular and chromosomal damage; and experimentally added nucleic acids are capable of activating both innate and adaptive immune systems and inducing a sterile inflammatory response. The possibility as to whether circulating nucleic acids may, likewise, have biological activities has not been explored. In this review we raise the question as to whether circulating nucleic acids may have damaging effects on the host and be implicated in ageing and diverse acute and chronic human pathologies.

  2. Influence of salt bridge interactions on the gas-phase stability of DNA/peptide complexes

    Science.gov (United States)

    Alves, Sandra; Woods, Amina; Delvolvé, Alice; Tabet, Jean Claude

    2008-12-01

    Negative ion mode electrospray ionization mass spectrometry was used to study DNA duplexes-peptide interaction. In the present study, we show that peptides that contain two adjacent basic residues interact noncovalently with DNA single strand or duplex. Fragmentation of the complexes between peptides containing basic residues and DNA were studied under collisions and showed unexpected dissociation pathways, as previously reported for peptide-peptide interactions. The binary complexes are dissociated either along fragmentation of the covalent bonds of the peptide backbone and/or along the single DNA strand backbone cleavage without disruption of noncovalent interaction, which demonstrates the strong binding of peptide to the DNA strand. Sequential MS/MS and MSn were further performed on ternary complexes formed between duplexes and peptides to investigate the nature of interaction. The CID spectra showed as major pathway the disruption of the noncovalent interactions and the formation of binary complexes and single-strand ions, directed by the nucleic acid gas-phase acidity. Indeed, a preferential formation of complexes with thymidine containing single strands is observed. An alternative pathway is also detected, in which complexes are dissociated along the covalent bond of the peptide and/or DNA according to the basicity. Our experimental data suggest the presence of strong salt bridge interactions between DNA and peptides containing basic residues.

  3. Identification of novel endogenous antisense transcripts by DNA microarray analysis targeting complementary strand of annotated genes

    Directory of Open Access Journals (Sweden)

    Kohama Chihiro

    2009-08-01

    Full Text Available Abstract Background Recent transcriptomic analyses in mammals have uncovered the widespread occurrence of endogenous antisense transcripts, termed natural antisense transcripts (NATs. NATs are transcribed from the opposite strand of the gene locus and are thought to control sense gene expression, but the mechanism of such regulation is as yet unknown. Although several thousand potential sense-antisense pairs have been identified in mammals, examples of functionally characterized NATs remain limited. To identify NAT candidates suitable for further functional analyses, we performed DNA microarray-based NAT screening using mouse adult normal tissues and mammary tumors to target not only the sense orientation but also the complementary strand of the annotated genes. Results First, we designed microarray probes to target the complementary strand of genes for which an antisense counterpart had been identified only in human public cDNA sources, but not in the mouse. We observed a prominent expression signal from 66.1% of 635 target genes, and 58 genes of these showed tissue-specific expression. Expression analyses of selected examples (Acaa1b and Aard confirmed their dynamic transcription in vivo. Although interspecies conservation of NAT expression was previously investigated by the presence of cDNA sources in both species, our results suggest that there are more examples of human-mouse conserved NATs that could not be identified by cDNA sources. We also designed probes to target the complementary strand of well-characterized genes, including oncogenes, and compared the expression of these genes between mammary cancerous tissues and non-pathological tissues. We found that antisense expression of 95 genes of 404 well-annotated genes was markedly altered in tumor tissue compared with that in normal tissue and that 19 of these genes also exhibited changes in sense gene expression. These results highlight the importance of NAT expression in the regulation

  4. Cross-catalytic peptide nucleic acid (PNA) replication based on templated ligation

    DEFF Research Database (Denmark)

    Singhal, Abhishek; Nielsen, Peter E

    2014-01-01

    We report the first PNA self-replicating system based on template directed cross-catalytic ligation, a process analogous to biological replication. Using two template PNAs and four pentameric precursor PNAs, all four possible carbodiimide assisted amide ligation products were detected and identif...... oligonucleotides are feasible, thereby strengthening the foundation for the discussion of a possible role for PNA (like) genetic material in the prebiotic evolution of life and lay the ground for further studies into evolution of such potentially prebiotic systems....

  5. Improved Synthesis Strategy for Peptide Nucleic Acids (PNA) appropriate for Cell-specific Fluorescence Imaging

    OpenAIRE

    Rüdiger Pipkorn, Manfred Wiessler, Waldemar Waldeck, Ute Hennrich, Kiyoshi Nokihara, Marcel Beining, Klaus Braun

    2012-01-01

    Progress in genomics and proteomics attended to the door for better understanding the recent rapid expanding complex research field of metabolomics. This trend in biomedical research increasingly focuses to the development of patient-specific therapeutic approaches with higher efficiency and sustainability. Simultaneously undesired adverse reactions are avoided. In parallel, the development of molecules for molecular imaging is required not only for the imaging of morphological structures but...

  6. Improved Synthesis Strategy for Peptide Nucleic Acids (PNA) appropriate for Cell-specific Fluorescence Imaging

    OpenAIRE

    Pipkorn, Rüdiger; Wiessler, Manfred; Waldeck, Waldemar; Hennrich, Ute; Nokihara, Kiyoshi; Beining, Marcel; Braun, Klaus

    2011-01-01

    Progress in genomics and proteomics attended to the door for better understanding the recent rapid expanding complex research field of metabolomics. This trend in biomedical research increasingly focuses to the development of patient-specific therapeutic approaches with higher efficiency and sustainability. Simultaneously undesired adverse reactions are avoided. In parallel, the development of molecules for molecular imaging is required not only for the imaging of morphological structures but...

  7. Tethered particle analysis of supercoiled circular DNA using peptide nucleic acid handles

    DEFF Research Database (Denmark)

    Norregaard, Kamilla; Andersson, Magnus; Nielsen, Peter Eigil;

    2014-01-01

    of both the dynamics of the molecule itself and of its interactions with a regulatory protein. Two bis-PNA clamps designed to bind with extremely high affinity to predetermined homopurine sequence sites in supercoiled DNA are prepared: one conjugated with digoxigenin for attachment to an anti...

  8. High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers

    OpenAIRE

    Hansen, Mads E.; Bentin, Thomas; Nielsen, Peter E.

    2009-01-01

    While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA–dsDNA triplexes—mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine substitution, in combination with (oligo)lysine or 9-aminoacridine conjugation, homopyrimidine PNA oligomers bind complementary dsDNA targets via triplex formation with (sub)nanomolar affinities (at pH 7....

  9. High-affinity triplex targeting of double stranded DNA using chemically modified peptide nucleic acid oligomers

    DEFF Research Database (Denmark)

    Hansen, Mads E; Bentin, Thomas; Nielsen, Peter E

    2009-01-01

    While sequence-selective dsDNA targeting by triplex forming oligonucleotides has been studied extensively, only very little is known about the properties of PNA-dsDNA triplexes-mainly due to the competing invasion process. Here we show that when appropriately modified using pseudoisocytosine...... substitution, in combination with (oligo)lysine or 9-aminoacridine conjugation, homopyrimidine PNA oligomers bind complementary dsDNA targets via triplex formation with (sub)nanomolar affinities (at pH 7.2, 150 mM Na(+)). Binding affinity can be modulated more than 1000-fold by changes in pH, PNA oligomer...... length, PNA net charge and/or by substitution of pseudoisocytosine for cytosine, and conjugation of the DNA intercalator 9-aminoacridine. Furthermore, 9-aminoacridine conjugation also strongly enhanced triplex invasion. Specificity for the fully matched target versus one containing single centrally...

  10. Enhanced peptide nucleic acid binding to supercoiled DNA: possible implications for DNA "breathing" dynamics

    DEFF Research Database (Denmark)

    Bentin, T; Nielsen, Peter E.

    1996-01-01

    efficient with supercoiled than with linear DNA. In the presence of 140 mM KCI, the PNA binding rate was reduced but, notably, highly dependent on template topology. Negative supercoiling (mean superhelix density, sigma approximately -0.051) increased the rate of binding by 2 orders of magnitude compared...... to that of relaxed DNA. The pseudo-first-order rate constant [k psi (sigma)] obeys an exponential function, k psi (sigma) = k psi (lin)e-sigma delta, where delta is a constant of 105 and k psi lin is the rate of PNA binding to linear DNA (sigma = 0). The activation energy [Ea(sigma)] was determined as approximately...... 93 and approximately 48 kJ mol-1 for PNA binding to linear and supercoiled DNA, respectively. The results are discussed in relation to the possible future use of PNA as an antigene agent and in the framework of DNA "breathing" dynamics....

  11. Water-absorbent polymer as a carrier for a discrete deposit of antisense oligodeoxynucleotides in the central nervous system.

    Science.gov (United States)

    Bannai, M; Ichikawa, M; Nishimura, F; Nishihara, M; Takahashi, M

    1998-09-01

    One of the problems of introducing antisense oligodeoxynucleotides (ODN) into the central nervous system (CNS) is their rapid disappearance from the target site due to their dispersion and diffusion, which results in poor uptake and/or retention in cells (M. Morris, A.B. Lucion, Antisense oligonucleotides in the study of neuroendocrine systems, J. Neuroendocrinol. 7 (1995) 493-500; S. Ogawa, H.E. Brown, H.J. Okano, D.W. Pfaff, Cellular uptake of intracerebrally administrated oligodeoxynucleotides in mouse brain, Regul. Pept. 59 (1995) 143-149) [2,5]. Recently, we adapted a new method using water-absorbent polymer (WAP; internally cross-linked starch-grafted-polyacrylates) as a carrier for antisense ODN. The polymer forms a hydro-gel after absorbing water which is chemically and biologically inert. In these studies, the polymer (powder-form) is fully swollen by physiological saline containing antisense ODN (0.2 micromol/ml) to make 80-fold volume gel. Hydro-gel (1 microliter) is injected into the target site, and water solutes are assumed to be diffused stoichiometrically into CNS from the surface of the gel. Histological studies indicate that 24 h after the injection, antisense ODN (5'biotinylated-S-oligos of 15 mer) are distributed to within 800 micrometer from the edge of the area where the gel is located and then gradually disappear from this area within days, but still remain within 300-micrometer distance 7 days later. Antisense ODN are effectively incorporated by all the cell types examined, i.e., neurons, astrocytes and microglias, and suppress the synthesis of the target protein. This method can be adapted to slow delivery of antisense ODN and other water soluble substances into the CNS. PMID:9767125

  12. Antisense PMO found in dystrophic dog model was effective in cells from exon 7-deleted DMD patient.

    Directory of Open Access Journals (Sweden)

    Takashi Saito

    Full Text Available BACKGROUND: Antisense oligonucleotide-induced exon skipping is a promising approach for treatment of Duchenne muscular dystrophy (DMD. We have systemically administered an antisense phosphorodiamidate morpholino oligomer (PMO targeting dystrophin exons 6 and 8 to a dog with canine X-linked muscular dystrophy in Japan (CXMD(J lacking exon 7 and achieved recovery of dystrophin in skeletal muscle. To date, however, antisense chemical compounds used in DMD animal models have not been directly applied to a DMD patient having the same type of exon deletion. We recently identified a DMD patient with an exon 7 deletion and tried direct translation of the antisense PMO used in dog models to the DMD patient's cells. METHODOLOGY/PRINCIPAL FINDINGS: We converted fibroblasts of CXMD(J and the DMD patient to myotubes by FACS-aided MyoD transduction. Antisense PMOs targeting identical regions of dog and human dystrophin exons 6 and 8 were designed. These antisense PMOs were mixed and administered as a cocktail to either dog or human cells in vitro. In the CXMD(J and human DMD cells, we observed a similar efficacy of skipping of exons 6 and 8 and a similar extent of dystrophin protein recovery. The accompanying skipping of exon 9, which did not alter the reading frame, was different between cells of these two species. CONCLUSION/SIGNIFICANCE: Antisense PMOs, the effectiveness of which has been demonstrated in a dog model, achieved multi-exon skipping of dystrophin gene on the FACS-aided MyoD-transduced fibroblasts from an exon 7-deleted DMD patient, suggesting the feasibility of systemic multi-exon skipping in humans.

  13. Nucleic acid therapy for lifespan prolongation: Present and future

    Indian Academy of Sciences (India)

    Wing-Fu Lai

    2011-09-01

    Lifespan prolongation is a common desire of the human race. With advances in biotechnology, the mechanism of aging has been gradually unraveled, laying the theoretical basis of nucleic acid therapy for lifespan prolongation. Regretfully, clinically applicable interventions do not exist without the efforts of converting theory into action, and it is the latter that has been far from adequately addressed at the moment. This was demonstrated by a database search on PubMed and Web of Science, from which only seven studies published between 2000 and 2010 were found to directly touch on the development of nucleic acid therapy for anti-aging and/or longevity enhancing purposes. In light of this, the objective of this article is to overview the current understanding of the intimate association between genes and longevity, and to bring the prospect of nucleic acid therapy for lifespan prolongation to light.

  14. Nucleic acid detection technologies and marker molecules in bacterial diagnostics.

    Science.gov (United States)

    Scheler, Ott; Glynn, Barry; Kurg, Ants

    2014-05-01

    There is a growing need for quick and reliable methods for microorganism detection and identification worldwide. Although traditional culture-based technologies are trustworthy and accurate at a relatively low cost, they are also time- and labor-consuming and are limited to culturable bacteria. Those weaknesses have created a necessity for alternative technologies that are capable for faster and more precise bacterial identification from medical, food or environmental samples. The most common current approach is to analyze the nucleic acid component of analyte solution and determine the bacterial composition according to the specific nucleic acid profiles that are present. This review aims to give an up-to-date overview of different nucleic acid target sequences and respective analytical technologies.

  15. Soni-removal of nucleic acids from inclusion bodies.

    Science.gov (United States)

    Neerathilingam, Muniasamy; Mysore, Sumukh; Gandham, Sai Hari A

    2014-05-23

    Inclusion bodies (IBs) are commonly formed in Escherichia coli due to over expression of recombinant proteins in non-native state. Isolation, denaturation and refolding of these IBs is generally performed to obtain functional protein. However, during this process IBs tend to form non-specific interactions with sheared nucleic acids from the genome, thus getting carried over into downstream processes. This may hinder the refolding of IBs into their native state. To circumvent this, we demonstrate a methodology termed soni-removal which involves disruption of nucleic acid-inclusion body interaction using sonication; followed by solvent based separation. As opposed to conventional techniques that use enzymes and column-based separations, soni-removal is a cost effective alternative for complete elimination of buried and/or strongly bound short nucleic acid contaminants from IBs.

  16. PeptideAtlas

    Data.gov (United States)

    U.S. Department of Health & Human Services — PeptideAtlas is a multi-organism, publicly accessible compendium of peptides identified in a large set of tandem mass spectrometry proteomics experiments. Mass...

  17. Multifunctional combinatorial-designed nanoparticles for nucleic acid therapy

    Science.gov (United States)

    Amiji, Mansoor M.

    2016-05-01

    Recent advances in biomedical sciences, especially in the field of human genetics, is increasingly considered to facilitate a new frontier in development of novel disease-modifying therapeutics. One of major challenges in the development of nucleic acid therapeutics is efficient and specific delivery of the molecules to the target tissue and cell upon systemic administration. In this report, I discuss our strategy to develop combinatorial-designed multifunctional nanoparticle assemblies based on natural biocompatible and biodegradable polymers for nucleic acid delivery in: (1) overcoming tumor drug resistance and (2) genetic modulation of macrophage functional phenotype from M1 to M2 in treatment of inflammatory diseases.

  18. PH dependent adhesive peptides

    Science.gov (United States)

    Tomich, John; Iwamoto, Takeo; Shen, Xinchun; Sun, Xiuzhi Susan

    2010-06-29

    A novel peptide adhesive motif is described that requires no receptor or cross-links to achieve maximal adhesive strength. Several peptides with different degrees of adhesive strength have been designed and synthesized using solid phase chemistries. All peptides contain a common hydrophobic core sequence flanked by positively or negatively charged amino acids sequences.

  19. Antimicrobial Peptides in 2014

    Directory of Open Access Journals (Sweden)

    Guangshun Wang

    2015-03-01

    Full Text Available This article highlights new members, novel mechanisms of action, new functions, and interesting applications of antimicrobial peptides reported in 2014. As of December 2014, over 100 new peptides were registered into the Antimicrobial Peptide Database, increasing the total number of entries to 2493. Unique antimicrobial peptides have been identified from marine bacteria, fungi, and plants. Environmental conditions clearly influence peptide activity or function. Human α-defensin HD-6 is only antimicrobial under reduced conditions. The pH-dependent oligomerization of human cathelicidin LL-37 is linked to double-stranded RNA delivery to endosomes, where the acidic pH triggers the dissociation of the peptide aggregate to release its cargo. Proline-rich peptides, previously known to bind to heat shock proteins, are shown to inhibit protein synthesis. A model antimicrobial peptide is demonstrated to have multiple hits on bacteria, including surface protein delocalization. While cell surface modification to decrease cationic peptide binding is a recognized resistance mechanism for pathogenic bacteria, it is also used as a survival strategy for commensal bacteria. The year 2014 also witnessed continued efforts in exploiting potential applications of antimicrobial peptides. We highlight 3D structure-based design of peptide antimicrobials and vaccines, surface coating, delivery systems, and microbial detection devices involving antimicrobial peptides. The 2014 results also support that combination therapy is preferred over monotherapy in treating biofilms.

  20. Peptide-Carrier Conjugation

    DEFF Research Database (Denmark)

    Hansen, Paul Robert

    2015-01-01

    To produce antibodies against synthetic peptides it is necessary to couple them to a protein carrier. This chapter provides a nonspecialist overview of peptide-carrier conjugation. Furthermore, a protocol for coupling cysteine-containing peptides to bovine serum albumin is outlined....

  1. DMPD: Plasmacytoid dendritic cells: sensing nucleic acids in viral infection andautoimmune diseases. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 18641647 Plasmacytoid dendritic cells: sensing nucleic acids in viral infection and... (.csml) Show Plasmacytoid dendritic cells: sensing nucleic acids in viral infection andautoimmune diseases.... PubmedID 18641647 Title Plasmacytoid dendritic cells: sensing nucleic acids in v

  2. Nucleic acid-binding glycoproteins which solubilize nucleic acids in dilute acid: re-examination of the Ustilago maydis glycoproteins

    Energy Technology Data Exchange (ETDEWEB)

    Unrau, P.; Champ, D.R.; Young, J.L.; Grant, C.E.

    1980-01-01

    Holloman reported the isolation from Ustilago maydis of a glycoprotein which prevented the precipitation of nucleic acids in cold 5% trichloroacetic acid. Two glycoprotein fractions from U. maydis with this nucleic acid-solubilizing activity were isolated in our laboratory using improved purification procedures. The activity was not due to nuclease contamination. The glycoproteins are distinguished by: their ability to bind to concanavalin A-Sepharose; their differential binding to double- and single-stranded deoxyribonucleic acid, and to ribonucleic acid; their molecular weights (46,000 and 69,000); and the relative amounts present in growing versus nongrowing cells. Both fractions required sulfhydryl-reducing conditions for optimal yields, specific activity, and stability. Nucleic acid binding was cooperative, the minimum number of glycoproteins required to make a native T7 DNA molecule soluble in dilute acid being estimated at 2 and 15, respectively.

  3. Antisense RNA: a genetic approach to cell resistance against Parvovirus. RNA antisentido: una aproximacion de resistencia genetica a Parvovirus

    Energy Technology Data Exchange (ETDEWEB)

    Ramirez Martinez, J.C.

    1992-01-01

    The Minute Virus of Mice (MVMp), an autonomous Parvovirus that replicates cytolytically in the A9 mouse fibroblast cell line, was interfered by constitutive expression of an antisense RNA targeted against the major non-structural NS-1 protein. Permanently transfected A9 clones expressing NS-1 antisense, showed increased proliferative capacity upon virus infection, and likewise cultures infected at low multiplicity by MVMp reached confluence overcoming virus growth. Correspondingly, an inhibition in virus multiplication was demonstrated by a significant lower virus production and plaque forming ability in clones expressing antisense RNa. At the molecular level, several fold reduction in viral DNA, RNA and proteins was quantitated by respective analysis of Southern, RNase protection and bidimensional gels. Remarkably, the accumulation of all three viral messengers(R1,R2,R3) was decreased both in the cytoplasm and in the nucleus, suggesting that antisense-mediated inhibition is primarily exerted at the level of viral transcription or nuclear post-transcriptional events. Thus, this system illustrates the possibility to create an antisense-mediated protective stage to highly cytotoxic viruses in permissive cells, by down-modulation the expression of a transactivator of virus genes. (author)180 refs., 25 figs.

  4. Antisense RNA: a genetic approach to cell resistance against Parvovirus; RNA antisentido: una aproximacion de resistencia genetica a Parvovirus

    Energy Technology Data Exchange (ETDEWEB)

    Ramirez Martinez, J.C.

    1992-12-31

    The Minute Virus of Mice (MVMp), an autonomous Parvovirus that replicates cytolytically in the A9 mouse fibroblast cell line, was interfered by constitutive expression of an antisense RNA targeted against the major non-structural NS-1 protein. Permanently transfected A9 clones expressing NS-1 antisense, showed increased proliferative capacity upon virus infection, and likewise cultures infected at low multiplicity by MVMp reached confluence overcoming virus growth. Correspondingly, an inhibition in virus multiplication was demonstrated by a significant lower virus production and plaque forming ability in clones expressing antisense RNa. At the molecular level, several fold reduction in viral DNA, RNA and proteins was quantitated by respective analysis of Southern, RNase protection and bidimensional gels. Remarkably, the accumulation of all three viral messengers(R1,R2,R3) was decreased both in the cytoplasm and in the nucleus, suggesting that antisense-mediated inhibition is primarily exerted at the level of viral transcription or nuclear post-transcriptional events. Thus, this system illustrates the possibility to create an antisense-mediated protective stage to highly cytotoxic viruses in permissive cells, by down-modulation the expression of a transactivator of virus genes. (author)180 refs., 25 figs.

  5. Identification and characterization of a cis-encoded antisense RNA associated with the replication process of Salmonella enterica serovar Typhi.

    Directory of Open Access Journals (Sweden)

    Isaac Dadzie

    Full Text Available Antisense RNAs that originate from the complementary strand of protein coding genes are involved in the regulation of gene expression in all domains of life. In bacteria, some of these antisense RNAs are transcriptional noise while others play a vital role to adapt the cell to changing environmental conditions. By deep sequencing analysis of transcriptome of Salmonella enterica serovar Typhi, a partial RNA sequence encoded in-cis to the dnaA gene was revealed. Northern blot and RACE analysis confirmed the transcription of this antisense RNA which was expressed mostly in the stationary phase of the bacterial growth and also under iron limitation and osmotic stress. Pulse expression analysis showed that overexpression of the antisense RNA resulted in a significant increase in the mRNA levels of dnaA, which will ultimately enhance their translation. Our findings have revealed that antisense RNA of dnaA is indeed transcribed not merely as a by-product of the cell's transcription machinery but plays a vital role as far as stability of dnaA mRNA is concerned.

  6. Inulin Derivatives Obtained Via Enhanced Microwave Synthesis for Nucleic Acid Based Drug Delivery.

    Science.gov (United States)

    Sardo, Carla; Craparo, Emanuela Fabiola; Fiorica, Calogero; Giammona, Gaetano; Cavallaro, Gennara

    2015-01-01

    A new class of therapeutic agents with a high potential for the treatment of different socially relevant human diseases is represented by Nucleic Acid Based Drugs (NABD), including small interfering RNAs (siRNA), decoy oligodeoxynucleotides (decoy ODN) and antisense oligonucleotides (ASOs). Although NABD can be engineered to be specifically directed against virtually any target, their susceptibility to nuclease degradation and the difficulty of delivery into target tissues severely limit their use in clinical practice and require the development of an appropriate nanostructured delivery system. For delivery of NABD, Inulin (Inu), a natural, water soluble and biocompatible polysaccharide, was derivatized by Spermine (Spm), a flexible molecule with four amine groups that, having pKa values in the range between 8-11, is mainly in the protonated form at pH 7.4. The synthesis of related copolymers (Inu-Spm) was performed by a two step reaction, using a method termed Enhanced Microwave Synthesis (EMS) which has the advantage, compared to conventional microwave reaction, that high amount of energy can be applied to the reaction system, by administering microwave irradiation and simultaneously controlling the temperature in the reaction vessel with cooled air. The synthesized inulin derivatives were characterized by FT-IR spectra and (1)H-NMR. INU-Spm derivatives with a degree of derivatization of about 14 % mol/mol were obtained. These polycations were tested to evaluate their ability to form non covalent complexes with genetic material (polyplexes). Agarose gel retardation assays showed that the obtained copolymers are able to electrostatically interact with DNA duplex to form polyplexes at different c/p weight ratios. Moreover, light scattering studies, performed to analyze size and z-potential of polyplexes, evidenced that copolymers are able to interact with genetic material leading to the formation of nanoscaled systems. In addition, biocompatibility of polyplexes

  7. Recent progress of nucleic acid probes based on small molecules%基于小分子的核酸结构探针最新研究进展

    Institute of Scientific and Technical Information of China (English)

    田沺; 肖珩; 翁小成; 王少儒; 周翔

    2012-01-01

    本文主要综述了基于小分子的核酸结构探针的最新研究进展.所探索的核酸结构主要有四链核酸(包括G-四链体以及i-motif)结构、三链核酸结构、左手螺旋DNA结构以及不规则核酸结构(包括突起结构以及环状结构)等;所探索的小分子包括过渡金属配合物、大环共轭化合物、环肽以及寡糖抗生素等.%This paper reviews the latest research progress of the nucleic acid probes based on small molecules. The related nucleic acid structures include quadruplex nucleic acids (including G-quadruplex and I-motif), triple-stranded nucleic acid structures, left-handed DNA helix structures, as well as irregular nucleic acid structures (bulge structures and loop structures). The related small molecules include transition metal complexes, the macrocyclic conjugated compounds, cyclic peptides, and oligosaccharide antibiotics.

  8. A real-time assay for monitoring nucleic acid cleavage by quadruplex formation

    OpenAIRE

    Kankia, Besik I.

    2006-01-01

    Direct and straightforward methods to follow nucleic acid cleavage are needed. A spectrophotometric quadruplex formation assay (QFA) was developed, which allows real-time monitoring of site-specific cleavage of nucleic acids. QFA was applied to study both protein and nucleic acid restriction enzymes, and was demonstrated to accurately determine Michaelis–Menten parameters for the cleavage reaction catalyzed by EcoRI. QFA can be used to study the mechanisms of protein–nucleic acid recognition....

  9. Complex organisation and structure of the ghrelin antisense strand gene GHRLOS, a candidate non-coding RNA gene

    Directory of Open Access Journals (Sweden)

    Herington Adrian C

    2008-10-01

    Full Text Available Abstract Background The peptide hormone ghrelin has many important physiological and pathophysiological roles, including the stimulation of growth hormone (GH release, appetite regulation, gut motility and proliferation of cancer cells. We previously identified a gene on the opposite strand of the ghrelin gene, ghrelinOS (GHRLOS, which spans the promoter and untranslated regions of the ghrelin gene (GHRL. Here we further characterise GHRLOS. Results We have described GHRLOS mRNA isoforms that extend over 1.4 kb of the promoter region and 106 nucleotides of exon 4 of the ghrelin gene, GHRL. These GHRLOS transcripts initiate 4.8 kb downstream of the terminal exon 4 of GHRL and are present in the 3' untranslated exon of the adjacent gene TATDN2 (TatD DNase domain containing 2. Interestingly, we have also identified a putative non-coding TATDN2-GHRLOS chimaeric transcript, indicating that GHRLOS RNA biogenesis is extremely complex. Moreover, we have discovered that the 3' region of GHRLOS is also antisense, in a tail-to-tail fashion to a novel terminal exon of the neighbouring SEC13 gene, which is important in protein transport. Sequence analyses revealed that GHRLOS is riddled with stop codons, and that there is little nucleotide and amino-acid sequence conservation of the GHRLOS gene between vertebrates. The gene spans 44 kb on 3p25.3, is extensively spliced and harbours multiple variable exons. We have also investigated the expression of GHRLOS and found evidence of differential tissue expression. It is highly expressed in tissues which are emerging as major sites of non-coding RNA expression (the thymus, brain, and testis, as well as in the ovary and uterus. In contrast, very low levels were found in the stomach where sense, GHRL derived RNAs are highly expressed. Conclusion GHRLOS RNA transcripts display several distinctive features of non-coding (ncRNA genes, including 5' capping, polyadenylation, extensive splicing and short open reading

  10. Release profile and stability evaluation of optimized chitosan/alginate nanoparticles as EGFR antisense vector

    Directory of Open Access Journals (Sweden)

    Ebrahim Azizi

    2010-06-01

    Full Text Available Ebrahim Azizi1,4, Alireza Namazi1, Ismaeil Haririan2,5, Shamileh Fouladdel1, Mohammad R Khoshayand3, Parisa Y Shotorbani6, Alireza Nomani1,7, Taraneh Gazori1,21Molecular Research Lab, Department of Pharmacology and Toxicology, 2Department of Pharmaceutics, 3Department of Food and Drug Control, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran; 4Department of Medical Biotechnology, School of Advanced Medical Technologies, Tehran University of Medical Sciences, Tehran, Iran; 5Biomaterials Research Center (BRC Tehran, Iran; 6Pharmaceutical Sciences Branch, Azad University, Tehran, Iran; 7Department of Pharmaceutics, Faculty of Pharmacy, Zanjan University of Medical Sciences, Zanjan, IranAbstract: Chitosan/alginate nanoparticles which had been optimized in our previous study using two different N/P ratios were chosen and their ability to release epidermal growth factor receptor (EGFR antisense was investigated. In addition, the stability of these nanoparticles in aqueous medium and after freeze-drying was investigated. In the case of both N/P ratios (5, 25, nanoparticles started releasing EGFR antisense as soon as they were exposed to the medium and the release lasted for approximately 50 hours. Nanoparticle size, shape, zeta potential, and release profile did not show any significant change after the freeze-drying process (followed by reswelling. The nanoparticles were reswellable again after freeze-drying in phosphate buffer with a pH of 7.4 over a period of six hours. Agarose gel electrophoresis of the nanoparticles with the two different N/P ratios showed that these nanoparticles could protect EGFR antisense molecules for six hours.Keywords: chitosan/alginate nanoparticles, release profile, freeze-drying, agarose gel electrophoresis

  11. 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.

  12. Antisense-mediated RNA targeting: versatile and expedient genetic manipulation in the brain

    Directory of Open Access Journals (Sweden)

    Ioannis eZalachoras

    2011-07-01

    Full Text Available A limiting factor in brain research still is the difficulty to evaluate in vivo the role of the increasing number of proteins implicated in neuronal processes. We discuss here the potential of antisense-mediated RNA targeting approaches. We mainly focus on those that manipulate splicing (exon skipping and exon inclusion, but will also briefly discuss mRNA targeting. Classic knockdown of expression by mRNA targeting is only one possible application of antisense oligonucleotides (AON in the control of gene function. Exon skipping and inclusion are based on the interference of AONs with splicing of pre-mRNAs. These are powerful, specific and particularly versatile techniques, which can be used to circumvent pathogenic mutations, shift splice variant expression, knock down proteins, or to create molecular models using in-frame deletions. Pre-mRNA targeting is currently used both as a research tool, e.g. in models for motor neuron disease, and in clinical trials for Duchenne muscular dystrophy and amyotrophic lateral sclerosis.AONs are particularly promising in relation to brain research, as the modified AONs are taken up extremely fast in neurons and glial cells with a long residence, and without the need for viral vectors or other delivery tools, once inside the blood brain barrier. In this review we cover 1. The principles of antisense-mediated techniques, chemistry and efficacy.2. The pros and cons of AON approaches in the brain compared to other techniques of interfering with gene function, such as transgenesis and short hairpin RNAs, in terms of specificity of the manipulation, spatial and temporal control over gene expression, toxicity and delivery issues.3. The potential applications for Neuroscience. We conclude that there is good evidence from animal studies that the CNS can be successfully targeted, but the potential of the diverse AON-based approaches appears to be under-recognized.

  13. Proliferative response of human prostate cancer cell to hormone inhibited by androgen receptor antisense RNA

    Institute of Scientific and Technical Information of China (English)

    江军; 王洛夫; 方玉华; 靳风烁; 靳文生

    2004-01-01

    Background The failure of endocrine treatment for advanced prostate cancer might be related to aberrant activation of androgen receptor (AR). Prostate cancer cell line LNCaP contains AR that can be activated by androgen, estrogen and progesterone. This study was set to investigate the effects of antisense AR RNA on growth of LNCaP cultured in medium containing varied concentrations of R1881, 17β-estradiol, and progesterone, respectively. Methods LNCaP cells transfected with antisense AR RNA retroviral vector pL-AR-SN were designated as LNCaPas-AR. LNCaP cells containing empty vector pLXSN served as LNCaPNeo. LNCaP and LNCaPNeo were taken as controls. In vitro cell growth assay, proliferative cells of LNCaP and tranfected LNCaPs were counted by typan staining when they cultured with synthetic androgen R1881, 17β-estradiol, and progesterone, respectively. Results Growth of LNCaPas-AR was inhibited significantly (P<0.05) compared with that of LNCaP and LNCaPNeo at 1 nmol/L R1881, 10 nmol/L 17β-estradiol, and 1 nmol/L progesterone, respectively. No difference was seen between LNCaP and LNCaPNeo(P>0.05). Microscopic observation showed that LNCaP and LNCaPNeo cells grew well, but only few LNCaPas-AR cells were alive. Conclusions Our observations indicate that antisense AR RNA retroviral vector pL-AR-SN could change androgen-independent characteristics of LNCaP cells, which might shed some novel insights into the treatment of androgen-independent prostate cancer.

  14. Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.

    Science.gov (United States)

    Miskew Nichols, Bailey; Aoki, Yoshitsugu; Kuraoka, Mutsuki; Lee, Joshua J A; Takeda, Shin'ichi; Yokota, Toshifumi

    2016-01-01

    Duchenne muscular dystrophy (DMD) is one of the most common lethal genetic diseases worldwide, caused by mutations in the dystrophin (DMD) gene. Exon skipping employs short DNA/RNA-like molecules called antisense oligonucleotides (AONs) that restore the reading frame and produce shorter but functional proteins. However, exon skipping therapy faces two major hurdles: limited applicability (up to only 13% of patients can be treated with a single AON drug), and uncertain function of truncated proteins. These issues were addressed with a cocktail AON approach. While approximately 70% of DMD patients can be treated by single exon skipping (all exons combined), one could potentially treat more than 90% of DMD patients if multiple exon skipping using cocktail antisense drugs can be realized. The canine X-linked muscular dystrophy (CXMD) dog model, whose phenotype is more similar to human DMD patients, was used to test the systemic efficacy and safety of multi-exon skipping of exons 6 and 8. The CXMD dog model harbors a splice site mutation in intron 6, leading to a lack of exon 7 in dystrophin mRNA. To restore the reading frame in CXMD requires multi-exon skipping of exons 6 and 8; therefore, CXMD is a good middle-sized animal model for testing the efficacy and safety of multi-exon skipping. In the current study, a cocktail of antisense morpholinos targeting exon 6 and exon 8 was designed and it restored dystrophin expression in body-wide skeletal muscles. Methods for transfection/injection of cocktail oligos and evaluation of the efficacy and safety of multi-exon skipping in the CXMD dog model are presented. PMID:27285612

  15. Mosaic protein and nucleic acid vaccines against hepatitis C virus

    Energy Technology Data Exchange (ETDEWEB)

    Yusim, Karina; Korber, Bette T. M.; Kuiken, Carla L.; Fischer, William M.

    2013-06-11

    The invention relates to immunogenic compositions useful as HCV vaccines. Provided are HCV mosaic polypeptide and nucleic acid compositions which provide higher levels of T-cell epitope coverage while minimizing the occurrence of unnatural and rare epitopes compared to natural HCV polypeptides and consensus HCV sequences.

  16. Watson-Crick hydrogen bonding of unlocked nucleic acids

    DEFF Research Database (Denmark)

    Langkjær, Niels; Wengel, Jesper; Pasternak, Anna

    2015-01-01

    We herein describe the synthesis of two new unlocked nucleic acid building blocks containing hypoxanthine and 2,6-diaminopurine as nucleobase moieties and their incorporation into oligonucleotides. The modified oligonucleotides were used to examine the thermodynamic properties of UNA against unmo...... unmodified oligonucleotides and the resulting thermodynamic data support that the hydrogen bonding face of UNA is Watson-Crick like....

  17. A DNA origami nanorobot controlled by nucleic acid hybridization

    KAUST Repository

    Torelli, Emanuela

    2014-03-20

    A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  18. Arrays of nucleic acid probes on biological chips

    Science.gov (United States)

    Chee, Mark; Cronin, Maureen T.; Fodor, Stephen P. A.; Huang, Xiaohua X.; Hubbell, Earl A.; Lipshutz, Robert J.; Lobban, Peter E.; Morris, MacDonald S.; Sheldon, Edward L.

    1998-11-17

    DNA chips containing arrays of oligonucleotide probes can be used to determine whether a target nucleic acid has a nucleotide sequence identical to or different from a specific reference sequence. The array of probes comprises probes exactly complementary to the reference sequence, as well as probes that differ by one or more bases from the exactly complementary probes.

  19. A nucleic acid dependent chemical photocatalysis in live human cells

    DEFF Research Database (Denmark)

    Arian, Dumitru; Cló, Emiliano; Gothelf, Kurt V;

    2010-01-01

    Only two nucleic acid directed chemical reactions that are compatible with live cells have been reported to date. Neither of these processes generate toxic species from nontoxic starting materials. Reactions of the latter type could be applied as gene-specific drugs, for example, in the treatment...

  20. A DNA origami nanorobot controlled by nucleic acid hybridization.

    Science.gov (United States)

    Torelli, Emanuela; Marini, Monica; Palmano, Sabrina; Piantanida, Luca; Polano, Cesare; Scarpellini, Alice; Lazzarino, Marco; Firrao, Giuseppe

    2014-07-23

    A prototype for a DNA origami nanorobot is designed, produced, and tested. The cylindrical nanorobot (diameter of 14 nm and length of 48 nm) with a switchable flap, is able to respond to an external stimulus and reacts by a physical switch from a disarmed to an armed configuration able to deliver a cellular compatible message. In the tested design the robot weapon is a nucleic acid fully contained in the inner of the tube and linked to a single point of the internal face of the flap. Upon actuation the nanorobot moves the flap extracting the nucleic acid that assembles into a hemin/G-quadruplex horseradish peroxidase mimicking DNAzyme catalyzing a colorimetric reaction or chemiluminescence generation. The actuation switch is triggered by an external nucleic acid (target) that interacts with a complementary nucleic acid that is beard externally by the nanorobot (probe). Hybridization of probe and target produces a localized structural change that results in flap opening. The flap movement is studied on a two-dimensional prototype origami using Förster resonance energy transfer and is shown to be triggered by a variety of targets, including natural RNAs. The nanorobot has potential for in vivo biosensing and intelligent delivery of biological activators.

  1. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2016-08-09

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  2. Polypeptides having cellulolytic enhancing activity and nucleic acids encoding same

    Energy Technology Data Exchange (ETDEWEB)

    Brown, Kimberly; Harris, Paul; Zaretsky, Elizabeth; Re, Edward; Vlasenko, Elena; McFarland, Keith; Lopez de Leon, Alfredo

    2014-09-30

    The present invention relates to isolated polypeptides having cellulolytic enhancing activity and isolated polynucleotides encoding the polypeptides. The invention also relates to nucleic acid constructs, vectors, and host cells comprising the polynucleotides as well as methods for producing and using the polypeptides.

  3. Effects of HSP70 Antisense Oligonucleotide on the Proliferation and Apoptosis of Human Hepatocellular Carcinoma Cells

    Institute of Scientific and Technical Information of China (English)

    杨雪; 贺海斌; 杨威; 宋涛; 郭成; 郑鑫; 刘青光

    2010-01-01

    The study investigated the effects of heat shock protein 70(HSP70) antisense oligonucleotide(ASODN) on the proliferation and apoptosis of a human hepatocellular carcinoma cell line(SMMC-7721 cells) in vitro.HSP70 oligonucleotide was transfected into SMMC-7721 cells by the mediation of SofastTM transfection reagent.Inhibition rate of SMMC-7721 cells was determined by using MTT method.Apoptosis rate and cell cycle distribution were measured by flow cytometry.Immunocytochemistry staining was used to observe th...

  4. Inhibition of Gene Expression in Escherichia coli by Antisense Phosphorodiamidate Morpholino Oligomers

    OpenAIRE

    Geller, B L; Deere, J. D.; Stein, D A; Kroeker, A. D.; Moulton, H. M.; Iversen, P. L.

    2003-01-01

    Antisense phosphorodiamidate morpholino oligomers (PMOs) were tested for the ability to inhibit gene expression in Escherichia coli. PMOs targeted to either a myc-luciferase reporter gene product or 16S rRNA did not inhibit luciferase expression or growth. However, in a strain with defective lipopolysaccharide (lpxA mutant), which has a leaky outer membrane, PMOs targeted to the myc-luciferase or acyl carrier protein (acpP) mRNA significantly inhibited their targets in a dose-dependent respon...

  5. Identification of an antisense transcript to ZIM2 in the primate lineage

    OpenAIRE

    Huang, Jennifer M.; Yu, Sungryul; Kim, Joomyeong

    2009-01-01

    In this study, we identified an antisense transcript to ZIM2 (zinc finger imprinted gene 2) in the human, called ZIM2as. Sequence analysis of the 110 kb region spanned by this transcript revealed a cluster of tandemly repeated sequence in the human, orangutan, and chimpanzee as well as a loss of approximately 70 kb from the corresponding region in the rhesus. The homologous region in most mammals contains a cluster of olfactory receptor (OLFR) genes, but this gene cluster has been lost from t...

  6. Antisense EGFR sequence reverses the growth properties of human liver carcinoma cell line BEL-7404 in vitro

    Institute of Scientific and Technical Information of China (English)

    XUYONGHUA; WANLIJIANG; SUFENGPENG; YINGHUACHEN

    1993-01-01

    A recombinant plasmid containing a full length human epidermal growth factor receptor (EGFR) cDNA sequence in antisense orientation was transferred into cells of a human liver carcinoma cell line BEL-7404. Compared with the control cell clone JX-0 transferred with the vector plasmid and the parent BEL-7404 cells, the antisense EGFR transferred cell clone JX-1 showed a decreased EGFR gene expression and reduced significantly the growth potential either in anchorage-dependent or anchorage-independent growth. Furthermore. JX-1 cells appeared to be distinctly dependent on serum concentration for monolayer growth. The results suggested that antisense EGFR could partly block the EGFR gene ex-pression and reverse the malignant growth properties of human liver carcinoma cells in vitro.

  7. Inhibition of HSP70 Gene Expression by Modified Antisense and Its Effects on Embryonic Sensitivity to Heat Shock

    Institute of Scientific and Technical Information of China (English)

    TIAN Wen-ru; DU Li-yin; HE Jian-bin; LI Shou-jun

    2004-01-01

    Experiments were performed to evaluate the efficiency of inhibition of HSP70 gene expression by antisense oligonucleotides complementary to the mRNA of HSP70 and to test the effects of inhibition of HSP70 gene expression on subsequent embryonic sensitivity to heat shock. The results showed that transfection of pre-implantation embryos at 4-cell stage with 5 μM antisense oligo had no effect on in vitro blastocyst development. However, transfection with 10 to 40 μM antisense oligo had reduced in vitro blastocyst development to 15, 10% and 0; For the embryos which exposed to 40 μM As arrested at the 16-cell stage, there was no blastocyst formation within the heat shock groups. In contrast, transfection had no effect on embryonic sensitivity to heat shock, above 25% of embryos developed to blastocyst stage in control groups.

  8. Characteristics of transgenic tomatoes antisensed for the ethylene receptor genes LeERT1 and LeERT2

    Institute of Scientific and Technical Information of China (English)

    WANG Zhong-feng; YING Tie-jin; ZHANG Ying; BAO Bi-li; HUANG Xiao-dan

    2006-01-01

    Two stable transformed lines containing antisense LeERT1 or LeERT2 sequences and their hybridized line were investigated to determine the effect of LeERT1 and LeERT2 specificity in the ethylene receptor family in tomato (Lycopersicon esculentum Mill.) on ethylene signaling. The transgenic line alel containing antisense LeERT1 displayed shorter length of seedling grown in the dark and adult plant in the light, severe epinastic petiole, and accelerated abscission of petiole explant and senescence of flower explant, compared with its wild type B1. The transgenic line ale2 containing antisense LeERT2 also exhibited shorter hypocotyls and slightly accelerated abscission. The phenotypes of cross line dale of LeERT1 and LeERT2 were close to alel in many aspects. These results suggested that LeERT1 probably plays a relatively important role in ethylene signaling of tomato growth and development.

  9. DMPD: Nucleic acid-sensing TLRs as modifiers of autoimmunity. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available 17082566 Nucleic acid-sensing TLRs as modifiers of autoimmunity. Deane JA, Bolland ...S. J Immunol. 2006 Nov 15;177(10):6573-8. (.png) (.svg) (.html) (.csml) Show Nucleic acid-sensing TLRs as mo...difiers of autoimmunity. PubmedID 17082566 Title Nucleic acid-sensing TLRs as modifiers of autoimmunity. Aut

  10. Anti-peptide aptamers recognize amino acid sequence and bind a protein epitope.

    OpenAIRE

    Xu, W; Ellington, A. D.

    1996-01-01

    In vitro selection of nucleic acid binding species (aptamers) is superficially similar to the immune response. Both processes produce biopolymers that can recognize targets with high affinity and specificity. While antibodies are known to recognize the sequence and conformation of protein surface features (epitopes), very little is known about the precise interactions between aptamers and their epitopes. Therefore, aptamers that could recognize a particular epitope, a peptide fragment of huma...

  11. Cell-penetrating peptides; chemical modification, mechanism of uptake and formulation development

    OpenAIRE

    Ezzat, Kariem

    2012-01-01

    Gene therapy holds the promise of revolutionizing the way we treat diseases. By using recombinant DNA and oligonucleotides (ONs), gene functions can be restored, altered or silenced according to the therapeutic need. However, gene therapy approaches require the delivery of large and charged nucleic acid-based molecules to their intracellular targets across the plasma membrane, which is inherently impermeable to such molecules. In this thesis, two chemically modified cell-penetrating peptides ...

  12. Effect of NHE1 antisense gene transfection on the biological behavior of SGC-7901 human gastric carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Hai-Feng Liu; Xiao-Chun Teng; Jing-Chen Zheng; Gang Chen; Xing-Wei Wang

    2008-01-01

    AIM: To study the effect of type 1 Na+/H+ exchanger (NHE1) antisense human gene transfection on the biological behavior of gastric carcinoma cell line SGC-7901.METHODS: Antisense NHE1 eukaryotic expression on vector pcDNA3.1 was constructed by recombinant DNA technique and transfected into gastric carcinoma cell line SGC-7901 with DOTAP liposome transfection method.Morphological changes of cells were observed with optic and electron microscopes. Changes in cell proliferative capacity, apoptosis, intracellular pH (pH1), cell cycle,clone formation in two-layer soft agar, and tumorigenicity in nude mice were examined.RESULTS: Antisense eukaryotic expressing vectors were successfully constructed and transfected into 5GC-7901.The transfectant obtained named 7901-antisense (7901-,45) stablely produced antisense NHE1. There was a significant difference between the pH1 of 7901-AS cells (6.77 ± 0.05) and that of 7901-zeo cells and SGC-7901 cells (7.24 ± 0.03 and 7.26 ± 0.03, P < 0.01). Compared with SGC-7901 and 7901-zeo cells, 7901-AS cells mostly showed cell proliferation inhibition, G1/Go phase arrest, increased cell apoptotic rate, recovery of contact inhibition, and density contact. The tumorigenicity in nude mice and cloning efficiency in the two-layer soft agar were dearly inhibited.CONCLUSION: NHE1 antisense gene significantly restrains the malignant behavior of human gastric carcinoma cells, suppresses cell growth and induces cell apoptosis, and partially reverses the malignant phenotypes of SGC-7901. These results suggest a potential role for human tumor gene therapy.

  13. Strategic down-regulation of DNA polymerase beta by antisense RNA sensitizes mammalian cells to specific DNA damaging agents.

    OpenAIRE

    Horton, J K; Srivastava, D K; Zmudzka, B Z; Wilson, S H

    1995-01-01

    Previously, mouse NIH 3T3 cells were stably transfected with human DNA polymerase beta (beta-pol) cDNA in the antisense orientation and under the control of a metallothionein promoter [Zmudzka, B.Z. and Wilson, S.H. (1990) Som. Cell Mol. Gen., 16, 311-320]. To assess the feasibility of enhancing the efficacy of chemotherapy by an antisense approach and to confirm a role for beta-pol in cellular DNA repair, we looked for increased sensitivity to DNA damaging agents under conditions where beta-...

  14. Inhibitory effect of human telomerase antisense oligodeoxyribonucleotides on the growth of gastric cancer cell lines in variant tumor pathological subtype

    Institute of Scientific and Technical Information of China (English)

    Jing Ye; Yun-Lin Wu; Shu Zhang; Zi Chen; Li-Xia Guo; Ruo-Yu Zhou; Hong Xie

    2005-01-01

    AIM: To investigate the inhibitory effect of specialized human telomerase antisense oligodeoxyribonucleotides on the growth of well (MKN-28), moderately (SGC-7901)and poorly (MKN-45) differentiated gastric cancer cell lines under specific conditions and its inhibition mechanism,and to observe the correlation between the growth inhibition ratio and the tumor pathologic subtype of gastric cancer cells.METHODS: Telomerase activity in three gastric cancer cell lines of variant tumor pathologic subtype was determined by modified TRAP assay before and after the specialized human telomerase antisense oligodeoxyribonucleotides were dealt with under specific conditions. Effect of antisense oligomer under specific conditions of the growth and viability of gastric cancer cell lines was explored by using trypan blue dye exclusion assay, and cell apoptosis was detected by cell morphology observation, flow cytometry and TUNEL assay.RESULTS: Telomerase activity was detected in well,moderately and poorly differentiated gastric cancer cell lines (the quantification expression of telomerase activity was 43.7TPG, 56.5TPG, 76.7TPG, respectively).Telomerase activity was controlled to 30.2TPG, 36.3TPG and 35.2TPG for MKN-28, SGC-7901 and MKN-45 cell lines respectively after treatment with human telomerase antisense oligomers at the concentration of 5 μmol/L, and was entirely inhibited at 10 μmol/L, against the template region of telomerase RNA component, whereas no inhibition effect was detected in missense oligomers (P<0.05). After treatment with antisense oligomers at different concentrations under specific conditions for 96 h, significant growth inhibition effects were found in MKN-45 and SGC-7901gastric cancer cell lines (the inhibition ratio was 40.89%and 71.28%), but not in MKN-28 cell lines (15.86%). The ratio of inactive SGC-7901 cells increased according to the prolongation of treatment from 48 to 96 h. Missense oligomers could not lead to the same effect (P<0

  15. Heat shock protein 70 antisense oligonucleotide inhibits cell growth and induces apoptosis in human gastric cancer cell line SGC-7901

    Institute of Scientific and Technical Information of China (English)

    Zhi-Gang Zhao; Wen-Lu Shen

    2005-01-01

    AIM: Heat shock protein (HSP)70 is over-expressed in human gastric cancer and plays an important role in the progression of this cancer. We investigated the effects of antisense HSP70 oligomer on human gastric cancer cell line SGC-7901, and its potential role in gene therapy for this cancer.METHODS: Human gastric cancer cell line SGC-7901 was treated in vitro with various concentrations of antisense HSP70 oligonucleotides at different intervals. Growth inhibition was determined as percentage by trypan blue dye exclusion test. Extracted DNA was electrophoresed on agarose gel, and distribution of cell cycle and kinetics of apoptosis induction were analyzed by propidium iodide DNA incorporation using flow cytometry, which was also used to detect the effects of antisense oligomer pretreatment on the subsequent apoptosis induced by heat shock in SGC-7901 cells. Proteins were extracted for simultaneous measurement of HSP70 expression level by SDS-PAGE Western blotting.RESULTS: The number of viable cells decreased in a doseand time-dependent manner, and ladder-like patterns of DNA fragments were observed in SGC-7901 cells treated with antisense HSP70 oligomers at a concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h, which were consistent with inter-nucleosomal DNA fragmentation. Flow cytometric analysis showed a dose- and time-dependent increase in apoptotic rate by HSP70 antisense oligomers. This response was accompanied with a decrease in the percentage of cells in the G1 and S phases of the cell cycle, suggesting inhibition of cell proliferation. In addition, flow cytometry also showed that pretreatment of SGC-7901 cells with HSP70 antisense oligomers enhanced the subsequent apoptosis induced by heat shock treatment. Western blotting demonstrated that HSP70 antisense oligomers inhibited HSP70 expression, which preceded apoptosis, and HSP70 was undetectable at the concentration of 10 μmol/L for 48 h or 8 μmol/L for 72 h.CONCLUSION: Antisense HSP70 oligomers

  16. Functional Capabilities of the Earliest Peptides and the Emergence of Life

    Directory of Open Access Journals (Sweden)

    Michael J. Russell

    2011-09-01

    Full Text Available Considering how biological macromolecules first evolved, probably within a marine environment, it seems likely the very earliest peptides were not encoded by nucleic acids, or at least not via the genetic code as we know it. An objective of the present work is to demonstrate that sequence-independent peptides, or peptides with variable and unreliable lengths and sequences, have the potential to perform a variety of chemically useful functions such as anion and cation binding and membrane and channel formation as well as simple types of catalysis. These functions tend to be performed with the assistance of the main chain CONH atoms rather than the more variable or limited side chain atoms of the peptides presumed to exist then.

  17. 靶向哺乳动物细胞线粒体的核酸转运%Targeting delivery nucleic acid into mammalian mitochondria

    Institute of Scientific and Technical Information of China (English)

    付爱玲

    2016-01-01

    Mitochondrial DNA (mtDNA)genome mutations and defects are the essential mechanism of a various of mitochondrial dysfunction associated with diseases. The studies of targeting de-livery nucleic acid into mammalian mitochondria can thoroughly correct mtDNA mutation, rescue mtDNA impairment and then reverse the progress of diseases. There’s obvious differences be-tween nucleic acid import pathway of mammalian mitochondria and gene transfection of nuclei. In this paper, the effective strat-egies of delivering DNA and RNA(tRNA,rRNA,mRNA and an-tisense RNA)into mitochondria have been reviewed, as well as the challenges and development.%线粒体 DNA(mitochondrial DNA, mtDNA)的遗传性突变和缺陷是多种线粒体功能失调相关疾病的根本原因。靶向线粒体递送核酸,可从根本上纠正 mtDNA 突变、挽救 mtD-NA 损伤、阻断疾病进程。哺乳细胞内线粒体的核酸转运途径与细胞核的基因转染大不相同。该文综述了向哺乳动物细胞线粒体递送 DNA 和 RNA(tRNA、rRNA、mRNA 和反义RNA)的有效策略,并对其存在问题和发展趋势做一阐述。

  18. Antisense suppression of LOX3 gene expression in rice endosperm enhances seed longevity.

    Science.gov (United States)

    Xu, Huibin; Wei, Yidong; Zhu, Yongsheng; Lian, Ling; Xie, Hongguang; Cai, Qiuhua; Chen, Qiushi; Lin, Zhongping; Wang, Zonghua; Xie, Huaan; Zhang, Jianfu

    2015-05-01

    Lipid peroxidation plays a major role in seed longevity and viability. In rice grains, lipid peroxidation is catalyzed by the enzyme lipoxygenase 3 (LOX3). Previous reports showed that grain from the rice variety DawDam in which the LOX3 gene was deleted had less stale flavour after grain storage than normal rice. The molecular mechanism by which LOX3 expression is regulated during endosperm development remains unclear. In this study, we expressed a LOX3 antisense construct in transgenic rice (Oryza sativa L.) plants to down-regulate LOX3 expression in rice endosperm. The transgenic plants exhibited a marked decrease in LOX mRNA levels, normal phenotypes and a normal life cycle. We showed that LOX3 activity and its ability to produce 9-hydroperoxyoctadecadienoic acid (9-HPOD) from linoleic acid were significantly lower in transgenic seeds than in wild-type seeds by measuring the ultraviolet absorption of 9-HPOD at 234 nm and by high-performance liquid chromatography. The suppression of LOX3 expression in rice endosperm increased grain storability. The germination rate of TS-91 (antisense LOX3 transgenic line) was much higher than the WT (29% higher after artificial ageing for 21 days, and 40% higher after natural ageing for 12 months). To our knowledge, this is the first report to demonstrate that decreased LOX3 expression can preserve rice grain quality during storage with no impact on grain yield, suggesting potential applications in agricultural production. PMID:25545811

  19. Lipid-Albumin Nanoparticles (LAN) for Therapeutic Delivery of Antisense Oligonucleotide against HIF-1α.

    Science.gov (United States)

    Li, Hong; Quan, Jishan; Zhang, Mengzi; Yung, Bryant C; Cheng, Xinwei; Liu, Yang; Lee, Young B; Ahn, Chang-Ho; Kim, Deog Joong; Lee, Robert J

    2016-07-01

    Lipid-albumin nanoparticles (LAN) were synthesized for delivery of RX-0047, an antisense oligonucleotide (ASO) against the hypoxia inducible factor-1 alpha (HIF-1α) to solid tumor. These lipid nanoparticles (LNs) incorporated a human serum albumin-pentaethylenehexamine (HSA-PEHA) conjugate, which is cationic and can form electrostatic complexes with negatively charged oligonucleotides. The delivery efficiency of LAN-RX-0047 was investigated in KB cells and a KB murine xenograft model. When KB cells were treated with LAN-RX-0047, significant HIF-1α downregulation and enhanced cellular uptake were observed compared to LN-RX-0047. LN-RX-0047 and LAN-RX-0047 showed similar cytotoxicity against KB cells with IC50 values of 19.3 ± 3.8 and 20.1 ± 4.2 μM, respectively. LAN-RX-0047 was shown to be taken up by the cells via the macropinocytosis and caveolae-mediated endocytosis pathways while LN-RX-0047 was taken up by cells via caveolae-mediated endocytosis. In the KB xenograft tumor model, LAN-RX-0047 exhibited tumor suppressive activity and significantly reduced intratumoral HIF-1α expression compared to LN-RX-0047. Furthermore, LAN-RX-0047 greatly increased survival time of mice bearing KB-1 xenograft tumors at doses of either 3 mg/kg or 16 mg/kg. These results indicated that LAN-RX-0047 is a highly effective vehicle for therapeutic delivery of antisense agents to tumor.

  20. Antisense mediated splicing modulation for inherited metabolic diseases: challenges for delivery.

    Science.gov (United States)

    Pérez, Belen; Vilageliu, Lluisa; Grinberg, Daniel; Desviat, Lourdes R

    2014-02-01

    In the past few years, research in targeted mutation therapies has experienced significant advances, especially in the field of rare diseases. In particular, the efficacy of antisense therapy for suppression of normal, pathogenic, or cryptic splice sites has been demonstrated in cellular and animal models and has already reached the clinical trials phase for Duchenne muscular dystrophy. In different inherited metabolic diseases, splice switching oligonucleotides (SSOs) have been used with success in patients' cells to force pseudoexon skipping or to block cryptic splice sites, in both cases recovering normal transcript and protein and correcting the enzyme deficiency. However, future in vivo studies require individual approaches for delivery depending on the gene defect involved, given the different patterns of tissue and organ expression. Herein we review the state of the art of antisense therapy targeting RNA splicing in metabolic diseases, grouped according to their expression patterns-multisystemic, hepatic, or in central nervous system (CNS)-and summarize the recent progress achieved in the field of in vivo delivery of oligonucleotides to each organ or system. Successful body-wide distribution of SSOs and preferential distribution in the liver after systemic administration have been reported in murine models for different diseases, while for CNS limited data are available, although promising results with intratechal injections have been achieved. PMID:24506780

  1. Search for antisense copies of beta-globin mRNA in anemic mouse spleen

    Directory of Open Access Journals (Sweden)

    Taylor John M

    2001-03-01

    Full Text Available Abstract Background Previous studies by Volloch and coworkers have reported that during the expression of high levels of β-globin mRNA in the spleen of anemic mice, they could also detect small but significant levels of an antisense (AS globin RNA species, which they postulated might have somehow arisen by RNA-directed RNA synthesis. For two reasons we undertook to confirm and possibly extend these studies. First, previous studies in our lab have focussed on what is an unequivocal example of host RNA-directed RNA polymerase activity on the RNA genome of human hepatitis delta virus. Second, if AS globin species do exist they could in turn form double-stranded RNA species which might induce post-transcriptional gene silencing, a phenomenon somehow provoked in eukaryotic cells by AS RNA sequences. Results We reexamined critical aspects of the previous globin studies. We used intraperitoneal injections of phenylhydrazine to induce anemia in mice, as demonstrated by the appearance and ultimate disappearance of splenomegaly. While a 30-fold increase in globin mRNA was detected in the spleen, the relative amount of putative AS RNA could be no more than 0.004%. Conclusions Contrary to earlier reports, induction of a major increase in globin transcripts in the mouse spleen was not associated with a detectable level of antisense RNA to globin mRNA.

  2. Ustilago maydis natural antisense transcript expression alters mRNA stability and pathogenesis.

    Science.gov (United States)

    Donaldson, Michael E; Saville, Barry J

    2013-07-01

    Ustilago maydis infection of Zea mays leads to the production of thick-walled diploid teliospores that are the dispersal agent for this pathogen. Transcriptome analyses of this model biotrophic basidiomycete fungus identified natural antisense transcripts (NATs) complementary to 247 open reading frames. The U. maydis NAT cDNAs were fully sequenced and annotated. Strand-specific RT-PCR screens confirmed expression and identified NATs preferentially expressed in the teliospore. Targeted screens revealed four U. maydis NATs that are conserved in a related fungus. Expression of NATs in haploid cells, where they are not naturally occurring, resulted in increased steady-state levels of some complementary mRNAs. The expression of one NAT, as-um02151, in haploid cells resulted in a twofold increase in complementary mRNA levels, the formation of sense-antisense double-stranded RNAs, and unchanged Um02151 protein levels. This led to a model for NAT function in the maintenance and expression of stored teliospore mRNAs. In testing this model by deletion of the regulatory region, it was determined that alteration in NAT expression resulted in decreased pathogenesis in both cob and seedling infections. This annotation and functional analysis supports multiple roles for U. maydis NATs in controlling gene expression and influencing pathogenesis.

  3. On Measuring miRNAs after Transient Transfection of Mimics or Antisense Inhibitors

    Science.gov (United States)

    Thomson, Daniel W.; Bracken, Cameron P.; Szubert, Jan M.; Goodall, Gregory J.

    2013-01-01

    The ability to alter microRNA (miRNA) abundance is crucial for studying miRNA function. To achieve this there is widespread use of both exogenous double-stranded miRNA mimics for transient over-expression, and single stranded antisense RNAs (antimiRs) for miRNA inhibition. The success of these manipulations is often assessed using qPCR, but this does not accurately report the level of functional miRNA. Here, we draw attention to this discrepancy, which is overlooked in many published reports. We measured the functionality of exogenous miRNA by comparing the total level of transfected miRNA in whole cell extracts to the level of miRNA bound to Argonaute following transfection and show that the supraphysiological levels of transfected miRNA frequently seen using qPCR do not represent the functional levels, because the majority of transfected RNA that is detected is vesicular and not accessible for loading into Argonaute as functionally active miRNAs. In the case of microRNA inhibition by transient transfection with antisense inhibitors, there is also the potential for discrepancy, because following cell lysis the abundant inhibitor levels from cellular vesicles can directly interfere with the PCR reaction used to measure miRNA level. PMID:23358900

  4. Cathepsin B antisense oligodeoxynucleotide suppresses invasive potential of MG-63 cells

    Institute of Scientific and Technical Information of China (English)

    He Maolin; Xiao Zengming; Li Shide; Chen Anmin

    2008-01-01

    Objective To study the biological effects of cathepsin B phosporotbioated antisense oligodeoxynucleotide on human osteosarcoma cell line MG-63 after transfection. Methods A 18-mer phosphorothioate antisense oligodeoxynucleotide (ASODN) targeted against the cathepsin B mRNA was transfected into the human osteosarcoma cell line MG-63 by lipofectamine 2000. The sense and nonsense oligodeoxynucleotides to cathepsin B and blank vector were used as controls. The expression of cathepsin B mRNA was examined by RT-PCR and the expression of cathepsin B was examined by Western blot. The invasive capability of MG-63 cells was evaluated by the boydern chamber assay. Results The expression of cathcpsin B was obviously inhibited in antlsense oligodeoxynucleotide treated cells compared with the control cells. The number of invading MG-63 cells was significantly lower in the ASODN-treated groups than that in the control groups. Conclusion The cathepsin B ASODN significantly inhibits the expression of cathepsin B and invasive ability of MG-63 cell in osteosarcoma.

  5. Identification of novel sense and antisense transcription at the TRPM2 locus in cancer

    Institute of Scientific and Technical Information of China (English)

    Ugo Orfanelli; Ann-Kathrin Wenke; Claudio Doglioni; Vincenzo Russo; Anja Katrin Bosserhoff; Giovanni Lavorgna

    2008-01-01

    It has been proposed that in cancer, where the bulk of the genome becomes hypomethylated, there is an increase in transcriptional noise that might lead to the generation of antisense transcripts that could affect the function of key oncosuppressor genes, ultimately leading to malignant transformation. Here, we describe the computational identifi-cation of a melanoma-enriched antisense transcript, TRPM2-AS, mapped within the locus of TRPM2, an ion channel capable of mediating susceptibility to cell death. Analysis of the TRPM2-AS genomic region indicated the presence in the same region of another tumor-enriched TRPM2 transcript, TRPM2-TE, located across a CpG island shared with TRPM2-AS. Quantitative PCR experiments confirmed that TRPM2-AS and TRPM2-TE transcripts were up-regu-lated in melanoma, and their activation was consistent with the methylation status of the shared CpG island. Func-tional knock-out of TRPM2-TE, as well as over-expression of wild-type TRPM2, increased melanoma susceptibility to apoptosis and necrosis. Finally, expression analysis in other cancer types indicated that TRPM2-AS and TRPM2-TE over-expression might have an even wider role than anticipated, reinforcing the relevance of our computational approach in identifying new potential therapeutic targets.

  6. On measuring miRNAs after transient transfection of mimics or antisense inhibitors.

    Directory of Open Access Journals (Sweden)

    Daniel W Thomson

    Full Text Available The ability to alter microRNA (miRNA abundance is crucial for studying miRNA function. To achieve this there is widespread use of both exogenous double-stranded miRNA mimics for transient over-expression, and single stranded antisense RNAs (antimiRs for miRNA inhibition. The success of these manipulations is often assessed using qPCR, but this does not accurately report the level of functional miRNA. Here, we draw attention to this discrepancy, which is overlooked in many published reports. We measured the functionality of exogenous miRNA by comparing the total level of transfected miRNA in whole cell extracts to the level of miRNA bound to Argonaute following transfection and show that the supraphysiological levels of transfected miRNA frequently seen using qPCR do not represent the functional levels, because the majority of transfected RNA that is detected is vesicular and not accessible for loading into Argonaute as functionally active miRNAs. In the case of microRNA inhibition by transient transfection with antisense inhibitors, there is also the potential for discrepancy, because following cell lysis the abundant inhibitor levels from cellular vesicles can directly interfere with the PCR reaction used to measure miRNA level.

  7. Antisense oligonucleotide targeting p53 increased apoptosis of MCF-7 cells induced by ionizing radiation

    Institute of Scientific and Technical Information of China (English)

    Li-cheng DAI; Xiang WANG; Xing YAO; Li-shan MIN; Fu-chu QIAN; Jian-fang HE

    2006-01-01

    Aim: To investigate the effect of antisense compounds (AS) targeting human p53 mRNA on radiosensitivity of MCF-7 cells. Methods: Western blotting and RT-PCR were used to analyze the protein content and mRNA level. Additionally, cell proliferation, cell cycle and cell apoptosis were all analyzed in irradiated or sham-irradiated cells. Results: Among the five antisense compounds (AS), AS3 was identified to efficiently inhibit p53 mRNA level and protein content. Interestingly, ASS transfer has little effect on cell proliferation in DU-145 cells (mutant p53) after ionizing radiation (IR). In contrast, a marked increase of cell apoptosis and growth inhibition were observed in MCF-7 cells (wild-type p53), suggesting that AS3 can increase radiosensitivity of MCF-7 cells. Additionally, it was also observed that the transfection of AS3 decreased the fraction of G1 phase cells, and increased the proportion of S phase cells compared to untreated cells 24 h after IR in MCF-7 cell lines. Conclusion: AS3 transfection increases MCF-7 cell apoptosis induced by 5 Gy-radiation, and this mechanism may be closely associated with abrogation of G1 phase arrest.

  8. Irradiation of Human Prostate Cancer Cells Increases Uptake of Antisense Oligodeoxynucleotide

    International Nuclear Information System (INIS)

    Purpose: To investigate whether irradiation before antisense Bcl-2 oligodeoxynucleotide (ODN) administration enhances tissue uptake, and whether periodic dosing enhances cellular uptake of fluorescently labeled ODN relative to constant dosing. Methods and Materials: PC-3-Bcl-2 cells (prostate cancer cell line engineered to overexpress Bcl-2) were subjected to increasing doses of irradiation (0-10 Gy) with or without increasing concentrations of fluorescently labeled antisense Bcl-2 ODN (G4243). The fluorescent signal intensity was quantified as the total grain area with commercial software. In addition, PC-3-Bcl-2 subcutaneous xenograft tumors were treated with or without irradiation in combination with various dosing schemas of G4243. The uptake of fluorescent G4243 in tumors was quantitated. Results: The uptake of G4243 was increased in prostate cancer cells exposed to low doses of irradiation both in vitro and in vivo. Irradiation before G4243 treatment resulted in increased fluorescent signal intensity in xenograft tumors compared with those irradiated after G4243 treatment. A single weekly dose of G4243 produced higher G4243 uptake in xenograft tumors than daily dosing, even when the total dose administered per week was held constant. Conclusions: These findings suggest that ionizing radiation increases the uptake of therapeutic ODN in target tissues and, thus, has potential to increase the efficacy of ODN in clinical applications

  9. Antitumor Effect of Antisense Ornithine Decarboxylase Adenovirus on Human Lung Cancer Cells

    Institute of Scientific and Technical Information of China (English)

    Hui TIAN; Lin LI; Xian-Xi LIU; Yan ZHANG

    2006-01-01

    Ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis, was found to increase in cancer cells, especially lung cancer cells. Some chemotherapeutic agents aimed at decreasing ODC gene expression showed inhibitory effects on cancer cells. In this study, we examined the effects of adenoviral transduced antisense ODC on lung cancer cells. An adenovirus carrying antisense ODC (rAd-ODC/Ex3as) was used to infect lung cancer cell line A-549. The 3-(4,5-me thylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was used to analyze the effect on cell growth. Expression of ODC and concentration of polyamines in cells were determined by Western blot analysis and high performance liquid chromatography. Terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling was used to analyze cell apoptosis. The expression of ODC in A-549 cells was reduced to 54%, and that of three polyamines was also decreased through the rAd-ODC/Ex3as treatment. Consequently, cell growth was substantially inhibited and terminal deoxynucleotidyl transferase-mediated biotin-dUTP nick-end labeling showed that rAd-ODC/Ex3as could lead to cell apoptosis, with apoptosis index of 46%. This study suggests that rAd-ODC/Ex3as has an antitumor effect on the human lung cancer cells.

  10. Small regulatory RNAs in lambdoid bacteriophages and phage-derived plasmids: Not only antisense.

    Science.gov (United States)

    Nejman-Faleńczyk, Bożena; Bloch, Sylwia; Licznerska, Katarzyna; Felczykowska, Agnieszka; Dydecka, Aleksandra; Węgrzyn, Alicja; Węgrzyn, Grzegorz

    2015-03-01

    Until recently, only two small regulatory RNAs encoded by lambdoid bacteriophages were known. These transcripts are derived from paQ and pO promoters. The former one is supposed to act as an antisense RNA for expression of the Q gene, encoding a transcription antitermination protein. The latter transcript, called oop RNA, was initially proposed to have a double role, in establishing expression of the cI gene and in providing a primer for DNA replication. Although the initially proposed mechanisms by which oop RNA could influence the choice between two alternative developmental pathways of the phage and the initiation of phage DNA replication were found not true, the pO promoter has been demonstrated to be important for both regulation of phage development and control of DNA replication. Namely, the pO-derived transcript is an antisense RNA for expression of the cII gene, and pO is a part of a dual promoter system responsible for regulation of initiation of DNA synthesis from the oriλ region. Very recent studies identified a battery of small RNAs encoded by lambdoid bacteriophages existing as prophages in chromosomes of enterohemorrhagic Escherichia coli strains. Some of them have very interesting functions, like anti-small RNAs.

  11. Efficient inhibition of human telomerase activity by antisense oligonucleotides sensitizes cancer cells to radiotherapy

    Institute of Scientific and Technical Information of China (English)

    Xue-mei JI; Cong-hua XIE; Ming-hao FANG; Fu-xiang ZHOU; Wen-jie ZHANG; Ming-sheng ZHANG; Yun-feng ZHOU

    2006-01-01

    Aim: To investigate the effect of the antisense oligonucleotides (ASODN) specific for human telomerase RNA (hTR) on radio sensitization and proliferation inhibition in human neurogliocytoma cells (U251). Methods: U251 cells were transfected with hTR ASODN or nonspecific oligonucleotides (NSODN). Before and after irradiation of 60Co-γray, telomerase activity was assayed by telomeric repeat amplification protocol (TRAP-PCR-ELISA), and DNA damage and repair were examined by the comet assay. The classical colony assay was used to plot the cell-survival curve, to detect the D0 value. Results: hTR antisense oligonucleotides could downregulate the telomerase activity, increase radiation induced DNA damage and reduce the subsequent repair. Furthermore, it could inhibit the proliferation and decrease the D0 value which demonstrates rising radiosensitivity. However, telomere length was unchanged over a short period of time. Conclusion: These findings suggest that an ASODN-based strategy may be used to develop telomerase inhibitors, which can efficiently sensitize radiotherapy.

  12. Antisense oligonucleotides targeting midkine induced apoptosis and increased chemosensitivity in hepatocellular carcinoma cells

    Institute of Scientific and Technical Information of China (English)

    Li-cheng DAI; Xiang WANG; Xing YAO; Yong-liang LU; Jin-liang PING; Jian-fang HE

    2006-01-01

    Aim: Overexpression of midkine (MK) has been observed in many malignancies. This aim of this study is to screen for suitable antisense oligonucleotides (ASODN) targeting MK in hepatocellular carcinoma (HCC) cells and evaluate its antitumor activity. Methods: Ten ASODN targeting MK were designed and synthesized. After transfection with ASODN, cell proliferation was analyzed with MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2//-tetrazolium, inner salt] assay. In addition, MK mRNA, protein levels, as well as apoptosis and caspase-3 activity were also examined in HepG2 cells. Cell proliferation was then analyzed after treatment with both ASODN and chemotherapeu-tic drugs. Results: In this experiment, the ASODN5 among the 10 ASODN showed higher inhibitory activity against proliferation of hepatocellular carcinoma cells in a dose-dependent manner. In HepG2 cells, ASODN5 could significantly reduce the MK mRNA level and protein content. After transfection with ASODN5 for 48 h, accompanied with a decline of survivin and Bcl-2 protein content, a remarkable increase of apoptosis and caspase-3 activity was observed in HepG2 cells. Furthermore, ASODN5 transfer can significantly increase chemosensitivity in HepG2 cells. Conclusion: Antisense oligonucleotides targeting MK shows therapeutic effects on HCC; ASODN5 has the possibility to be developed as an effective antitumor agent.

  13. Antisense oligodeoxynucleotides targeting the serine/threonine kinase Pim-2 inhibited proliferation of DU-145 cells

    Institute of Scientific and Technical Information of China (English)

    Jin-ming DAI; Shu-qun ZHANG; Wei ZHANG; Ru-xian LIN; Zong-zheng JI; Sheng-qi WANG

    2005-01-01

    Aim: To investigate the effect of antisense oligodeoxynucleotides (ASODN) targeting Pim-2 on cell proliferation of DU-145 cells. Methods: Three ASODN targeting Pim-2 were designed and synthesized. After transfection with ASODN, cell proliferation was analyzed using an MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] assay. In addition, Pim-2 mRNA, protein levels, and cell cycles were examined. Results:The ASODN designed and synthesized by our laboratory significantly reduced Pim-2 mRNA level and protein content in DU-145 cells. After transfection with ASODN for 48 h, a marked reduction in cell viability was observed in DU- 145 cells in a dose-dependent manner. No remarkable apoptosis occurred in cells treated with ASODN compared with control cells. However, it should be noted that G1 phase arrest was clearly observed in ASODN-treated cells. Conclusion: ASODN targeting Pim-2 resulted in a marked reduction in DU-145 cell proliferation, and induction of G1 phase cell cycle arrest is one of the important mechanisms for ASODN to reduce cell growth. Moreover, antisense inhibition of Pim-2 expression provides a new promising therapy target for prostate cancer.

  14. Plant signalling peptides

    OpenAIRE

    Wiśniewska, Justyna; Trejgell, Alina; Tretyn, Andrzej

    2003-01-01

    Biochemical and genetic studies have identified peptides that play crucial roles in plant growth and development, including defence mechanisms in response to wounding by pests, the control of cell division and expansion, and pollen self-incompatibility. The first two signalling peptides to be described in plants were tomato systemin and phytosulfokine (PSK). There is also biochemical evidence that natriuretic peptide-like molecules, immunologically-relatedt o those found ...

  15. Polycyclic peptide therapeutics.

    Science.gov (United States)

    Baeriswyl, Vanessa; Heinis, Christian

    2013-03-01

    Owing to their excellent binding properties, high stability, and low off-target toxicity, polycyclic peptides are an attractive molecule format for the development of therapeutics. Currently, only a handful of polycyclic peptides are used in the clinic; examples include the antibiotic vancomycin, the anticancer drugs actinomycin D and romidepsin, and the analgesic agent ziconotide. All clinically used polycyclic peptide drugs are derived from natural sources, such as soil bacteria in the case of vancomycin, actinomycin D and romidepsin, or the venom of a fish-hunting coil snail in the case of ziconotide. Unfortunately, nature provides peptide macrocyclic ligands for only a small fraction of therapeutic targets. For the generation of ligands of targets of choice, researchers have inserted artificial binding sites into natural polycyclic peptide scaffolds, such as cystine knot proteins, using rational design or directed evolution approaches. More recently, large combinatorial libraries of genetically encoded bicyclic peptides have been generated de novo and screened by phage display. In this Minireview, the properties of existing polycyclic peptide drugs are discussed and related to their interesting molecular architectures. Furthermore, technologies that allow the development of unnatural polycyclic peptide ligands are discussed. Recent application of these technologies has generated promising results, suggesting that polycyclic peptide therapeutics could potentially be developed for a broad range of diseases. PMID:23355488

  16. Design of amphiphilic oligopeptides as models for fine tuning peptide assembly with plasmid DNA.

    Science.gov (United States)

    Goparaju, Geetha N; Gupta, Pardeep K

    2014-08-01

    We discuss the design of novel amphiphilic oligopeptides with hydrophobic and cationic amino acids to serve as models to understand peptide-DNA assembly. Biophysical and thermodynamic characterization of interaction of these amphiphilic peptides with plasmid DNA is presented. Peptides with at least +4 charges favor stable complex formation. Surface potential is dependent on the type of hydrophobic amino acid for a certain charge. Thermodynamically it is a spontaneous interaction between most of the peptides and plasmid DNA. Lys(7) and Tyr peptides with +4/+5 charges indicate cooperative binding with pDNA without saturation of interaction while Val(2)-Gly-Lys(4), Val-Gly-Lys(5), and Phe-Gly-Lys(5) lead to saturation of interaction indicating condensed pDNA within the range of N/Ps studied. We show that the biophysical properties of DNA-peptide complexes could be modulated by design and the peptides presented here could be used as building blocks for creating DNA-peptide complexes for various biomedical applications, mainly nucleic acid delivery.

  17. Fluorescent Probes for Nucleic Acid Visualization in Fixed and Live Cells

    Directory of Open Access Journals (Sweden)

    Alexandre S. Boutorine

    2013-12-01

    Full Text Available This review analyses the literature concerning non-fluorescent and fluorescent probes for nucleic acid imaging in fixed and living cells from the point of view of their suitability for imaging intracellular native RNA and DNA. Attention is mainly paid to fluorescent probes for fluorescence microscopy imaging. Requirements for the target-binding part and the fluorophore making up the probe are formulated. In the case of native double-stranded DNA, structure-specific and sequence-specific probes are discussed. Among the latest, three classes of dsDNA-targeting molecules are described: (i sequence-specific peptides and proteins; (ii triplex-forming oligonucleotides and (iii polyamide oligo(N-methylpyrrole/N-methylimidazole minor groove binders. Polyamides seem to be the most promising targeting agents for fluorescent probe design, however, some technical problems remain to be solved, such as the relatively low sequence specificity and the high background fluorescence inside the cells. Several examples of fluorescent probe applications for DNA imaging in fixed and living cells are cited. In the case of intracellular RNA, only modified oligonucleotides can provide such sequence-specific imaging. Several approaches for designing fluorescent probes are considered: linear fluorescent probes based on modified oligonucleotide analogs, molecular beacons, binary fluorescent probes and template-directed reactions with fluorescence probe formation, FRET donor-acceptor pairs, pyrene excimers, aptamers and others. The suitability of all these methods for living cell applications is discussed.

  18. Electroporation-enhanced delivery of nucleic acid vaccines.

    Science.gov (United States)

    Broderick, Kate E; Humeau, Laurent M

    2015-02-01

    The naked delivery of nucleic acid vaccines is notoriously inefficient, and an enabling delivery technology is required to direct efficiently these constructs intracellularly. A delivery technology capable of enhancing nucleic acid uptake in both cells in tissues and in culture is electroporation (EP). EP is a physical delivery mechanism that increases the permeability of mammalian cell membranes and allows the trafficking of large macromolecules into the cell. EP has now been used extensively in the clinic and been shown to be an effective method to increase both the uptake of the construct and the breadth and magnitude of the resulting immune responses. Excitingly, 2014 saw the announcement of the first EP-enhanced DNA vaccine Phase II trial demonstrating clinical efficacy. This review seeks to introduce the reader to EP as a technology to enhance the delivery of DNA and RNA vaccines and highlight several published clinical trials using this delivery modality.

  19. Nucleic acids encoding antifungal polypeptides and uses thereof

    Science.gov (United States)

    Altier, Daniel J.; Ellanskaya, I. A.; Gilliam, Jacob T.; Hunter-Cevera, Jennie; Presnail, James K; Schepers, Eric; Simmons, Carl R.; Torok, Tamas; Yalpani, Nasser

    2010-11-02

    Compositions and methods for protecting a plant from a pathogen, particularly a fungal pathogen, are provided. Compositions include an amino acid sequence, and variants and fragments thereof, for an antipathogenic polypeptide that was isolated from a fungal fermentation broth. Nucleic acid molecules that encode the antipathogenic polypeptides of the invention, and antipathogenic domains thereof, are also provided. A method for inducing pathogen resistance in a plant using the nucleotide sequences disclosed herein is further provided. The method comprises introducing into a plant an expression cassette comprising a promoter operably linked to a nucleotide sequence that encodes an antipathogenic polypeptide of the invention. Compositions comprising an antipathogenic polypeptide or a transformed microorganism comprising a nucleic acid of the invention in combination with a carrier and methods of using these compositions to protect a plant from a pathogen are further provided. Transformed plants, plant cells, seeds, and microorganisms comprising a nucleotide sequence that encodes an antipathogenic polypeptide of the invention are also disclosed.

  20. Nucleic Acid Engineering: RNA Following the Trail of DNA.

    Science.gov (United States)

    Kim, Hyejin; Park, Yongkuk; Kim, Jieun; Jeong, Jaepil; Han, Sangwoo; Lee, Jae Sung; Lee, Jong Bum

    2016-02-01

    The self-assembly feature of the naturally occurring biopolymer, DNA, has fascinated researchers in the fields of materials science and bioengineering. With the improved understanding of the chemical and structural nature of DNA, DNA-based constructs have been designed and fabricated from two-dimensional arbitrary shapes to reconfigurable three-dimensional nanodevices. Although DNA has been used successfully as a building block in a finely organized and controlled manner, its applications need to be explored. Hence, with the myriad of biological functions, RNA has recently attracted considerable attention to further the application of nucleic acid-based structures. This Review categorizes different approaches of engineering nucleic acid-based structures and introduces the concepts, principles, and applications of each technique, focusing on how DNA engineering is applied as a guide to RNA engineering.