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Sample records for abundant oligonucleotides common

  1. Analysis of common mitochondrial DNA mutations by allele-specific oligonucleotide and Southern blot hybridization.

    Tang, Sha; Halberg, Michelle C; Floyd, Kristen C; Wang, Jing

    2012-01-01

    Mitochondrial disorders are clinically and genetically heterogeneous. There are a set of recurrent point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases, including MELAS (mitochondrial encephalopathy, lactic acidosis, stroke-like episodes), MERRF (myoclonic epilepsy and ragged red fibers), LHON (Leber's hereditary optic neuropathy), NARP (neuropathy, ataxia, retinitis pigmentosa), and Leigh syndrome. Most of the pathogenic mtDNA point mutations are present in the heteroplasmic state, meaning that the wild-type and mutant-containing mtDNA molecules are coexisting. Clinical heterogeneity may be due to the degree of mutant load (heteroplasmy) and distribution of heteroplasmic mutations in affected tissues. Additionally, Kearns-Sayre syndrome and Pearson syndrome are caused by large mtDNA deletions. In this chapter, we describe a multiplex PCR/allele-specific oligonucleotide (ASO) hybridization method for the screening of 13 common point mutations. This method allows the detection of low percentage of mutant heteroplasmy. In addition, a nonradioactive Southern blot hybridization protocol for the analysis of mtDNA large deletions is also described. PMID:22215554

  2. Commonly Rare and Rarely Common: Comparing Population Abundance of Invasive and Native Aquatic Species

    Hansen, Gretchen J. A.; M Jake Vander Zanden; Michael J Blum; Clayton, Murray K.; Hain, Ernie F.; Jennifer Hauxwell; Marit Izzo; Matthew S Kornis; Peter B. McIntyre; Alison Mikulyuk; Erika Nilsson; Julian D Olden; Monica Papeş; Sapna Sharma

    2013-01-01

    Invasive species are leading drivers of environmental change. Their impacts are often linked to their population size, but surprisingly little is known about how frequently they achieve high abundances. A nearly universal pattern in ecology is that species are rare in most locations and abundant in a few, generating right-skewed abundance distributions. Here, we use abundance data from over 24,000 populations of 17 invasive and 104 native aquatic species to test whether invasive species diffe...

  3. Commonly Rare and Rarely Common: Comparing Population Abundance of Invasive and Native Aquatic Species

    Hansen, Gretchen J. A.; Vander Zanden, M. Jake; Blum, Michael J.; Clayton, Murray K.; Hain, Ernie F.; Hauxwell, Jennifer; Izzo, Marit; Kornis, Matthew S.; McIntyre, Peter B.; Mikulyuk, Alison; Nilsson, Erika; Olden, Julian D.; Papeş, Monica; Sharma, Sapna

    2013-01-01

    Invasive species are leading drivers of environmental change. Their impacts are often linked to their population size, but surprisingly little is known about how frequently they achieve high abundances. A nearly universal pattern in ecology is that species are rare in most locations and abundant in a few, generating right-skewed abundance distributions. Here, we use abundance data from over 24,000 populations of 17 invasive and 104 native aquatic species to test whether invasive species differ from native counterparts in statistical patterns of abundance across multiple sites. Invasive species on average reached significantly higher densities than native species and exhibited significantly higher variance. However, invasive and native species did not differ in terms of coefficient of variation, skewness, or kurtosis. Abundance distributions of all species were highly right skewed (skewness>0), meaning both invasive and native species occurred at low densities in most locations where they were present. The average abundance of invasive and native species was 6% and 2%, respectively, of the maximum abundance observed within a taxonomic group. The biological significance of the differences between invasive and native species depends on species-specific relationships between abundance and impact. Recognition of cross-site heterogeneity in population densities brings a new dimension to invasive species management, and may help to refine optimal prevention, containment, control, and eradication strategies. PMID:24194883

  4. Commonly rare and rarely common: comparing population abundance of invasive and native aquatic species.

    Gretchen J A Hansen

    Full Text Available Invasive species are leading drivers of environmental change. Their impacts are often linked to their population size, but surprisingly little is known about how frequently they achieve high abundances. A nearly universal pattern in ecology is that species are rare in most locations and abundant in a few, generating right-skewed abundance distributions. Here, we use abundance data from over 24,000 populations of 17 invasive and 104 native aquatic species to test whether invasive species differ from native counterparts in statistical patterns of abundance across multiple sites. Invasive species on average reached significantly higher densities than native species and exhibited significantly higher variance. However, invasive and native species did not differ in terms of coefficient of variation, skewness, or kurtosis. Abundance distributions of all species were highly right skewed (skewness>0, meaning both invasive and native species occurred at low densities in most locations where they were present. The average abundance of invasive and native species was 6% and 2%, respectively, of the maximum abundance observed within a taxonomic group. The biological significance of the differences between invasive and native species depends on species-specific relationships between abundance and impact. Recognition of cross-site heterogeneity in population densities brings a new dimension to invasive species management, and may help to refine optimal prevention, containment, control, and eradication strategies.

  5. Livestock grazing intensity affects abundance of Common shrews (Sorex araneus) in two meadows in Denmark

    Schmidt, Niels Martin; Olsen, Henrik; Leirs, Herwig

    2009-01-01

    habitat type for a large number of animal species in today's fragmented and intensively cultivated landscape of Europe. Here we focus on the population characteristics of Common shrews Sorex araneus in relation to livestock grazing intensity in two wet meadows in western Denmark. Results: High grazing...... intensity had a significant negative effect on Common shrew number High grazing intensity had a significant negative effect on Common shrew number compared to low grazing intensity and no grazing. Common shrew abundance was generally, but  not significantly, higher on the low grazing intensity plots than...... on the ungrazed controls. No differences in body mass, sex ratio, or reproductive output between Common shrew individuals from the various grazing treatments were found. Conclusion: No negative effects of low intensity grazing on Common shrew abundance were No negative effects of low intensity grazing on Common...

  6. Response of rare, common and abundant bacterioplankton to anthropogenic perturbations in a Mediterranean coastal site.

    Baltar, Federico; Palovaara, Joakim; Vila-Costa, Maria; Salazar, Guillem; Calvo, Eva; Pelejero, Carles; Marrasé, Cèlia; Gasol, Josep M; Pinhassi, Jarone

    2015-06-01

    Bacterioplankton communities are made up of a small set of abundant taxa and a large number of low-abundant organisms (i.e. 'rare biosphere'). Despite the critical role played by bacteria in marine ecosystems, it remains unknown how this large diversity of organisms are affected by human-induced perturbations, or what controls the responsiveness of rare compared to abundant bacteria. We studied the response of a Mediterranean bacterioplankton community to two anthropogenic perturbations (i.e. nutrient enrichment and/or acidification) in two mesocosm experiments (in winter and summer). Nutrient enrichment increased the relative abundance of some operational taxonomic units (OTUs), e.g. Polaribacter, Tenacibaculum, Rhodobacteraceae and caused a relative decrease in others (e.g. Croceibacter). Interestingly, a synergistic effect of acidification and nutrient enrichment was observed on specific OTUs (e.g. SAR86). We analyzed the OTUs that became abundant at the end of the experiments and whether they belonged to the rare (1% relative abundance) fractions. Most of the abundant OTUs at the end of the experiments were abundant, or at least common, in the original community of both experiments, suggesting that ecosystem alterations do not necessarily call for rare members to grow. PMID:26032602

  7. Abundance of common species, not species richness, drives delivery of a real-world ecosystem service.

    Winfree, Rachael; Fox, Jeremy W; Williams, Neal M; Reilly, James R; Cariveau, Daniel P

    2015-07-01

    Biodiversity-ecosystem functioning experiments have established that species richness and composition are both important determinants of ecosystem function in an experimental context. Determining whether this result holds for real-world ecosystem services has remained elusive, however, largely due to the lack of analytical methods appropriate for large-scale, associational data. Here, we use a novel analytical approach, the Price equation, to partition the contribution to ecosystem services made by species richness, composition and abundance in four large-scale data sets on crop pollination by native bees. We found that abundance fluctuations of dominant species drove ecosystem service delivery, whereas richness changes were relatively unimportant because they primarily involved rare species that contributed little to function. Thus, the mechanism behind our results was the skewed species-abundance distribution. Our finding that a few common species, not species richness, drive ecosystem service delivery could have broad generality given the ubiquity of skewed species-abundance distributions in nature. PMID:25959973

  8. Cytokines and therapeutic oligonucleotides.

    Hartmann, G; Bidlingmaier, M; Eigler, A; Hacker, U; Endres, S

    1997-12-01

    Therapeutic oligonucleotides - short strands of synthetic nucleic acids - encompass antisense and aptamer oligonucleotides. Antisense oligonucleotides are designed to bind to target RNA by complementary base pairing and to inhibit translation of the target protein. Antisense oligonucleotides enable specific inhibition of cytokine synthesis. In contrast, aptamer oligonucleotides are able to bind directly to specific proteins. This binding depends on the sequence of the oligonucleotide. Aptamer oligonucleotides with CpG motifs can exert strong immunostimulatory effects. Both kinds of therapeutic oligonucleotides - antisense and aptamer oligonucleotides - provide promising tools to modulate immunological functions. Recently, therapeutic oligonucleotides have moved towards clinical application. An antisense oligonucleotide directed against the proinflammatory intercellular adhesion molecule 1 (ICAM-1) is currently being tested in clinical trials for therapy of inflammatory disease. Immunostimulatory aptamer oligonucleotides are in preclinical development for immunotherapy. In the present review we summarize the application of therapeutic oligonucleotides to modulate immunological functions. We include technological aspects as well as current therapeutic concepts and clinical studies. PMID:9740353

  9. The effect of oilseed rape occurrence on main prey abundance and breeding success of the Common Buzzard Buteo buteo

    Panek, Marek; Husek, Jan

    2014-01-01

    Capsule The occurrence of oilseed rape increased main prey abundance and breeding success of Common Buzzards. Aims We tested whether the occurrence of oilseed rape influences the abundance of Common Voles, i.e. the main prey of Common Buzzards and so also nesting activity and breeding success of Common Buzzards. Methods The study was carried out in 2005–2012 in a 38 km2 area in western Poland, where oilseed rape plantations (12–106 ha) covered 18% of the agricultural land. The number ...

  10. Numerical Response of the Common Buzzard Buteo Buteo to The Changes In Abundance Of Small Mammals

    Tóth László

    2014-06-01

    Full Text Available I investigated the numerical response of the Common Buzzard to variations in density of small mammals. The study was carried out at the Hortobágy region in 2000-2001. During nest visiting periods clutch size, number of hatched and fledged young were recorded. Population of small mammals were also monitored by live-trapping. Effect of weather on the survival of overwintering rodents was also investigated. There was significant difference in clutch size between 2000 and 2001 (means 2.3 and 3.1. It can be explained by the remarkable differences in abundance of small mammal populations between the two years. The density of rodents was very low (9 specimen/ha in 2000. During 2001 the amount of small mammals has increased more than eightfold (76 specimen/ha. In February and March, 2000 there were 4 short mild periods alternating with 4 freezing periods, when distribution of significant precipitation (6-8 mm rainfall in each coincided with the mild periods. Thus the overwintering population almost extincted from the area because the tunnel complexes of voles are repeatedly flooded and huge part of the animals died, resulting very low density during the breeding season. In 2001 there was no such alternating periods, mild weather started 3 weeks earlier, thus voles overwintered successfully and their numbers increased rapidly producing a peak during the breeding season.

  11. Abundance gradients in low surface brightness spirals: clues on the origin of common gradients in galactic discs

    Bresolin, Fabio

    2015-01-01

    We acquired spectra of 141 HII regions in ten late-type low surface brightness galaxies (LSBGs). The analysis of the chemical abundances obtained from the nebular emission lines shows that metallicity gradients are a common feature of LSBGs, contrary to previous claims concerning the absence of such gradients in this class of galaxies. The average slope, when expressed in units of the isophotal radius, is found to be significantly shallower in comparison to galaxies of high surface brightness. This result can be attributed to the reduced surface brightness range measured across their discs, when combined with a universal surface mass density-metallicity relation. With a similar argument we explain the common abundance gradient observed in high surface brightness galaxy (HSBG) discs and its approximate dispersion. This conclusion is reinforced by our result that LSBGs share the same common abundance gradient with HSBGs, when the slope is expressed in terms of the exponential disc scale length.

  12. A common scaling rule for abundance, energetics, and production of parasitic and free-living species

    Hechinger, Ryan F.; Lafferty, Kevin D.; Dobson, Andy P.; Brown, James H.; Kuris, Armand M.

    2011-01-01

    The metabolic theory of ecology uses the scaling of metabolism with body size and temperature to explain the causes and consequences of species abundance. However, the theory and its empirical tests have never simultaneously examined parasites alongside free-living species. This is unfortunate because parasites represent at least half of species diversity. We show that metabolic scaling theory could not account for the abundance of parasitic or free-living species in three estuarine food webs until accounting for trophic dynamics. Analyses then revealed that the abundance of all species uniformly scaled with body mass to the - 3/4 power. This result indicates "production equivalence," where biomass production within trophic levels is invariant of body size across all species and functional groups: invertebrate or vertebrate, ectothermic or endothermic, and free-living or parasitic.

  13. Working with Oligonucleotide Arrays.

    Carvalho, Benilton S

    2016-01-01

    Preprocessing microarray data consists of a number of statistical procedures that convert the observed intensities into quantities that represent biological events of interest, like gene expression and allele-specific abundances. Here, we present a summary of the theory behind microarray data preprocessing for expression, whole transcriptome and SNP designs and focus on the computational protocol used to obtain processed data that will be used on downstream analyses. We describe the main features of the oligo Bioconductor package, an application designed to support oligonucleotide microarrays using the R statistical environment and the infrastructure provided by Bioconductor, allowing the researcher to handle probe-level data and interface with advanced statistical tools under a simplified framework. We demonstrate the use of the package by preprocessing data originated from three different designs. PMID:27008013

  14. Numerical Response of the Common Buzzard Buteo Buteo to The Changes In Abundance Of Small Mammals

    Tóth László

    2014-01-01

    I investigated the numerical response of the Common Buzzard to variations in density of small mammals. The study was carried out at the Hortobágy region in 2000-2001. During nest visiting periods clutch size, number of hatched and fledged young were recorded. Population of small mammals were also monitored by live-trapping. Effect of weather on the survival of overwintering rodents was also investigated. There was significant difference in clutch size between 2000 and 2001 (means 2.3 and 3.1)...

  15. The Origin of the Metal-Poor Common Proper Motion Pair HD 134439/134440: Insights from New Elemental Abundances

    Chen, Yu; Boesgaard, Ann M

    2014-01-01

    The low [alpha/Fe] ratio in the metal-poor ([Fe/H]= -1.50) common proper motion pair HD 134439 and HD 134440 has been variously attributed to chemical evolution in an extragalactic environment with an irregular star formation history, planetessimal accretion, and formation in an environment with an unusually high dust-to-gas ratio. We explore these various putative origins using CNO, Be, Ag, and Eu abundances derived from high-resolution near-UV Keck/HIRES spectroscopy. While we confirm a previously suggested correlation between elemental abundance ratios and condensation temperature at the 95% confidence level, these ratios lie within the continuum of values manifested by extant dSph data. We argue that the most plausible origin of our stars' distinctive abundance distribution relative to the Galactic halo field is formation in an environment chemically dominated by products of Type II SN of low progenitor mass; such a progenitor mass bias has been previously suggested as an explanation of low alpha-element ...

  16. Peptide-LNA oligonucleotide conjugates

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

    2013-01-01

    Although peptide-oligonucleotide conjugates (POCs) are well-known for nucleic acids delivery and therapy, reports on internal attachment of peptides to oligonucleotides are limited in number. To develop a convenient route for preparation of internally labeled POCs with improved biomedical...

  17. Fully automated parallel oligonucleotide synthesizer

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

    2001-01-01

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

  18. VirOligo: a database of virus-specific oligonucleotides

    Onodera, Kenji; Melcher, Ulrich

    2002-01-01

    VirOligo is a database of virus-specific oligonucleotides. The VirOligo database consists of two tables, Common data and Oligo data. The Oligo data table contains PCR primers and hybridization probes used for detection of viral nucleic acids and the Common data table contains the experimental conditions used in their detection. Each oligonucleotide entry contains links to PubMed, GenBank, NCBI Taxonomy databases and BLAST. As of July 2001, the VirOligo database contains a complete listing of ...

  19. Oligonucleotide recombination in corynebacteria without the expression of exogenous recombinases.

    Krylov, Alexander A; Kolontaevsky, Egor E; Mashko, Sergey V

    2014-10-01

    Brevibacterium lactofermentum and Corynebacterium glutamicum are important biotechnology species of the genus Corynebacterium. The single-strand DNA annealing protein (SSAP)-independent oligonucleotide-mediated recombination procedure was successfully applied to the commonly used wild-type strains B. lactofermentum AJ1511 and C. glutamicum ATCC13032. When the rpsL gene was used as a target, the optimized protocol yielded up to (1.4±0.3)×10(3) and (6.7±1.3)×10(3) streptomycin-resistant colonies per 10(8) viable cells for the corresponding strains. We tested the influence of several parameters that are known to enhance the efficiency of oligonucleotide-mediated recombination in other bacterial species. Among them, increasing the concentration of oligonucleotides and targeting the lagging strand of the chromosome have proven to have positive effects on both of the tested species. No difference in the efficiency of recombination was observed between the oligonucleotides phosphorothiorated at the 5' ends and the unmodified oligonucleotides or between the oligonucleotides with four mutated nucleotides and those with one mutated nucleotide. The described approach demonstrates that during the adaptation of the recombineering technique, testing SSAP-independent oligonucleotide-mediated recombination could be a good starting point. Such testing could decrease the probability of an incorrect interpretation of the effect of exogenous protein factors (such as SSAP and/or corresponding exonucleases) due to non-optimal experimental conditions. In addition, SSAP-independent recombination itself could be useful in combination with suitable selection/enrichment methods. PMID:25087479

  20. Gene expression profiling in peanut using high density oligonucleotide microarrays

    Burow Mark

    2009-06-01

    Full Text Available Abstract Background Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology. Results We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B, oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes. Conclusion The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues.

  1. Thermodynamics of Oligonucleotide Duplex Melting

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-01-01

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

  2. Stable dye-labelled oligonucleotide-nanoparticle conjugates for nucleic acid detection

    Barrett, Lee; Dougan, Jennifer A.; Faulds, Karen; Graham, Duncan

    2011-08-01

    Metallic nanoparticles functionalized with oligonucleotides are used for a number of nucleic acid detection strategies. However, oligonucleotide-nanoparticle conjugates suffer from a lack of stability when exposed to certain conditions associated with DNA detection assays. In this study, we report the synthesis of thiol and thioctic acid-modified oligonucleotide gold nanoparticle (OGNs) conjugates functionalized with a dye label and varying spacer groups. The thioctic acid-modified conjugates exhibit increased stability when treated with dithiothreitol (DTT) compared to the more commonly used thiol modification. When the dye labelled oligonucleotide nanoparticle conjugates are exposed to the same conditions there is a pronounced increase in the stability for both thioctic acid and thiol modified sequences. These results open up the possibility of simply using a dye label to enhance the stability of oligonucleotide-nanoparticle conjugates in DNA detection assays where the enhanced stability of the conjugate system can be advantageous in more complex biological environments.

  3. Gene cloning based on long oligonucleotide probes

    The most commonly used technique for gene cloning has been to utilize oligonucleotide probe based on protein sequence data. Of course this approach requires characterized and purified protein so that at least a portion of amino acid sequence can be determined and used to infer the corresponding DNA sequence. Based on the amino acid sequence information, either short or long oligonucleotide probes can be synthesized chemically. Long probes are typically 30-100 nucleotides long and are a single sequence based on a best guess for each codon. The long probe approach was first used to screen for three different genes: bovine trypsin inhibitor, human insulin-like growth factor I, and human factor IX. There are three advantages of long probes. (1) Any stretch of amino acid sequence 10 or longer can be used. (2) The amino acid sequence need not be absolutely correct. (3) These probes can be used to screen high-complexity libraries with fewer false positives. In spite of the uncertainties over codon selection, the long probe approach is currently the method of choice in screening for genes based on protein sequence data

  4. [Study toward practical use of oligonucleotide therapeutics].

    Inoue, Takao; Yoshida, Tokuyuki

    2014-01-01

    Over the past decade, oligonucleotide-based therapeutics such as antisense oligonucleotides and small interfering RNAs (siRNAs) have been developed extensively. For example, mipomersen (Kynamro; ISIS Pharmaceuticals), which is a second-generation antisense oligonucleotide administered by subcutaneous injection, has recently been approved by the FDA for the treatment of homozygous familial hypercholesterolemia. On the other hands, methods for the evaluation of quality, efficacy and safety of oligonucleotide therapeutics have not been fully discussed. Furthermore, the regulatory guidance specific for oligonucleotide therapeutics has not been established yet. Under these circumstances, we started to collaborate with Osaka University and PMDA to discuss regulatory science focused on oligonucleotide therapeutics. Through the collaboration, we would like to propose the possible design of quality evaluation and preclinical safety-evaluation of oligonucleotide therapeutics. PMID:25707197

  5. A group 6 late embryogenesis abundant protein from common bean is a disordered protein with extended helical structure and oligomer-forming properties.

    Rivera-Najera, Lucero Y; Saab-Rincón, Gloria; Battaglia, Marina; Amero, Carlos; Pulido, Nancy O; García-Hernández, Enrique; Solórzano, Rosa M; Reyes, José L; Covarrubias, Alejandra A

    2014-11-14

    Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association. PMID:25271167

  6. Targeted Delivery of a Splice-Switching Oligonucleotide by Cationic Polyplexes of RGD-Oligonucleotide Conjugate

    Ming, Xin; Feng, Lan

    2012-01-01

    Nanoparticle-based delivery has become an important strategy to advance therapeutic oligonucleotides into clinical reality. Delivery by nanocarriers can enhance access of oligonucleotides to their pharmacological targets within cells; preferably, targeting ligands are incorporated into nanoparticles for targeting oligonucleotides to disease sites, often by conjugation to delivery carriers. In this study, a splice-switching oligonucleotide (SSO) was conjugated to a bivalent RGD peptide, and th...

  7. Common Carp Abundance, Biomass, and Removal from Dewey and Clear Lakes on the Valentine National Wildlife Refuge: Does Trapping and Removing Carp Payoff?

    US Fish and Wildlife Service, Department of the Interior — Common carp Cyprinus carpio is a nonnative invasive nuisance species to North America. Many authors have documented the detrimental affects of common carp invasions...

  8. Rapid bacterial identification using evanescent-waveguide oligonucleotide microarray classification.

    Francois, Patrice; Charbonnier, Yvan; Jacquet, Jean; Utinger, Dominic; Bento, Manuela; Lew, Daniel; Kresbach, Gerhard M; Ehrat, Markus; Schlegel, Werner; Schrenzel, Jacques

    2006-06-01

    Bacterial identification relies primarily on culture-based methodologies and requires 48-72 h to deliver results. We developed and used i) a bioinformatics strategy to select oligonucleotide signature probes, ii) a rapid procedure for RNA labelling and hybridization, iii) an evanescent-waveguide oligoarray with exquisite signal/noise performance, and iv) informatics methods for microarray data analysis. Unique 19-mer signature oligonucleotides were selected in the 5'-end of 16s rDNA genes of human pathogenic bacteria. Oligonucleotides spotted onto a Ta(2)O(5)-coated microarray surface were incubated with chemically labelled total bacterial RNA. Rapid hybridization and stringent washings were performed before scanning and analyzing the slide. In the present paper, the eight most abundant bacterial pathogens representing >54% of positive blood cultures were selected. Hierarchical clustering analysis of hybridization data revealed characteristic patterns, even for closely related species. We then evaluated artificial intelligence-based approaches that outperformed conventional threshold-based identification schemes on cognate probes. At this stage, the complete procedure applied to spiked blood cultures was completed in less than 6 h. In conclusion, when coupled to optimal signal detection strategy, microarrays provide bacterial identification within a few hours post-sampling, allowing targeted antimicrobial prescription. PMID:16216356

  9. A short review of the distribution of short-beaked common dolphins (Delphinus delphis in the central and eastern North Atlantic with an abundance estimate for part of this area

    Ana Cañadas

    2013-10-01

    Full Text Available This paper uses data from 3 programmes: (1 the North Atlantic Sightings Surveys (NASS surveys undertaken throughout much of the central and eastern North Atlantic north of about 40° N in 1987, 1989, 1995 and 2001; (2 the MICA-93 programme; and (3 the north eastern Atlantic segment of the Small Cetacean Abundance in the North Sea (SCANS survey in 1994. The data from all surveys were used to examine the distribution of common dolphins in the NE Atlantic. No sightings were made north of 57° N. An initial attempt to examine distribution against 4 potential non biological explanatory variables was made. A simple interpretation of the preliminary analyses presented here is that the primary areas for groups of common dolphins were in waters over 15° C and depths of 400-1,000 m (there does appear a link with shelf features, between around 49°-55° N especially between 20°-30°W. An illustrative example of spatial modelling is presented. Only for 1 year (and part of the total survey area were there sufficient data to attempt to estimate abundance: 1995. The estimated abundance in the W Block of the NASS-95 Faroese survey was 273,159 (cv=0.26; 95% CI=153,392-435,104 short-beaked common dolphins. This estimate is corrected for animals missed on the trackline (g(0 and for responsive movement.

  10. Maximum abundant isotopes correlation

    The neutron excess of the most abundant isotopes of the element shows an overall linear dependence upon the neutron number for nuclei between neutron closed shells. This maximum abundant isotopes correlation supports the arguments for a common history of the elements during nucleosynthesis. (Auth.)

  11. Synthesis of 5'-Aldehyde Oligonucleotide.

    Lartia, Rémy

    2016-01-01

    Synthesis of oligonucleotide ending with an aldehyde functional group at their 5'-end (5'-AON) is possible for both DNA (5'-AODN) and RNA (5'-AORN) series irrespectively of the nature of the last nucleobase. The 5'-alcohol of on-support ODN is mildly oxidized under Moffat conditions. Transient protection of the resulting aldehyde by N,N'-diphenylethylenediamine derivatives allows cleavage, deprotection, and RP-HPLC purification of the protected 5'-AON. Finally, 5'-AON is deprotected by usual acetic acid treatment. In the aggregates, 5'-AON can be now synthesized and purified as routinely as non-modified ODNs, following procedures similar to the well-known "DMT-On" strategy. PMID:26967469

  12. Differentiation of regions with atypical oligonucleotide composition in bacterial genomes

    Reva Oleg N

    2005-10-01

    Full Text Available Abstract Background Complete sequencing of bacterial genomes has become a common technique of present day microbiology. Thereafter, data mining in the complete sequence is an essential step. New in silico methods are needed that rapidly identify the major features of genome organization and facilitate the prediction of the functional class of ORFs. We tested the usefulness of local oligonucleotide usage (OU patterns to recognize and differentiate types of atypical oligonucleotide composition in DNA sequences of bacterial genomes. Results A total of 163 bacterial genomes of eubacteria and archaea published in the NCBI database were analyzed. Local OU patterns exhibit substantial intrachromosomal variation in bacteria. Loci with alternative OU patterns were parts of horizontally acquired gene islands or ancient regions such as genes for ribosomal proteins and RNAs. OU statistical parameters, such as local pattern deviation (D, pattern skew (PS and OU variance (OUV enabled the detection and visualization of gene islands of different functional classes. Conclusion A set of approaches has been designed for the statistical analysis of nucleotide sequences of bacterial genomes. These methods are useful for the visualization and differentiation of regions with atypical oligonucleotide composition prior to or accompanying gene annotation.

  13. A Tandem Oligonucleotide Approach for SNP-Selective RNA Degradation Using Modified Antisense Oligonucleotides

    Magner, Dorota; Biala, Ewa; Lisowiec-Wachnicka, Jolanta; Kierzek, Elzbieta; Kierzek, Ryszard

    2015-01-01

    Antisense oligonucleotides have been studied for many years as a tool for gene silencing. One of the most difficult cases of selective RNA silencing involves the alleles of single nucleotide polymorphisms, in which the allele sequence is differentiated by a single nucleotide. A new approach to improve the performance of allele selectivity for antisense oligonucleotides is proposed. It is based on the simultaneous application of two oligonucleotides. One is complementary to the mutated form of...

  14. Abundance distribution of common and rare plant species of Brazilian savannas along a seasonality gradient Distribuição de abundâncias de espécies de plantas comuns e raras de savanas brasileiras ao longo de um gradiente de estacionalidade

    Igor Aurélio Silva

    2010-06-01

    Full Text Available We examined the species abundance distribution (SAD of plant communities in: (1 a wet grassland, waterlogged throughout most of the year; (2 a seasonal savanna, with an annual dry season; and (3 a hyperseasonal savanna, with alternating drought and waterlogging over the year. We searched for differences in the abundance distributions of all species, as well as of the common and rare species. We tested whether the SADs fitted the lognormal, log-series, power fraction, and random assortment models. We found that environmental constraints may reduce the evenness of plant communities and change the SADs in savannas. We observed a lognormal abundance distribution in the wet grassland and a random abundance distribution in the hyperseasonal cerrado. The SAD of the seasonal savanna did not follow any model. The common species in the three communities were better fitted by the lognormal model. The rare species in the wet grassland and the hyperseasonal cerrado were better fitted by the random assortment model. The SAD of the rare species of the seasonal savanna did not follow any model. Seasonality seems to modify the lognormal distribution of the overall plant community, generating abundance distributions indistinguishable from random. However, differential community structuring between common and rare species may not be affected by seasonality. The different signatures of the abundance distributions of common and rare plants indicate that composite models are better predictors for SADs in savannas.Examinamos as distribuições de abundâncias de espécies (DAEs de comunidades de plantas em: (1 um campo úmido, alagado durante a maior parte do ano; (2 uma savana estacional, com uma estação seca anual; e (3 uma savana hiper-estacional, com uma estação seca e um alagamento alternantes durante o ano. Procuramos por diferenças na distribuição de abundância de todas as espécies, bem como das espécies comuns e raras. Testamos se as DAEs se

  15. Antisense oligonucleotides as therapeutics for malignant diseases.

    Ho, P T; Parkinson, D R

    1997-04-01

    The continued progress in our understanding of the biology of neoplasia and in the identification, cloning, and sequencing of genes critical to tumor cell function permits the exploitation of this information to develop specific agents that may directly modulate the function of these genes or their protein products. Antisense oligonucleotides are being investigated as a potential therapeutic modality that takes direct advantage of molecular sequencing. The antisense approach uses short oligonucleotides designed to hybridize to a target mRNA transcript through Watson-Crick base pairing. The formation of this oligonucleotide: RNA heteroduplex results in mRNA inactivation and consequent inhibition of synthesis of the protein product. A fundamental attraction of the antisense approach is that this method potentially may be applied to any gene product, in theory, for the treatment of malignant and non-malignant diseases. However, this simple and attractive model has proven to be much more complex in practice. A number of important challenges in the preclinical development of antisense oligonucleotides have been identified, including stability, sequence length, cellular uptake, target sequence selection, appropriate negative controls, oligonucleotide: protein interactions, and cost of manufacture. Although the biological activity of an oligonucleotide against its molecular target is theoretically sequence-dependent, the animal pharmacokinetics and toxicology of phosphorothioate analogues directed against vastly disparate gene products appear relatively non-sequence-specific. In oncology, a number of clinical trials have been initiated with antisense oligonucleotides directed against molecular targets including: p53; bcl-2; raf kinase; protein kinase C-alpha; c-myb. The experience gained from these early clinical trials will be applicable to the next generation of antisense agents in development. These may include molecules with novel backbones or other structural

  16. Construction and Evaluation of Desulfovibrio vulgaris Whole-Genome Oligonucleotide Microarrays

    Z. He; Q. He; L. Wu; M.E. Clark; J.D. Wall; Jizhong Zhou; Matthew W. Fields

    2004-03-17

    ,4-cyclodiphosphate (MECDP) synthase. Spermidines are polyamines that are typically abundant in rapidly dividing cells and are essential growth factors in eukaryotic organisms. Polyamines are thought to stabilize DNA by the association of the amino groups with the phosphate residues of DNA and can also enhance tRNA and ribosome stability. The MECDP synthase enzyme is essential in Escherichia coli and participates in the nonmevalonate pathway of isoprenoid biosynthesis, a critical pathway present in some bacteria and apicomplexans but distinct from that used by mammals. Several of the highly up-regulated ORFs were annotated as conserved hypothetical proteins. Interestingly, an ORF that was predicted to contain a flocculin repeat domain was almost 9-fold up-regulated in stationary phase cells compared to logarithmically growing cells. The flocculin domain is commonly observed in fungi, and is thought to play a role during flocculation (non-sexual aggregation of single-cell microorganisms). These preliminary results have identified possible responses of D. vulgaris cells to stationary phase growth and suggest that polyamine production as well as cell aggregation and/or extracellular polymer production are responses of D. vulgaris during stationary phase. The initial microarray results indicate that the recently produced oligonucleotide microarrays are functional. We are currently optimizing growth conditions in order to culture D. vulgaris cells in the presence of uranium(VI) and to monitor whole-genome expression levels.

  17. Profound effect of normalization on detection of differentially expressed genes in oligonucleotide microarray data analysis

    Hoffmann, Reinhard; Seidl, Thomas; Dugas, Martin

    2002-01-01

    Background Oligonucleotide microarrays measure the relative transcript abundance of thousands of mRNAs in parallel. A large number of procedures for normalization and detection of differentially expressed genes have been proposed. However, the relative impact of these methods on the detection of differentially expressed genes remains to be determined. Results We have employed four different normalization methods and all possible combinations with three different statistical algorithms for det...

  18. Oligonucleotide-based therapy for neurodegenerative diseases.

    Magen, Iddo; Hornstein, Eran

    2014-10-10

    Molecular genetics insight into the pathogenesis of several neurodegenerative diseases, such as Alzheimer׳s disease, Parkinson׳s disease, Huntington׳s disease and amyotrophic lateral sclerosis, encourages direct interference with the activity of neurotoxic genes or the molecular activation of neuroprotective pathways. Oligonucleotide-based therapies are recently emerging as an efficient strategy for drug development and these can be employed as new treatments of neurodegenerative states. Here we review advances in this field in recent years which suggest an encouraging assessment that oligonucleotide technologies for targeting of RNAs will enable the development of new therapies and will contribute to preservation of brain integrity. PMID:24727531

  19. Common Tern - Avian Average Annual Abundance

    National Oceanic and Atmospheric Administration, Department of Commerce — The data represent predicted number of individuals of each listed seabird species per standardized survey segment (15 minute travel time at 10 knots = approx. 2.5...

  20. Common Eider - Avian Average Annual Abundance

    National Oceanic and Atmospheric Administration, Department of Commerce — The data represent predicted number of individuals of each listed seabird species per standardized survey segment (15 minute travel time at 10 knots = approx. 2.5...

  1. Common Loon - Avian Average Annual Abundance

    National Oceanic and Atmospheric Administration, Department of Commerce — The data represent predicted number of individuals of each listed seabird species per standardized survey segment (15 minute travel time at 10 knots = approx. 2.5...

  2. Radio-marking and in vivo imagery of oligonucleotides

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

  3. Synthesis of oligonucleotide conjugates carrying viologen and fluorescent compounds

    Alvira, Margarita; Quinn, Susan J.; Aviñó, Anna; Fitzmaurice, Donald; Eritja Casadellà, Ramón

    2008-01-01

    The preparation of oligonucleotide conjugates carrying viologen and fluorescein is described. Reaction of the appropriate carboxyl derivatives with oligonucleotides carrying aliphatic amino groups gave the desired compounds. A simple method for the introduction of the amino group at the 5'-end of the oligonucleotides is reported.

  4. In vivo imaging of oligonucleotidic aptamers

    Tavitian, B.; Boisgard, R. [Inserm U803, Laboratoire d' Imagerie Moleculaire Experimentale, Service hospitalier Frederic joliot, Intitut d' Imagerie Biomedicale, CEA, Orsay (France); Duconge, F.; Dolle, F. [Groupe de Radiochimie, Laboratoire d' Imagerie Moleculaire Experimentale, Service hospitalier Frederic joliot, Intitut d' Imagerie Biomedicale, CEA, Orsay (France)

    2009-07-01

    In this chapter we present the methods developed in our laboratory for in vivo imaging of oligonucleotidic aptamers. These methods relate to (i) the labelling of aptamers with fluorine-18, a positron emitter (ii) Positron Emission Tomography imaging of laboratory animals with [({sup 18})F]aptamers and (iii) labelling with fluorescent dyes and optical imaging of aptamers in mice. (authors)

  5. Triplex-forming ability of modified oligonucleotides

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

    2007-01-01

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

  6. Comparison of small molecules and oligonucleotides that target a toxic, non-coding RNA.

    Costales, Matthew G; Rzuczek, Suzanne G; Disney, Matthew D

    2016-06-01

    Potential RNA targets for chemical probes and therapeutic modalities are pervasive in the transcriptome. Oligonucleotide-based therapeutics are commonly used to target RNA sequence. Small molecules are emerging as a modality to target RNA structures selectively, but their development is still in its infancy. In this work, we compare the activity of oligonucleotides and several classes of small molecules that target the non-coding r(CCUG) repeat expansion (r(CCUG)(exp)) that causes myotonic dystrophy type 2 (DM2), an incurable disease that is the second-most common cause of adult onset muscular dystrophy. Small molecule types investigated include monomers, dimers, and multivalent compounds synthesized on-site by using RNA-templated click chemistry. Oligonucleotides investigated include phosphorothioates that cleave their target and vivo-morpholinos that modulate target RNA activity via binding. We show that compounds assembled on-site that recognize structure have the highest potencies amongst small molecules and are similar in potency to a vivo-morpholino modified oligonucleotide that targets sequence. These studies are likely to impact the design of therapeutic modalities targeting other repeats expansions that cause fragile X syndrome and amyotrophic lateral sclerosis, for example. PMID:27117425

  7. Electrochemical behavior of G-rich oligonucleotides

    Havran, Luděk; Vidláková, Pavlína; Pivoňková, Hana; Kejnovská, Iva; Vorlíčková, Michaela; Fojta, Miroslav

    Beijing, 2009. s. 1. [The 60th Annual Meeting of the International Society of Electrochemistry. 16.08.2009-21.08.2009, Beijing] R&D Projects: GA AV ČR(CZ) IAA400040903; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : oligonucleotides * electrochemical behavior * DNA secondary structures Subject RIV: BO - Biophysics

  8. Electrochemical behavior of oligonucleotides containing guanine stretches

    Havran, Luděk; Vidláková, Pavlína; Pivoňková, Hana; Kejnovská, Iva; Vorlíčková, Michaela; Fojta, Miroslav

    Jětřichovice, 2009. s. 33. ISBN 978-80-254-3997-5. [XXIX. Moderní elektrochemické metody. 25.05.2009-29.05.2009, Jetřichovice] R&D Projects: GA AV ČR(CZ) IAA400040903; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : oligonucleotides * electrochemical behavior * DNA secondary structures Subject RIV: BO - Biophysics

  9. Application of heteronuclear couplings to conformational analysis of oligonucleotides

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

    1994-12-01

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

  10. AFM phase lag mapping for protein-DNA oligonucleotide complexes

    Atomic force microscope phase lag imaging of protein-DNA oligonucleotide complexes has been performed to visualize the immobilized oligonucleotides on the protein surface. In normal sample conditions, neither the topographic nor phase lag images show any discriminate signals for the immobilized oligonucleotides. Use of a highly humid incubator, controls the surface humidity of the sample. Thereby, the phase lag image reveals the oligonucleotide location by the local difference of tip adhesion distribution. The resultant phase lag image shows extremely strong signals in the center of the protein surface, indicating the location of the oligonucleotides with resolution better than 20 nm. The signal frequency was strongly influenced by the used oligonucleotide concentration in the range 5 nM-50 μM

  11. Dendritische Polythioetherliganden zur Synthese von Goldnanopartikel-Oligonucleotid-Monokonjugaten

    Mönninghoff, Sven

    2009-01-01

    Basierend auf der Entwicklung des Rubigoldes als thermostabilem Marker für Biomoleküle wurden in dieser Arbeit Polythioether-Liganden zur Synthese von monofunktionalen Cluster-Oligonucleotid-Konjugaten (COK) entwickelt. Zum Aufbau der Liganden wurden 2,4,6-substituierte Phenole als zentrales Strukturmotiv eingesetzt. Durch Konjugation der Ligandensysteme an Oligonucleotide wurden Ligand-Oligonucleotid-Konjugate (LOK) hergestellt, die durch Reduktion von Au (III)-Ionen in Anwesenhe...

  12. Investigations of oligonucleotide usage variance within and between prokaryotes

    Bohlin, J.; Skjerve, E.; Ussery, David

    2008-01-01

    different DNA 'word-sizes' and explore how oligonucleotide frequencies differ in coding and non-coding regions. In addition, we used oligonucleotide frequencies to investigate DNA composition and how DNA sequence patterns change within and between prokaryotic organisms. Among the results found was that...... prokaryotic chromosomes can be described by hexanucleotide frequencies, suggesting that prokaryotic DNA is predominantly short range correlated, i. e., information in prokaryotic genomes is encoded in short oligonucleotides. Oligonucleotide usage varied more within AT-rich and host-associated genomes than in...

  13. Antisense Oligonucleotide-Mediated Exon Skipping for Duchenne Muscular Dystrophy: Progress and Challenges.

    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...

  14. Optically Triggered Immune Response through Photocaged Oligonucleotides

    Govan, Jeane M.; Young, Douglas D.; Lively, Mark O.

    2015-01-01

    Bacterial and viral CpG oligonculeotides are unmethylated cytosine-phosphate-guanosine dinucleotide sequences and trigger an innate immune response through activation of the toll-like receptor 9 (TLR9). We have developed synthetic photocaged CpGs via site-specific incorporation of nitropiperonyloxymethyl (NPOM)-caged thymidine residues. These oligonucleotides enable the optical control of TLR9 function and thereby provide light-activation of an immune response. We provide a proof-of-concept model by applying a reporter assay in live cells and by quantification of endogenous production of interleukin 6. PMID:26034339

  15. Bioconjugation of oligonucleotides for treating liver fibrosis.

    Ye, Zhaoyang; Houssein, Houssam S Hajj; Mahato, Ram I

    2007-01-01

    Liver fibrosis results from chronic liver injury due to hepatitis B and C, excessive alcohol ingestion, and metal ion overload. Fibrosis culminates in cirrhosis and results in liver failure. Therefore, a potent antifibrotic therapy is urgently needed to reverse scarring and eliminate progression to cirrhosis. Although activated hepatic stellate cells (HSCs) remain the principle cell type responsible for liver fibrosis, perivascular fibroblasts of portal and central veins as well as periductular fibroblasts are other sources of fibrogenic cells. This review will critically discuss various treatment strategies for liver fibrosis, including prevention of liver injury, reduction of inflammation, inhibition of HSC activation, degradation of scar matrix, and inhibition of aberrant collagen synthesis. Oligonucleotides (ODNs) are short, single-stranded nucleic acids, which disrupt expression of target protein by binding to complementary mRNA or forming triplex with genomic DNA. Triplex forming oligonucleotides (TFOs) provide an attractive strategy for treating liver fibrosis. A series of TFOs have been developed for inhibiting the transcription of alpha1(I) collagen gene, which opens a new area for antifibrotic drugs. There will be in-depth discussion on the use of TFOs and how different bioconjugation strategies can be utilized for their site-specific delivery to HSCs or hepatocytes for enhanced antifibrotic activities. Various insights developed in individual strategy and the need for multipronged approaches will also be discussed. PMID:18154454

  16. Novel oligonucleotides modified with acyclic nucleoside phosphonate units

    Janeba, Zlatko; Hocková, Dana; Kaiser, Martin Maxmilian; Ménová, Petra; Novák, Pavel; Rosenbergová, Šárka; Páv, Ondřej; Pohl, Radek; Poštová Slavětínská, Lenka; Rosenberg, Ivan

    Newport : Gordon Research Conference, 2015. [Nucleosides, Nucleotides & Oligonucleotides. Gordon Research Conference 2015. 28.06.2015-03.07.2015, Newport] R&D Projects: GA ČR GA13-26526S Institutional support: RVO:61388963 Keywords : nucleotide analogues * modified oligonucleotides Subject RIV: CC - Organic Chemistry

  17. Voltage-gated calcium channel and antisense oligonucleotides thereto

    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.

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

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

    2005-01-01

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

  19. Use of nanoparticles to deliver immunomodulatory oligonucleotides.

    Klinman, Dennis M; Sato, Takashi; Shimosato, Takeshi

    2016-07-01

    Synthetic oligonucleotides (ODNs) containing unmethylated 'CpG motifs' stimulate the innate immune system to produce cytokines, chemokines, and polyreactive antibodies. CpG ODNs have shown promise as vaccine adjuvants and for the treatment of infectious diseases and cancer. The immunostimulatory activity of CpG ODNs is inhibited by DNA-containing 'suppressive' motifs. ODNs expressing suppressive motifs (Sup ODNs) reduce ongoing immune reactions and show promise in the treatment of autoimmune and inflammatory diseases. This work reviews recent progress in the use of nanoparticles as carriers of CpG and Sup ODNs to target their delivery to the GI tract and lungs. WIREs Nanomed Nanobiotechnol 2016, 8:631-637. doi: 10.1002/wnan.1382 For further resources related to this article, please visit the WIREs website. PMID:26663867

  20. Hole hopping rates in single strand oligonucleotides

    Borrelli, Raffaele; Capobianco, Amedeo; Peluso, Andrea

    2014-08-01

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

  1. Preparation and application of triple helix forming oligonucleotides and single strand oligonucleotide donors for gene correction.

    Alam, Rowshon; Thazhathveetil, Arun Kalliat; Li, Hong; Seidman, Michael M

    2014-01-01

    Strategies for site-specific modulation of genomic sequences in mammalian cells require two components. One must be capable of recognizing and activating a specific target sequence in vivo, driving that site into an exploitable repair pathway. Information is transferred to the site via participation in the pathway by the second component, a donor nucleic acid, resulting in a permanent change in the target sequence. We have developed biologically active triple helix forming oligonucleotides (TFOs) as site-specific gene targeting reagents. These TFOs, linked to DNA reactive compounds (such as a cross-linking agent), activate pathways that can engage informational donors. We have used the combination of a psoralen-TFO and single strand oligonucleotide donors to generate novel cell lines with directed sequence changes at the target site. Here we describe the synthesis and purification of bioactive psoralen-linked TFOs, their co-introduction into mammalian cells with donor nucleic acids, and the identification of cells with sequence conversion of the target site. We have emphasized details in the synthesis and purification of the oligonucleotides that are essential for preparation of reagents with optimal activity. PMID:24557899

  2. Ca2+ enrichment in culture medium potentiates effect of oligonucleotides.

    Hori, Shin-Ichiro; Yamamoto, Tsuyoshi; Waki, Reiko; Wada, Shunsuke; Wada, Fumito; Noda, Mio; Obika, Satoshi

    2015-10-30

    Antisense and RNAi-related oligonucleotides have gained attention as laboratory tools and therapeutic agents based on their ability to manipulate biological events in vitro and in vivo. We show that Ca(2+) enrichment of medium (CEM) potentiates the in vitro activity of multiple types of oligonucleotides, independent of their net charge and modifications, in various cells. In addition, CEM reflects in vivo silencing activity more consistently than conventional transfection methods. Microscopic analysis reveals that CEM provides a subcellular localization pattern of oligonucleotides resembling that obtained by unassisted transfection, but with quantitative improvement. Highly monodispersed nanoparticles ~100 nm in size are found in Ca(2+)-enriched serum-containing medium regardless of the presence or absence of oligonucleotides. Transmission electron microscopy analysis reveals that the 100-nm particles are in fact an ensemble of much smaller nanoparticles (ϕ ∼ 15 nm). The presence of these nanoparticles is critical for the efficient uptake of various oligonucleotides. In contrast, CEM is ineffective for plasmids, which are readily transfected via the conventional calcium phosphate method. Collectively, CEM enables a more accurate prediction of the systemic activity of therapeutic oligonucleotides, while enhancing the broad usability of oligonucleotides in the laboratory. PMID:26101258

  3. Design and analysis of mismatch probes for long oligonucleotide microarrays

    Deng, Ye; He, Zhili; Van Nostrand, Joy D.; Zhou, Jizhong

    2008-08-15

    Nonspecific hybridization is currently a major concern with microarray technology. One of most effective approaches to estimating nonspecific hybridizations in oligonucleotide microarrays is the utilization of mismatch probes; however, this approach has not been used for longer oligonucleotide probes. Here, an oligonucleotide microarray was constructed to evaluate and optimize parameters for 50-mer mismatch probe design. A perfect match (PM) and 28 mismatch (MM) probes were designed for each of ten target genes selected from three microorganisms. The microarrays were hybridized with synthesized complementary oligonucleotide targets at different temperatures (e.g., 42, 45 and 50 C). In general, the probes with evenly distributed mismatches were more distinguishable than those with randomly distributed mismatches. MM probes with 3, 4 and 5 mismatched nucleotides were differentiated for 50-mer oligonucleotide probes hybridized at 50, 45 and 42 C, respectively. Based on the experimental data generated from this study, a modified positional dependent nearest neighbor (MPDNN) model was constructed to adjust the thermodynamic parameters of matched and mismatched dimer nucleotides in the microarray environment. The MM probes with four flexible positional mismatches were designed using the newly established MPDNN model and the experimental results demonstrated that the redesigned MM probes could yield more consistent hybridizations. Conclusions: This study provides guidance on the design of MM probes for long oligonucleotides (e.g., 50 mers). The novel MPDNN model has improved the consistency for long MM probes, and this modeling method can potentially be used for the prediction of oligonucleotide microarray hybridizations.

  4. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    Nolan, John P.; White, P. Scott

    2006-12-26

    Methods for rapidly detecting single or multiple sequence alleles in a sample nucleic acid are described. Provided are all of the oligonucleotide pairs capable of annealing specifically to a target allele and discriminating among possible sequences thereof, and ligating to each other to form an oligonucleotide complex when a particular sequence feature is present (or, alternatively, absent) in the sample nucleic acid. The design of each oligonucleotide pair permits the subsequent high-level PCR amplification of a specific amplicon when the oligonucleotide complex is formed, but not when the oligonucleotide complex is not formed. The presence or absence of the specific amplicon is used to detect the allele. Detection of the specific amplicon may be achieved using a variety of methods well known in the art, including without limitation, oligonucleotide capture onto DNA chips or microarrays, oligonucleotide capture onto beads or microspheres, electrophoresis, and mass spectrometry. Various labels and address-capture tags may be employed in the amplicon detection step of multiplexed assays, as further described herein.

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

    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

  6. Optical Characterization of Oligonucleotide DNA Influenced by Magnetic Fields

    Seyedeh Maryam Banihashemian

    2013-09-01

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

  7. Sequence-dependent theory of oligonucleotide hybridization kinetics

    Marimuthu, Karthikeyan [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Chakrabarti, Raj, E-mail: raj@pmc-group.com, E-mail: rajc@andrew.cmu.edu [Department of Chemical Engineering and Center for Advanced Process Decision-Making, Carnegie Mellon University, Pittsburgh, Pennsylvania 15213 (United States); Division of Fundamental Research, PMC Advanced Technology, Mount Laurel, New Jersey 08054 (United States)

    2014-05-07

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

  8. Oligonucleotide-based theranostic nanoparticles in cancer therapy.

    Shahbazi, Reza; Ozpolat, Bulent; Ulubayram, Kezban

    2016-05-01

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

  9. The nonenzymatic template-directed ligation of oligonucleotides

    A.V. Lutay

    2006-01-01

    Full Text Available The nonenzymatic template-directed ligation of oligonucleotides containing 2',3'-cyclic phosphate was investigated in the presence of divalent cations. Ligation of the oligonucleotides readily occurred in the presence of Mg2+, Mn2+, Co2+, Zn2+, Pb2+. Efficacy of the metal ion catalysts inversely correlated with pKa values of the metal-bound water molecules. The intermolecular transesterification reaction yielded at least 95% of 2',5'-phosphodiester bonds independently on the nature of the metal ion. Relatively high reaction yields (up to 15% suggest, that RNA fragmentation to oligonucleotides with 2',3'-cyclic phosphates, followed by reactions of those oligonucleotides could provide a source of new RNA molecules under prebiotic conditions.

  10. Synthesis and applications of oligonucleotides containing 2'-modified nucleosides

    Shelbourne, Montserrat

    2012-01-01

    This thesis describes the synthesis and applications of chemically modified oligonucleotides, principally those containing modifications at the 2?-position of ribose. One application is their use in triplex-forming oligonucleotides (TFOs). DNA triplexes are formed by the binding of a TFO to a DNA duplex. TFOs are potential therapeutic agents against cancer and viral infections. TFOs containing 2?-aminoethoxy-T and 5-MeC were shown by UV melting studies to strongly stabilise parallel triple...

  11. Electron migration in oligonucleotides upon γ-irradiation in solution

    Electron migration in irradiated solutions of DNA was investigated using 5-bromouracil synthetically incorporated into oligonucleotides of defined base composition as a molecular indicator of electron interactions. Solvated electrons interact quantitatively with 5-bromouracil, leading to a highly reactive 5-yl radical which can abstract an adjacent hydrogen atom to yield uracil. Yields of uracil, or loss of 5-bromouracil, from irradiated oligonucleotide samples were measured using gas chromatography-mass spectrometric analysis of their trimethylsilylated acid hydrolysates. (author)

  12. Portable System for Microbial Sample Preparation and Oligonucleotide Microarray Analysis

    Bavykin, Sergei G.; Akowski, James P.; Zakhariev, Vladimir M.; Barsky, Viktor E.; Perov, Alexander N.; Mirzabekov, Andrei D.

    2001-01-01

    We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the ...

  13. Sequencing of oligonucleotide phosphorothioates based on solid-supported desulfurization.

    Wyrzykiewicz, T K; Cole, D L

    1994-01-01

    We described a solid-supported desulfurization procedure allowing easy access to the sequence analysis of oligonucleotide phosphorothioates. The described method is based upon selective removal of the 2-cyanoethyl phosphate protecting groups, followed by iodine-promoted desulfurization of the resulting phosphorothioate diesters. Automatic oxidation of oligonucleotide phosphorothioates, anchored via an ester linkage to a standard solid support (LCAA/CPG), is combined with Maxam-Gilbert solid-s...

  14. Oligonucleotide conjugates - Candidates for gene silencing therapeutics.

    Gooding, Matt; Malhotra, Meenakshi; Evans, James C; Darcy, Raphael; O'Driscoll, Caitriona M

    2016-10-01

    The potential therapeutic and diagnostic applications of oligonucleotides (ONs) have attracted great attention in recent years. The capability of ONs to selectively inhibit target genes through antisense and RNA interference mechanisms, without causing un-intended sideeffects has led them to be investigated for various biomedical applications, especially for the treatment of viral diseases and cancer. In recent years, many researchers have focused on enhancing the stability and target specificity of ONs by encapsulating/complexing them with polymers or lipid chains to formulate nanoparticles/nanocomplexes/micelles. Also, chemical modification of nucleic acids has emerged as an alternative to impart stability to ONs against nucleases and other degrading enzymes and proteins found in blood. In addition to chemically modifying the nucleic acids directly, another strategy that has emerged, involves conjugating polymers/peptide/aptamers/antibodies/proteins, preferably to the sense strand (3'end) of siRNAs. Conjugation to the siRNA not only enhances the stability and targeting specificity of the siRNA, but also allows for the development of self-administering siRNA formulations, with a much smaller size than what is usually observed for nanoparticle (∼200nm). This review concentrates mainly on approaches and studies involving ON-conjugates for biomedical applications. PMID:27521696

  15. Oligonucleotide and Long Polymeric DNA Encoding

    Miller, E; Mariella Jr., R P; Christian, A T; Gardner, S N; Williams, J M

    2003-11-24

    This report summarizes the work done at Lawrence Livermore National Laboratory for the Oligonucleotide and Long Polymeric DNA Encoding project, part of the Microelectronic Bioprocesses Program at DARPA. The goal of the project was to develop a process by which long (circa 10,000 base-pair) synthetic DNA molecules could be synthesized in a timely and economic manner. During construction of the long molecule, errors in DNA sequence occur during hybridization and/or the subsequent enzymatic process. The work done on this project has resulted in a novel synthesis scheme that we call the parallel pyramid synthesis protocol, the development of a suit of computational tools to minimize and quantify errors in the synthesized DNA sequence, and experimental proof of this technique. The modeling consists of three interrelated modules: the bioinformatics code which determines the specifics of parallel pyramid synthesis for a given chain of long DNA, the thermodynamics code which tracks the products of DNA hybridization and polymerase extension during the later steps in the process, and the kinetics model which examines the temporal and spatial processes during one thermocycle. Most importantly, we conducted the first successful syntheses of a gene using small starting oligomers (tetramers). The synthesized sequence, 813 base pairs long, contained a 725 base pair gene, modified green fluorescent protein (mGFP), which has been shown to be a functional gene by cloning into cells and observing its green fluorescent product.

  16. Antisense Oligonucleotide Therapy for Inherited Retinal Dystrophies.

    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

  17. Optimizing antisense oligonucleotides using phosphorodiamidate morpholino oligomers.

    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

  18. Hole hopping rates in single strand oligonucleotides

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

    2014-08-31

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

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

    Tripathy, Sushant

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

  20. Abundance of introduced species at home predicts abundance away in herbaceous communities

    Firn, Jennifer; Moore, Joslin L.; MacDougall, Andrew S.; Borer, Elizabeth T.; Seabloom, Eric W.; HilleRisLambers, Janneke; Harpole, W. Stanley; Cleland, Elsa E.; Brown, Cynthia S.; Knops, Johannes M.H.; Prober, Suzanne M.; Pyke, David A.; Farrell, Kelly A.; Bakker, John D.; O'Halloran, Lydia R.; Adler, Peter B.; Collins, Scott L.; D'Antonio, Carla M.; Crawley, Michael J.; Wolkovich, Elizabeth M.; La Pierre, Kimberly J.; Melbourne, Brett A.; Hautier, Yann; Morgan, John W.; Leakey, Andrew D.B.; Kay, Adam; McCulley, Rebecca; Davies, Kendi F.; Stevens, Carly J.; Chu, Cheng-Jin; Holl, Karen D.; Klein, Julia A.; Fay, Phillip A.; Hagenah, Nicole; Kirkman, Kevin P.; Buckley, Yvonne M.

    2011-01-01

    Many ecosystems worldwide are dominated by introduced plant species, leading to loss of biodiversity and ecosystem function. A common but rarely tested assumption is that these plants are more abundant in introduced vs. native communities, because ecological or evolutionary-based shifts in populations underlie invasion success. Here, data for 26 herbaceous species at 39 sites, within eight countries, revealed that species abundances were similar at native (home) and introduced (away) sites - grass species were generally abundant home and away, while forbs were low in abundance, but more abundant at home. Sites with six or more of these species had similar community abundance hierarchies, suggesting that suites of introduced species are assembling similarly on different continents. Overall, we found that substantial changes to populations are not necessarily a pre-condition for invasion success and that increases in species abundance are unusual. Instead, abundance at home predicts abundance away, a potentially useful additional criterion for biosecurity programmes.

  1. Triplex Forming Oligonucleotides against Type α 1(I) Collagen attenuates Liver Fibrosis induced by Bile Duct ligation

    Panakanti, Ravikiran; Pratap, Akshay; Yang, Ningning; JACKSON, JOHN S.; Mahato, Ram I.

    2010-01-01

    Liver fibrosis is a consequence of chronic liver disorders which lead to the accumulation of extracellular matrix (ECM). Particularly, there is an increased accumulation of collagen in the fibrotic liver. We have therefore used a triplex forming oligonucleotide (TFO) against the type α 1 (I) collagen and evaluated, whether it can attenuate liver fibrosis induced by common bile duct ligation (CBDL) in rats. There was a significant decrease in hydroxyproline levels and Masson’s trichrome staini...

  2. Inhibition of hepatitis B virus in vitro by antisense oligonucleotides

    A series of antisense phosphorothioate oligodeoxynucleotides against hepatitis B virus (HBV) were synthesized and evaluated for their antiviral effect in Hep-G2 cells transfected with HBV genome. The inhibitor effect of the tested antisense oligonucleotides was sequence-specific, dose- and time-dependent, and synergistic for certain combinations. In virus-inhibitory concentrations the oligonucleotides were harmless to 2.2.15 cells. The most effective antisense oligonucleotides were found directed against the HBV mRNA transcribed from the cap site of SP II promoter, the portion of polyadenylation signal and the initiation region of gene S, with an inhibition of the HBsAg and HBeAg production by 85 - 95 % and 50 - 60 %, respectively. To our surprise, antisense oligonucleotides directed against three key sites of HBV X gene blocked the expression of HBsAg, HBeAg and HBxAg. This fact might be related to the trans-activation of HBV X protein. Using radioisotope labelling, we demonstrated that Lipofectin promoted the cellular uptake and antiviral effect of antisense oligomers in 2.2.15 cells. These results suggest a therapeutic potential of antisense oligonucleotides in the treatment of patients chronically infected with HBV. (author)

  3. Stability measurements of antisense oligonucleotides by capillary gel electrophoresis.

    Bruin, G J; Börnsen, K O; Hüsken, D; Gassmann, E; Widmer, H M; Paulus, A

    1995-08-11

    The approach of using antisense oligonucleotides as potential drugs is based on hybridization of a short chemically-modified oligonucleotide with complementary cellular DNA or RNA sequences. A critical question is the stability of chemically modified antisense oligonucleotides in cellular environments. In a model system, resistance against various nucleases was evaluated by capillary gel electrophoresis (CGE). For some of the samples, matrix assisted laser desorption and ionization mass spectrometry (MALDI-MS) was used as an additional analytical tool to perform stability measurements. Using CGE, the enzymatic degradation of single nucleotides from the oligomer can be followed after different incubation times. 10% T polyacrylamide gels give baseline resolution for oligonucleotides ranging between 5 and 30 bases in length. The kinetic influence of a specific nuclease concentration and the antisense oligonucleotide structure on the cleavage reaction are discussed. Also, a simple desalting method to improve the injection efficiency and sensitivity of the method are described. Examples of measurements of chemically modified antisense 19-mers are presented. PMID:7581844

  4. Climate and local abundance in freshwater fishes

    Knouft, Jason H; Anthony, Melissa M.

    2016-01-01

    Identifying factors regulating variation in numbers of individuals among populations across a species' distribution is a fundamental goal in ecology. A common prediction, often referred to as the abundant-centre hypothesis, suggests that abundance is highest near the centre of a species' range. However, because of the primary focus on the geographical position of a population, this framework provides little insight into the environmental factors regulating local abundance. While range-wide va...

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

    Goyenvalle Aurélie

    2011-02-01

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

  6. Oligonucleotide probes for the screening of recombinant DNA libraries

    Oligonucleotides of unique sequence are also useful for screening recombinant DNA libraries. The hybridization specificity of oligonucleotide probes allows one to use unique sequences probes to screen for genomic clones or cDNAs encoding a specific number of a multigene family, to screen for a new allele when the sequence of one allele is known, to screen for a specific region of a gene, to screen for specific mutants created by site-directed mutagenesis, or to screen libraries with probes whose sequence represents a consensus coding sequence. This chapter deals with procedures for the use of oligonucleotides as hybridization probes including probe design, labeling, and hybridization to colonies, phage plaques, DNA, and RNA

  7. Delivery of RNAi-Based Oligonucleotides by Electropermeabilization

    Muriel Golzio

    2013-04-01

    Full Text Available For more than a decade, understanding of RNA interference (RNAi has been a growing field of interest. The potent gene silencing ability that small oligonucleotides have offers new perspectives for cancer therapeutics. One of the present limits is that many biological barriers exist for their efficient delivery into target cells or tissues. Electropermeabilization (EP is one of the physical methods successfully used to transfer small oligonucleotides into cells or tissues. EP consists in the direct application of calibrated electric pulses to cells or tissues that transiently permeabilize the plasma membranes, allowing efficient in vitro and in vivo. cytoplasmic delivery of exogenous molecules. The present review reports on the type of therapeutic RNAi-based oligonucleotides that can be electrotransferred, the mechanism(s of their electrotransfer and the technical settings for pre-clinical purposes.

  8. Optimizing the design of oligonucleotides for homology directed gene targeting.

    Judith Miné-Hattab

    Full Text Available BACKGROUND: Gene targeting depends on the ability of cells to use homologous recombination to integrate exogenous DNA into their own genome. A robust mechanistic model of homologous recombination is necessary to fully exploit gene targeting for therapeutic benefit. METHODOLOGY/PRINCIPAL FINDINGS: In this work, our recently developed numerical simulation model for homology search is employed to develop rules for the design of oligonucleotides used in gene targeting. A Metropolis Monte-Carlo algorithm is used to predict the pairing dynamics of an oligonucleotide with the target double-stranded DNA. The model calculates the base-alignment between a long, target double-stranded DNA and a probe nucleoprotein filament comprised of homologous recombination proteins (Rad51 or RecA polymerized on a single strand DNA. In this study, we considered different sizes of oligonucleotides containing 1 or 3 base heterologies with the target; different positions on the probe were tested to investigate the effect of the mismatch position on the pairing dynamics and stability. We show that the optimal design is a compromise between the mean time to reach a perfect alignment between the two molecules and the stability of the complex. CONCLUSION AND SIGNIFICANCE: A single heterology can be placed anywhere without significantly affecting the stability of the triplex. In the case of three consecutive heterologies, our modeling recommends using long oligonucleotides (at least 35 bases in which the heterologous sequences are positioned at an intermediate position. Oligonucleotides should not contain more than 10% consecutive heterologies to guarantee a stable pairing with the target dsDNA. Theoretical modeling cannot replace experiments, but we believe that our model can considerably accelerate optimization of oligonucleotides for gene therapy by predicting their pairing dynamics with the target dsDNA.

  9. Silver and Cyanine Staining of Oligonucleotides in Polyacrylamide Gel

    Tang, Weizhong; Zhou, Huafu; Li, Wei

    2015-01-01

    To explore why some oligonucleotides in denaturing polyacrylamide gel could not be silver-stained, 134 different oligonucleotides were analyzed using denaturing polyacrylamide gel electrophoresis stained with silver and asymmetric cyanine. As a result, we found that the sensitivity of oligos (dA), (dC), (dG) and (dT) to silver staining could be ranged as (dA) > (dG) > (dC) > (dT) from high to low. It was unexpected that oligo (dT) was hard to be silver-stained. Moreover, the silver staining o...

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

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

    2010-11-15

    Surface modification at the nanometer scale is a challenge for the future of molecular electronics. In particular, the precise anchoring and electrical addressing of biological scaffolds such as complex DNA nanonetworks is of importance for generating bio-directed assemblies of nano-objects for nanocircuit purposes. Herein, we consider the individual modification of nanoelectrodes with different oligonucleotide sequences by an electrochemically driven co-polymerization process of pyrrole and modified oligonucleotide sequences bearing pyrrole monomers. We demonstrate that this one-step technique presents the advantages of simplicity, localization of surface modification, mechanical, biological and chemical stability of the coatings, and high lateral resolution. PMID:20973021

  11. Introduction of radiolabeled therapeutic oligonucleotides as nanonuclear explosive gene therapy

    The synthetic oligonucleotide technology is also at early trial points in human testing against HIV, leukemia, Herpes virus, and other diseases, whose outcome will remain for the future. The current status of these varied approaches is presented in later parts in this article: What are therapeutic oligonucleotides?, Why Auger-emitters are useful in gene therapy?, What is the synergistic effect on combining Auger emitter and Triplex-forming ODN?, How have TFO researches evolved from the starting point?, In which areas of clinical research will this research illuminating?

  12. Enzymatic Synthesis of Modified Oligonucleotides by PEAR Using Phusion and KOD DNA Polymerases

    Wang, Xuxiang; Zhang, Jianye; Li, Yingjia; Chen, Gang; Wang, Xiaolong

    2015-01-01

    Antisense synthetic oligonucleotides have been developed as potential gene-targeted therapeutics. We previously reported polymerase–endonuclease amplification reaction (PEAR) for amplification of natural and 5′-O-(1-thiotriphosphate) (S)-modified oligonucleotides. Here, we extended the PEAR technique for enzymatic preparation of 2′-deoxy-2′-fluoro-(2′-F) and 2′-F/S double-modified oligonucleotides. The result showed that KOD and Phusion DNA polymerase could synthesize oligonucleotides with on...

  13. Reversal of phenotypes in MECP2 duplication mice using genetic rescue or antisense oligonucleotides.

    Sztainberg, Yehezkel; Chen, Hong-mei; Swann, John W; Hao, Shuang; Tang, Bin; Wu, Zhenyu; Tang, Jianrong; Wan, Ying-Wooi; Liu, Zhandong; Rigo, Frank; Zoghbi, Huda Y

    2015-12-01

    Copy number variations have been frequently associated with developmental delay, intellectual disability and autism spectrum disorders. MECP2 duplication syndrome is one of the most common genomic rearrangements in males and is characterized by autism, intellectual disability, motor dysfunction, anxiety, epilepsy, recurrent respiratory tract infections and early death. The broad range of deficits caused by methyl-CpG-binding protein 2 (MeCP2) overexpression poses a daunting challenge to traditional biochemical-pathway-based therapeutic approaches. Accordingly, we sought strategies that directly target MeCP2 and are amenable to translation into clinical therapy. The first question that we addressed was whether the neurological dysfunction is reversible after symptoms set in. Reversal of phenotypes in adult symptomatic mice has been demonstrated in some models of monogenic loss-of-function neurological disorders, including loss of MeCP2 in Rett syndrome, indicating that, at least in some cases, the neuroanatomy may remain sufficiently intact so that correction of the molecular dysfunction underlying these disorders can restore healthy physiology. Given the absence of neurodegeneration in MECP2 duplication syndrome, we propose that restoration of normal MeCP2 levels in MECP2 duplication adult mice would rescue their phenotype. By generating and characterizing a conditional Mecp2-overexpressing mouse model, here we show that correction of MeCP2 levels largely reverses the behavioural, molecular and electrophysiological deficits. We also reduced MeCP2 using an antisense oligonucleotide strategy, which has greater translational potential. Antisense oligonucleotides are small, modified nucleic acids that can selectively hybridize with messenger RNA transcribed from a target gene and silence it, and have been successfully used to correct deficits in different mouse models. We find that antisense oligonucleotide treatment induces a broad phenotypic rescue in adult

  14. Analysis of oligonucleotide array experiments with repeated measures using mixed models

    Getchell Thomas V

    2004-12-01

    Full Text Available Abstract Background Two or more factor mixed factorial experiments are becoming increasingly common in microarray data analysis. In this case study, the two factors are presence (Patients with Alzheimer's disease or absence (Control of the disease, and brain regions including olfactory bulb (OB or cerebellum (CER. In the design considered in this manuscript, OB and CER are repeated measurements from the same subject and, hence, are correlated. It is critical to identify sources of variability in the analysis of oligonucleotide array experiments with repeated measures and correlations among data points have to be considered. In addition, multiple testing problems are more complicated in experiments with multi-level treatments or treatment combinations. Results In this study we adopted a linear mixed model to analyze oligonucleotide array experiments with repeated measures. We first construct a generalized F test to select differentially expressed genes. The Benjamini and Hochberg (BH procedure of controlling false discovery rate (FDR at 5% was applied to the P values of the generalized F test. For those genes with significant generalized F test, we then categorize them based on whether the interaction terms were significant or not at the α-level (αnew = 0.0033 determined by the FDR procedure. Since simple effects may be examined for the genes with significant interaction effect, we adopt the protected Fisher's least significant difference test (LSD procedure at the level of αnew to control the family-wise error rate (FWER for each gene examined. Conclusions A linear mixed model is appropriate for analysis of oligonucleotide array experiments with repeated measures. We constructed a generalized F test to select differentially expressed genes, and then applied a specific sequence of tests to identify factorial effects. This sequence of tests applied was designed to control for gene based FWER.

  15. Fabrication of oligonucleotide microarray for the detection of Japanese encephalitis virus

    HAI YAN ZHANG; WEN LI MA; XIAO MING ZHANG; WEN LING ZHENG

    2006-01-01

    A low-density oligonucleotide microarray was used for the detection of Japanese encephalitis virus (JEV), combining with restriction display PCR labeling method. The hybridization targets were amplified from 6 plasmids containing several JEV gene fragments. Corresponding oligonucleotide probe spots were detected unambiguously. We claim that the oligonucleotide microarray technology is feasible and may have potential for clinical laboratory application.

  16. Intracellular Uptake of Modified Oligonucleotide Studied by Two Fluorescence Techniques

    Kočišová, E.; Praus, P.; Rosenberg, Ivan; Seksek, O.; Sureau, F.; Štěpánek, J.; Turpin, P. Y.

    2004-01-01

    Roč. 74, - (2004), s. 110-114. ISSN 0006-3525 R&D Projects: GA ČR GA203/01/1166; GA ČR GP202/03/D118 Institutional research plan: CEZ:AV0Z4055905 Keywords : cellular uptake * antisense oligonucleotide * flluorescence microimagigng Subject RIV: CC - Organic Chemistry Impact factor: 2.863, year: 2004

  17. Oligonucleotide-directed mutagenesis for precision gene editing.

    Sauer, Noel J; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-02-01

    Differences in gene sequences, many of which are single nucleotide polymorphisms, underlie some of the most important traits in plants. With humanity facing significant challenges to increase global agricultural productivity, there is an urgent need to accelerate the development of these traits in plants. oligonucleotide-directed mutagenesis (ODM), one of the many tools of Cibus' Rapid Trait Development System (RTDS(™) ) technology, offers a rapid, precise and non-transgenic breeding alternative for trait improvement in agriculture to address this urgent need. This review explores the application of ODM as a precision genome editing technology, with emphasis on using oligonucleotides to make targeted edits in plasmid, episomal and chromosomal DNA of bacterial, fungal, mammalian and plant systems. The process of employing ODM by way of RTDS technology has been improved in many ways by utilizing a fluorescence conversion system wherein a blue fluorescent protein (BFP) can be changed to a green fluorescent protein (GFP) by editing a single nucleotide of the BFP gene (CAC→TAC; H66 to Y66). For example, dependent on oligonucleotide length, applying oligonucleotide-mediated technology to target the BFP transgene in Arabidopsis thaliana protoplasts resulted in up to 0.05% precisely edited GFP loci. Here, the development of traits in commercially relevant plant varieties to improve crop performance by genome editing technologies such as ODM, and by extension RTDS, is reviewed. PMID:26503400

  18. Systematic design of mouse Vh gene family-specific oligonucleotides

    Seijen, AM; Seijen, HG; Bos, NA

    2001-01-01

    Kabat's database has often been used to design mouse Vh gene-specific 5 ' primers. The emphasis was mostly on constructing a universal (degenerate) 5 ' primer or 5 ' primer set, which would be able to match every mouse Vh gene. We were interested in finding oligonucleotides that could be used as pri

  19. Solid-phase-supported synthesis of morpholinoglycine oligonucleotide mimics

    Tatyana V. Abramova

    2014-05-01

    Full Text Available An efficient solid-phase-supported peptide synthesis (SPPS of morpholinoglycine oligonucleotide (MorGly mimics has been developed. The proposed strategy includes a novel specially designed labile linker group containing the oxalyl residue and the 2-aminomethylmorpholino nucleoside analogues as first subunits.

  20. DNA oligonucleotide conformations: high resolution NMR studies

    The present work describes a DNA double-helix model, which is well comparable with the models derived from fibre-diffraction studies. The model has a mononucleotide repeat with torsion angles in accordance with average geometries as derived from 1H NMR studies. Special attention was paid to reduce the number of short H-H nonbonding contacts, which are abundantly present in the 'classical' fibre-diffraction models. Chapter 3 describes the first complete assignment of a 1H NMR spectrum of a DNA tetramer, d(TAAT). Preliminary conformational data derived from the spectral parameters recorded at 27 0C are given. A more detailed analysis employing temperature-dependence studies is given in Chapter 4. (Auth.)

  1. Hf Transition Probabilities and Abundances

    Lawler, J E; Labby, Z E; Sneden, C; Cowan, J J; Ivans, I I

    2006-01-01

    Radiative lifetimes from laser-induced fluorescence measurements, accurate to about +/- 5 percent, are reported for 41 odd-parity levels of Hf II. The lifetimes are combined with branching fractions measured using Fourier transform spectrometry to determine transition probabilities for 150 lines of Hf II. Approximately half of these new transition probabilities overlap with recent independent measurements using a similar approach. The two sets of measurements are found to be in good agreement for measurements in common. Our new laboratory data are applied to refine the hafnium photospheric solar abundance and to determine hafnium abundances in 10 metal-poor giant stars with enhanced r-process abundances. For the Sun we derive log epsilon (Hf) = 0.88 +/- 0.08 from four lines; the uncertainty is dominated by the weakness of the lines and their blending by other spectral features. Within the uncertainties of our analysis, the r-process-rich stars possess constant Hf/La and Hf/Eu abundance ratios, log epsilon (Hf...

  2. Innovative instrumentation for microarray scanning and analysis: application for characterization of oligonucleotide duplexes behavior.

    Khomyakova, E B; Dreval, E V; Tran-Dang, M; Potier, M C; Soussaline, F P

    2004-05-01

    Accuracy in microarray technology requires new approaches to microarray reader development. A microarray reader system (optical scanning array or OSA reader) based on automated microscopy with large field of view, high speed 3 axis scanning at multiple narrow-band spectra of excitation light has been developed. It allows fast capture of high-resolution, multi-fluorescence images and is characterized by a linear dynamic range and sensitivity comparable to commonly used photo-multiplier tube (PMT)-based laser scanner. Controlled by high performance software, the instrument can be used for scanning and quantitative analysis of any type of dry microarray. Studies implying temperature-controlled hybridization chamber containing a microarray can also be performed. This enables the registration of kinetics and melting curves. This feature is required in a wide range of on-chip chemical and enzymatic reactions including on-chip PCR amplification. We used the OSA reader for the characterization of hybridization and melting behaviour of oligonucleotide:oligonucleotide duplexes on three-dimensional Code Link slides. PMID:15209342

  3. Oligonucleotide Functionalised Microbeads: Indispensable Tools for High-Throughput Aptamer Selection

    Lewis A. Fraser

    2015-12-01

    Full Text Available The functionalisation of microbeads with oligonucleotides has become an indispensable technique for high-throughput aptamer selection in SELEX protocols. In addition to simplifying the separation of binding and non-binding aptamer candidates, microbeads have facilitated the integration of other technologies such as emulsion PCR (ePCR and Fluorescence Activated Cell Sorting (FACS to high-throughput selection techniques. Within these systems, monoclonal aptamer microbeads can be individually generated and assayed to assess aptamer candidate fitness thereby helping eliminate stochastic effects which are common to classical SELEX techniques. Such techniques have given rise to aptamers with 1000 times greater binding affinities when compared to traditional SELEX. Another emerging technique is Fluorescence Activated Droplet Sorting (FADS whereby selection does not rely on binding capture allowing evolution of a greater diversity of aptamer properties such as fluorescence or enzymatic activity. Within this review we explore examples and applications of oligonucleotide functionalised microbeads in aptamer selection and reflect upon new opportunities arising for aptamer science.

  4. Biophysical and RNA Interference Inhibitory Properties of Oligonucleotides Carrying Tetrathiafulvalene Groups at Terminal Positions

    Sónia Pérez-Rentero

    2013-01-01

    Full Text Available Oligonucleotide conjugates carrying a single functionalized tetrathiafulvalene (TTF unit linked through a threoninol molecule to the 3′ or 5′ ends were synthesized together with their complementary oligonucleotides carrying a TTF, pyrene, or pentafluorophenyl group. TTF-oligonucleotide conjugates formed duplexes with higher thermal stability than the corresponding unmodified oligonucleotides and pyrene- and pentafluorophenyl-modified oligonucleotides. TTF-modified oligonucleotides are able to bind to citrate-stabilized gold nanoparticles (AuNPs and produce stable gold AuNPs functionalized with oligonucleotides. Finally, TTF-oligoribonucleotides have been synthesized to produce siRNA duplexes carrying TTF units. The presence of the TTF molecule is compatible with the RNA interference mechanism for gene inhibition.

  5. Precision Chemical Abundance Measurements

    Yong, David; Grundahl, Frank; Meléndez, Jorge;

    2012-01-01

    This talk covers preliminary work in which we apply a strictly differential line-by-line chemical abundance analysis to high quality UVES spectra of the globular cluster NGC 6752. We achieve extremely high precision in the measurement of relative abundance ratios. Our results indicate that the ob...

  6. Common Cold

    ... coughing - everyone knows the symptoms of the common cold. It is probably the most common illness. In ... people in the United States suffer 1 billion colds. You can get a cold by touching your ...

  7. Generating highly labeled oligonucleotides for DNA-protein interaction

    We developed a new strategy to prepare double-stranded oligonucleotides containing recognition sites for specific binding proteins to examine DNA-protein interactions in various assays (gel mobility shift, UV-crosslinking, and affinity chromatography). The advantages of our procedures are as follows. Only one strand needs to be synthesized using a commercial oligonucleotide synthesizer. The probes can be labeled to a high specific activity and the exact position of labeling can be chosen, which is necessary for UV-crosslinking studies. Furthermore, multimeric binding sites for efficient DNA affinity chromatography can easily be generated. It is also possible to precisely place modified bases without the need for chemical precursors. Using this protocol, more detailed information about the binding protein factors and their behavior in interaction with recognition sites can be obtained

  8. One-oligonucleotide method for constructing vectors for RNA interference

    Carlos Fabian FLORES-JASSO; Ines VELAZQUEZ-QUESADA; Carlos LANDA-SOLIS; Andres A GUTIERREZ; Luis VACA

    2005-01-01

    Aim: To develop an easy, fast, automated, and inexpensive method for constructing short-hairpin-RNA cassettes for RNAi studies. Methods: Using single oligonucleotides, a variety of DNA cassettes for RNAi vectors were constructed in only few minutes in an automated manner. The cassettes, targeting the eGFP,were cloned into plasmids driven by RNA polymerase Ⅲ promoter H 1. Then, the plasmids were transfected into HeLa cells that were later infected with a recombinant adenovirus encoding the eGFP gene. The level of eGFP fluorescence was evaluated by confocal imaging and flow cytometry. Results: The plasmids constructed with the DNA cassettes made by the one-oligonucleotide method inhibited eGFP with different potencies, ranging from 55% to 75%. Conclusion: By using the method reported here, it is possible to simultaneously construct hundreds of different DNA cassettes for RNAi experiments in an inexpensive, automated way. This method will facilitate functional genomics studies on mammalian cells.

  9. Fluorescence quenching of TMR by guanosine in oligonucleotides

    QU Peng; CHEN XuDong; ZHOU XiaoXue; LI Xun; ZHAO XinSheng

    2009-01-01

    Nucleotide-specific fluorescence quenching in fluorescently labeled DNA has many applications in biotechnology. We have studied the inter- and intra-molecular quenching of tetramethylrhodamine (TMR) by nucleotides to better understand their quenching mechanism and influencing factors. In agreement with previous work, dGMP can effectively quench TMR, while the quenching of TMR by other nucleotides is negligible. The Stern-Volmer plot between TMR and dGMP delivers a bimolecular quenching constant of Ks= 52.3 M~(-1). The fluorescence of TMR in labeled oligonucleotides decreases efficiently through photoinduced electron transfer by guanosine. The quenching rate constant between TMR and guanosine was measured using fluorescence correlation spectroscopy (FCS). In addition, our data show that the steric hindrance by bases around guanosine has significant effect on the G-quenching. The availability of these data should be useful in designing fluorescent oligonucleotides and understanding the G-quenching process.

  10. Fluorescence quenching of TMR by guanosine in oligonucleotides

    2009-01-01

    Nucleotide-specific fluorescence quenching in fluorescently labeled DNA has many applications in biotechnology. We have studied the inter-and intra-molecular quenching of tetramethylrhodamine (TMR) by nucleotides to better understand their quenching mechanism and influencing factors. In agreement with previous work, dGMP can effectively quench TMR, while the quenching of TMR by other nucleotides is negligible. The Stern-Volmer plot between TMR and dGMP delivers a bimolecular quenching constant of Ks=52.3 M-1. The fluorescence of TMR in labeled oligonucleotides decreases efficiently through photoinduced electron transfer by guanosine. The quenching rate constant between TMR and guanosine was measured using fluorescence correlation spectroscopy (FCS). In addition, our data show that the steric hindrance by bases around guanosine has significant effect on the G-quenching. The availability of these data should be useful in designing fluorescent oligonucleotides and understanding the G-quenching process.

  11. Chiral phosphonate internucleotide linkage: A promising modification for chimeric oligonucleotides?

    Liboska, Radek; Petrová, Magdalena; Pohl, Radek; Buděšínský, Miloš; Hurychová, Vladimíra; Rosenberg, Ivan

    -, č. 52 (2008), s. 317-318. ISSN 0261-3166. [Joint Symposium of the International Roundtable on Nucleosides, Nucleotides and Nucleic Acids /18./ and the International Symposium on Nucleic Acid Chemistry /35./. Kyoto, 08.09.2008-12.09.2008] R&D Projects: GA MŠk(CZ) LC06061 Grant ostatní: EMIL-FP6(XE) 503569 Institutional research plan: CEZ:AV0Z40550506 Keywords : oligonucleotide phosphonate Subject RIV: CC - Organic Chemistry

  12. The strand transfer oligonucleotide inhibitors of HIV-integrase

    Snášel, Jan; Rosenberg, Ivan; Pačes, Ondřej; Pichová, Iva

    2009-01-01

    Roč. 24, č. 1 (2009), s. 241-246. ISSN 1475-6366 R&D Projects: GA MŠk 1M0508; GA ČR GA203/05/0827; GA ČR GP203/05/P557; GA AV ČR IAA4055304 Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV-1 integrase * inhibition * phosphonate oligonucleotides Subject RIV: CE - Biochemistry Impact factor: 1.496, year: 2009

  13. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

    Jinho Kim; Olsen, Timothy R.; Jing Zhu; Hilton, John P.; Kyung-Ae Yang; Renjun Pei; Stojanovic, Milan N.; Qiao Lin

    2016-01-01

    We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling ...

  14. Molecular Selection, Modification and Development of Therapeutic Oligonucleotide Aptamers

    Yuanyuan Yu; Chao Liang; Quanxia Lv; Defang Li; Xuegong Xu; Baoqin Liu; Aiping Lu; Ge Zhang

    2016-01-01

    Monoclonal antibodies are the dominant agents used in inhibition of biological target molecules for disease therapeutics, but there are concerns of immunogenicity, production, cost and stability. Oligonucleotide aptamers have comparable affinity and specificity to targets with monoclonal antibodies whilst they have minimal immunogenicity, high production, low cost and high stability, thus are promising inhibitors to rival antibodies for disease therapy. In this review, we will compare the det...

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

    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...

  16. Oligonucleotide-based strategies to combat polyglutamine diseases

    Fiszer, Agnieszka; Krzyzosiak, Wlodzimierz J.

    2014-01-01

    Considerable advances have been recently made in understanding the molecular aspects of pathogenesis and in developing therapeutic approaches for polyglutamine (polyQ) diseases. Studies on pathogenic mechanisms have extended our knowledge of mutant protein toxicity, confirmed the toxicity of mutant transcript and identified other toxic RNA and protein entities. One very promising therapeutic strategy is targeting the causative gene expression with oligonucleotide (ON) based tools. This straig...

  17. Phosphorothioate Antisense Oligonucleotides Induce the Formation of Nuclear Bodies

    Lorenz, Peter; Baker, Brenda F.; Bennett, C. Frank; Spector, David L.

    1998-01-01

    Antisense oligonucleotides are powerful tools for the in vivo regulation of gene expression. We have characterized the intracellular distribution of fluorescently tagged phosphorothioate oligodeoxynucleotides (PS-ONs) at high resolution under conditions in which PS-ONs have the potential to display antisense activity. Under these conditions PS-ONs predominantly localized to the cell nucleus where they accumulated in 20–30 bright spherical foci designated phosphorothioate bodies (PS bodies), w...

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

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

  19. On the rapid deprotection of synthetic oligonucleotides and analogs.

    Polushin, N N; Morocho, A M; Chen, B. C.; Cohen, J. S.

    1994-01-01

    The efficiency of oligodeoxynucleotide deprotection is greatly enhanced using a combination of: (a) ethanolamine, and especially a mixture of hydrazine, ethanolamine and methanol, in place of the usual aqueous ammonia; (b) tert-butylphenoxyacetyl amino protecting groups, and (c) oxalyl link between the first nucleotide and the polymeric support. The extent of base modification, particularly of C, is shown to be extremely low, and the quality of deprotected oligonucleotides is as high as in th...

  20. Oligonucleotide-mediated gene editing of Apolipoprotein A-I.

    Disterer, P

    2008-01-01

    Apolipoprotein A-I (ApoA-I) is the major protein constituent of high density lipoprotein (HDL) and controls reverse cholesterol transport, an important process in preventing atherosclerosis. A natural point mutation, ApoA-lMiiano (ApoA-Im) enhances the atheroprotective potential of HDL. Here, I attempt to introduce this specific modification into the genome of mammalian cells using the gene therapy strategy of oligonucleotide-mediated gene editing. I showed successful APOA-I gene editing in r...

  1. Repair of DNA lesions associated with triplex-forming oligonucleotides

    Chin, Joanna Y; Glazer, Peter M.

    2009-01-01

    Triplex-forming oligonucleotides (TFOs) are gene targeting tools that can bind in the major groove of duplex DNA in a sequence-specific manner. When bound to DNA, TFOs can inhibit gene expression, can position DNA-reactive agents to specific locations in the genome, or can induce targeted mutagenesis and recombination. There is evidence that third strand binding, alone or with an associated cross-link, is recognized and metabolized by DNA repair factors, particularly the nucleotide excision r...

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

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

    2013-05-15

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

  3. Oligonucleotides modified with acyclic nucleoside phosphonate (HPEP) units

    Kaiser, Martin Maxmilian; Novák, Pavel; Rosenbergová, Šárka; Poštová Slavětínská, Lenka; Rosenberg, Ivan; Janeba, Zlatko

    Praha: Institute of Organic Chemistry and Biochemistry AS CR, v. v. i, 2014 - (Hocek, M.), s. 293-294. (Collection Symposium Series. 14). ISBN 978-80-86241-50-0. [Symposium on Chemistry of Nucleic Acid Components /16./. Český Krumlov (CZ), 08.06.2014-13.06.2014] R&D Projects: GA TA ČR TA03010598 Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonate * HPEP * oligonucleotides Subject RIV: CC - Organic Chemistry

  4. Cationic carbosilane dendrimers and oligonucleotide binding: an energetic affair

    Marson, D.; Laurini, E.; Posocco, P.; Fermeglia, M.; Pricl, S.

    2015-02-01

    Generation 2 cationic carbosilane dendrimers hold great promise as internalizing agents for gene therapy as they present low toxicity and retain and internalize the genetic material as an oligonucleotide or siRNA. In this work we carried out complete in silico structural and energetical characterization of the interactions of a set of G2 carbosilane dendrimers, showing different affinity towards two single strand oligonucleotide (ODN) sequences in vitro. Our simulations predict that these four dendrimers and the relevant ODN complexes are characterized by similar size and shape, and that the molecule-specific ODN binding ability can be rationalized only by considering a critical molecular design parameter: the normalized effective binding energy ΔGbind,eff/Neff, i.e. the performance of each active individual dendrimer branch directly involved in a binding interaction.Generation 2 cationic carbosilane dendrimers hold great promise as internalizing agents for gene therapy as they present low toxicity and retain and internalize the genetic material as an oligonucleotide or siRNA. In this work we carried out complete in silico structural and energetical characterization of the interactions of a set of G2 carbosilane dendrimers, showing different affinity towards two single strand oligonucleotide (ODN) sequences in vitro. Our simulations predict that these four dendrimers and the relevant ODN complexes are characterized by similar size and shape, and that the molecule-specific ODN binding ability can be rationalized only by considering a critical molecular design parameter: the normalized effective binding energy ΔGbind,eff/Neff, i.e. the performance of each active individual dendrimer branch directly involved in a binding interaction. Electronic supplementary information (ESI) available: Additional figures and tables. See DOI: 10.1039/c4nr04510f

  5. Antitumor Activity of a Novel Antisense Oligonucleotide Against Akt1

    Yoon, Heejeong; Kim, Deog Joong; Ahn, Eun Hyun; Gellert, Ginelle C.; Shay, Jerry W.; Ahn, Chang-Ho; Lee, Young Bok

    2009-01-01

    The AKT pathway is an important therapeutic target for cancer drug discovery as it functions as a main point for transducing extracellular and intracellular oncogenic signals. Moreover, alternations of the AKT pathway have been found in a wide range of cancers. In the present study, we found that an Akt1 antisense oligonucleotide (Akt1 AO) significantly downregulated the expression of AKT1 at both the mRNA and protein levels and inhibited cellular growth at nanomolar concentrations in various...

  6. Long range clustering of oligonucleotides containing the CG signal

    Katsaloulis, P.; T. Theoharis; A. Provata

    2009-01-01

    Abstract The distance distributions between successive occurrences of the same oligonucleotides in chromosomal DNA are studied, in different classes of higher eucaryotic organisms. A two-parameter modeling is undertaken and applied on the distance distribution of quintuplets (sequences of size five bps) and hexaplets (sequences of size six bps); the first parameter k refers to the short range exponential decay of the distributions, whereas the second parameter m refers to the power...

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

    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.

  8. Particle-Based Microarrays of Oligonucleotides and Oligopeptides

    Alexander Nesterov-Mueller

    2014-10-01

    Full Text Available In this review, we describe different methods of microarray fabrication based on the use of micro-particles/-beads and point out future tendencies in the development of particle-based arrays. First, we consider oligonucleotide bead arrays, where each bead is a carrier of one specific sequence of oligonucleotides. This bead-based array approach, appearing in the late 1990s, enabled high-throughput oligonucleotide analysis and had a large impact on genome research. Furthermore, we consider particle-based peptide array fabrication using combinatorial chemistry. In this approach, particles can directly participate in both the synthesis and the transfer of synthesized combinatorial molecules to a substrate. Subsequently, we describe in more detail the synthesis of peptide arrays with amino acid polymer particles, which imbed the amino acids inside their polymer matrix. By heating these particles, the polymer matrix is transformed into a highly viscous gel, and thereby, imbedded monomers are allowed to participate in the coupling reaction. Finally, we focus on combinatorial laser fusing of particles for the synthesis of high-density peptide arrays. This method combines the advantages of particles and combinatorial lithographic approaches.

  9. Properties of hybrid TiO2 -oligonucleotide nanocomposites

    Recent advancements in hybrid nanotechnology involving nucleic acids and inorganic molecules/structures are predominantly oriented towards cellular imaging or DNA microarray development or for nanoconstruction (such as assembly of ordered patterns of nanocrystals). Nevertheless, in this evolving field there is little work reported yet about the development of nanoparticles that can be used to manipulate biological materials in a novel way. We have synthesized TiO2 -DNA nanocomposites as new vehicles for biotechnology that express new biochemical properties, in an attempt to develop them into nanodevices that would be able to enter cells and conduct their functions in vivo and in situ. Absorption of light and ionizing radiation leads to charge separation in TiO2 , and nanocrystallites modified by the presence of oligonucleotides exhibit semiconducting through both constituents. In such a system charge pairs are instantaneously separated and electropositive holes accumulate on the oligonucleotide DNA, leading to a photo-/radio- catalytic DNA endonuclease reaction. In addition, hybrid nanocomposites of 4.5 nm TiO2 nanoparticles covalently attached to DNA oligonucleotides can be transferred across cellular and nuclear membranes using standard transfection techniques and retained inside mammalian cells. Therefore, TiO2 nanoparticles-biopolymer nanocomposites integrate intrinsic biological/ electrochemical (DNA) and photoelectrical (TiO2 ) properties of the biomolecule and inorganic components, which makes them suitable for development of new tools for biology and medicine

  10. Testing the feasibility of DNA typing for human identification by PCR and an oligonucleotide ligation assay

    Delahunty, C.; Ankener, W.; Deng, Qiang [Univ. of Washington, Seattle, WA (United States)] [and others

    1996-06-01

    The use of DNA typing in human genome analysis is increasing and finding widespread application in the area of forensic and paternity testing. In this report, we explore the feasibility of typing single nucleotide polymorphisms (SNPs) by using a semiautomated method for analyzing human DNA samples. In this approach, PCR is used to amplify segments of human DNA containing a common SNP. Allelic nucleotides in the amplified product are then typed by a calorimetric implementation of the oligonucleotide ligation assay (OLA). The results of the combined assay, PCR/OLA, are read directly by a spectrophotometer; the absorbances are compiled and the genotypes are automatically determined. A panel of 20 markers has been developed for DNA typing and has been tested using a sample panel from the CEPH pedigrees (CEPH parents). The results of this typing, as well as the potential to apply this method to larger populations, are discussed. 62 refs., 2 figs., 4 tabs.

  11. Climate and local abundance in freshwater fishes.

    Knouft, Jason H; Anthony, Melissa M

    2016-06-01

    Identifying factors regulating variation in numbers of individuals among populations across a species' distribution is a fundamental goal in ecology. A common prediction, often referred to as the abundant-centre hypothesis, suggests that abundance is highest near the centre of a species' range. However, because of the primary focus on the geographical position of a population, this framework provides little insight into the environmental factors regulating local abundance. While range-wide variation in population abundance associated with environmental conditions has been investigated in terrestrial species, the relationship between climate and local abundance in freshwater taxa across species' distributions is not well understood. We used GIS-based temperature and precipitation data to determine the relationships between climatic conditions and range-wide variation in local abundance for 19 species of North American freshwater fishes. Climate predicted a portion of the variation in local abundance among populations for 18 species. In addition, the relationship between climatic conditions and local abundance varied among species, which is expected as lineages partition the environment across geographical space. The influence of local habitat quality on species persistence is well documented; however, our results also indicate the importance of climate in regulating population sizes across a species geographical range, even in aquatic taxa. PMID:27429769

  12. Discrimination of oligonucleotides of different lengths with a wild-type aerolysin nanopore.

    Cao, Chan; Ying, Yi-Lun; Hu, Zheng-Li; Liao, Dong-Fang; Tian, He; Long, Yi-Tao

    2016-08-01

    Protein nanopores offer an inexpensive, label-free method of analysing single oligonucleotides. The sensitivity of the approach is largely determined by the characteristics of the pore-forming protein employed, and typically relies on nanopores that have been chemically modified or incorporate molecular motors. Effective, high-resolution discrimination of oligonucleotides using wild-type biological nanopores remains difficult to achieve. Here, we show that a wild-type aerolysin nanopore can resolve individual short oligonucleotides that are 2 to 10 bases long. The sensing capabilities are attributed to the geometry of aerolysin and the electrostatic interactions between the nanopore and the oligonucleotides. We also show that the wild-type aerolysin nanopores can distinguish individual oligonucleotides from mixtures and can monitor the stepwise cleavage of oligonucleotides by exonuclease I. PMID:27111839

  13. Scalable amplification of strand subsets from chip-synthesized oligonucleotide libraries

    Schmidt, Thorsten L.; Beliveau, Brian J.; Uca, Yavuz O.; Theilmann, Mark; Da Cruz, Felipe; Wu, Chao-Ting; Shih, William M.

    2015-01-01

    Synthetic oligonucleotides are the main cost factor for studies in DNA nanotechnology, genetics and synthetic biology, which all require thousands of these at high quality. Inexpensive chip-synthesized oligonucleotide libraries can contain hundreds of thousands of distinct sequences, however only at sub-femtomole quantities per strand. Here we present a selective oligonucleotide amplification method, based on three rounds of rolling-circle amplification, that produces nanomole amounts of single-stranded oligonucleotides per millilitre reaction. In a multistep one-pot procedure, subsets of hundreds or thousands of single-stranded DNAs with different lengths can selectively be amplified and purified together. These oligonucleotides are used to fold several DNA nanostructures and as primary fluorescence in situ hybridization probes. The amplification cost is lower than other reported methods (typically around US$ 20 per nanomole total oligonucleotides produced) and is dominated by the use of commercial enzymes. PMID:26567534

  14. Statistical Algorithms for Long DNA Sequences: Oligonucleotide Distributions and Homogeneity Maps

    P. Katsaloulis

    2005-01-01

    Full Text Available The statistical properties of oligonucleotide appearances within long DNA sequences often reveal useful characteristics of the corresponding DNA areas. Two algorithms to statistically analyze oligonucleotide appearances within long DNA sequences in genome banks are presented. The first algorithm determines statistical indices for arbitrary length oligonucleotides within arbitrary length DNA sequences. The critical exponent μ of the distance distribution between consecutive occurrences of the same oligonucleotide is calculated and its value is shown to characterize the functionality of the oligonucleotide. The second algorithm searches for areas with variable homogeneity, based on the density of oligonucleotides. The two algorithms have been applied to representative eucaryotes (the animal Mus musculusand the plant Arabidopsis thaliana and interesting results were obtained, confirmed by biological observations. All programs are open source and publicly available on our web site.

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

    Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

    2016-03-01

    Spermatozoa could introduce exogenous oligonucleotides of interest to the oocyte. The most important reason of low efficiency of sperm mediated gene transfer (SMGT) is low uptake of exogenous DNA by spermatozoa. The aim of this study was to evaluate the effects of static magnetic field on exogenous oligonucleotide uptake of spermatozoa using magnetofection method. Magnetic nanoparticles (MNPs) associated with the labeled oligonucleotides were used to increase the efficiency of exogenous oligonucleotide uptake by rooster spermatozoa. We used high-field/high-gradient magnet (NdFeB) to enhance and accelerate exogenous DNA sedimentation at the spermatozoa surface. Flow cytometry analysis was performed to measure viability and percentage of exogenous oligonucleotide uptake by sperm. Flow cytometry analysis showed a significant increase in exogenous oligonucleotide uptake by rooster spermatozoa (Pgene transfer.

  16. Nucleoside phosphonic acids in oligonucleotide chemistry: Structural diversity versus properties of isopolar phosphonate internucleotide linkages

    Hurychová, Vladimíra; Kóšiová, Ivana; Kovačková, Soňa; Králíková, Šárka; Liboska, Radek; Pačes, Ondřej; Páv, Ondřej; Petrová, Magdalena; Rejman, Dominik; Rosenberg, Ivan; Točík, Zdeněk

    Berlin : Oligonucleotide Therapeutic Society, 2007. s. 244-245. [Annual Meeting of the Oligonucleotide Therapeutics Society /3./. 04.10.2007-06.10.2007, Berlin] R&D Projects: GA MŠk(CZ) LC06077; GA MŠk(CZ) LC06061; GA ČR GA203/05/0827; GA ČR GA202/05/0628 Institutional research plan: CEZ:AV0Z40550506 Keywords : phosphonic acids * oligonucleotides * isopolar phosphonate * internucleotide linkage Subject RIV: CC - Organic Chemistry

  17. Recruitment of transcription factors to the target site by triplex-forming oligonucleotides.

    Svinarchuk, F; Nagibneva, I; Cherny, D; Ait-Si-Ali, S; Pritchard, L.L.; Robin, P.; Malvy, C; Harel-Bellan, A; Chern, D

    1997-01-01

    Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a hairpin-TFO able to recruit transcription factors to a target DNA. The designed oligonucleotide contains a triplex-forming sequence, linked through a nucleotide loop to a double-stranded hairpin including the SRE enhancer of the c-fos gene promoter. We show here that this oligonucleotide can specifically recognise its DNA...

  18. Cationic oligonucleotides can mediate specific inhibition of gene expression in Xenopus oocytes.

    Bailey, C P; Dagle, J M; Weeks, D L

    1998-01-01

    Base-specific hydrogen bonding between an oligonucleotide and the purines in the major groove of a DNA duplex provide an approach to selective inhibition of gene expression. Oligonucleotide-mediated triplex formation in vivo may be enhanced by a number of different chemical modifications. We have previously described an in vitro analysis of triplex formation using oligonucleotides containing internucleoside phosphate linkages modified with the cation N , N -diethyl-ethylenediamine (DEED). Whe...

  19. Coupling Strategies for the Synthesis of Peptide-Oligonucleotide Conjugates for Patterned Synthetic Biomineralization

    Joshua D. Carter; LaBean, Thomas H.

    2011-01-01

    This work describes preparation strategies for peptide-oligonucleotide conjugates that combine the self-assembling behavior of DNA oligonucleotides with the molecular recognition capabilities of peptides. The syntheses include a solution-phase fragment coupling reaction and a solid-phase fragment coupling strategy where the oligonucleotide has been immobilized on DEAE Sepharose. The yield of four coupling reagents is evaluated, two reagents in water, EDC (1-ethyl-3-(3-dimethylaminopropyl) car...

  20. Oligonucleotide-directed mutagenesis by microscale 'shot-gun' gene synthesis.

    Grundström, T; Zenke, W M; Wintzerith, M; Matthes, H W; Staub, A; Chambon, P

    1985-01-01

    We describe a rapid and efficient microscale method for in vitro site-directed mutagenesis by gene synthesis. Mutants are constructed by "shot-gun ligation" of overlapping synthetic oligonucleotides yielding double stranded synthetic DNA of more than 120 nucleotides in length. The terminal oligonucleotides of the DNA segment to be synthesized are designed to create sticky ends complementary to unique restriction sites of a polylinker present in an M13 vector. The oligonucleotides are hybridiz...

  1. TmPrime: fast, flexible oligonucleotide design software for gene synthesis

    Bode, Marcus; Khor, Samuel; Ye, Hongye; Li, Mo-Huang; Ying, Jackie Y.

    2009-01-01

    Herein we present TmPrime, a computer program to design oligonucleotide sets for gene assembly by both ligase chain reaction (LCR) and polymerase chain reaction (PCR). TmPrime offers much flexibility with no constraints on the gene and oligonucleotide lengths. The program divides the long input DNA sequence based on the input desired melting temperature, and dynamically optimizes the length of oligonucleotides to achieve homologous melting temperatures. The output reports the melting temperat...

  2. Orion A helium abundance

    The 22.4-GHz (H,He)66-alpha and 36.5-GHz (H,He)56-alpha radio recombination lines have been observed at several Jaffe-Pankonin positions in the central part of the Orion A source. The measured relative abundance of ionized helium increases with distance, averaging 11.6 percent at peripheral points. The observed behavior is interpreted by a blister-type model nebula, which implies that Orion A has a true He abundance of 12 percent, is moving with a radial velocity of 5 km/sec, and is expanding. 18 references

  3. Scavenger Receptor-Mediated Delivery of Antisense Mini-Exon Phosphorothioate Oligonucleotide to Leishmania-Infected Macrophages: SELECTIVE AND EFFICIENT ELIMINATION OF THE PARASITE

    Chaudhuri, Gautam

    1997-01-01

    Targeted delivery of a 17-mer antisense phosphorothioate oligodeoxyribonucleotide, complementary to the common 5′-end of every mRNA of the parasite cells, to the phagolysosomes of cultured murine macrophages infected with Leishmania mexicana amazonensis selectively and efficiently eliminated the parasite cells without causing any detectable harm to the host cells. The antisense mini-exon oligonucleotide (ASM) was encapsulated into liposomes coated with maleylated bovine serum albumin (MBSA), ...

  4. Common learning

    Cripps, M.; Ely, J.; Mailath, G.; Samuelson, L.

    2007-01-01

    Consider two agents who learn the value of an unknown parameter by observing a sequence of private signals. The signals are independent and identically distributed across time but not necessarily across agents. We show that when each agent's signal space is finite, the agents will commonly learn the value of the parameter, that is, that the true value of the parameter will become approximate common knowledge. The essential step in this argument is to express the expectation of one agent's sig...

  5. Testing Relationships between Energy and Vertebrate Abundance

    Understanding what drives variation in the abundance of organisms is fundamental to evolutionary ecology and wildlife management. Yet despite its importance, there is still great uncertainty about the main factors influencing variation in vertebrate abundance across taxa. We believe valuable knowledge and increased predictive power could be gained by taking into account both the intrinsic factors of species and the extrinsic factors related to environmental surroundings in the commonly cited RQ model, which provides a simple conceptual framework valid at both the interspecific and the intraspecific scales. Approaches comparing studies undertaken at different spatial and taxonomic scales could be key to our ability to better predict abundance, and thanks to the increased availability of population size data, global geographic datasets, and improved comparative methods, there might be unprecedented opportunities to (1) gain a greater understanding of vertebrate abundance patterns and (2) test existing theories on free-ranging animals.

  6. Oligonucleotide microarray for subtyping of influenza A viruses

    Klotchenko, S. A.; Vasin, A. V.; Sandybaev, N. T.; Plotnikova, M. A.; Chervyakova, O. V.; Smirnova, E. A.; Kushnareva, E. V.; Strochkov, V. M.; Taylakova, E. T.; Egorov, V. V.; Koshemetov, J. K.; Kiselev, O. I.; Sansyzbay, A. R.

    2012-02-01

    Influenza is one of the most widespread respiratory viral diseases, infecting humans, horses, pigs, poultry and some other animal populations. Influenza A viruses (IAV) are classified into subtypes on the basis of the surface hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) glycoproteins. The correct determination of IAV subtype is necessary for clinical and epidemiological studies. In this article we propose an oligonucleotide microarray for subtyping of IAV using universal one-step multisegment RT-PCR fluorescent labeling of viral gene segments. It showed to be an advanced approach for fast detection and identification of IAV.

  7. A convenient and efficient purification method for chemically labeled oligonucleotides.

    Hwang, Jihee; Kang, Junhee; Kim, Seong Keun; Kim, Younggyu

    2013-05-01

    We developed an efficient, cost-effective, and rapid purification method for chemically-labeled oligonucleotides that requires less time than conventional procedures such as ethanol precipitation or size-exclusion chromatography. Based on the hydrophilic and hydrophobic properties of DNA and amine-reactive fluorophores, we show that n-butanol saturated with distilled water may be used to remove unreacted fluorophores by sequestering them in the organic phase, while labeled DNA remains in the aqueous phase. This phase extraction method is simple, fast, and allows for processing multiple samples simultaneously, a necessity for high-throughput labeling strategies. PMID:23662899

  8. Elimination and adsorptive transfer techniques in an oligonucleotide analysis

    Jelen, František; Trnková, L.; Kouřilová, Alena; Kejnovská, Iva; Vorlíčková, Michaela

    Xi' an, 2009. P12. [International Symposium on Frontiers of Electrochemical Science and Technology . 12.08.2009-15.08.2009, Xi' an] R&D Projects: GA AV ČR(CZ) IAA400040804; GA AV ČR(CZ) IAA100040701; GA AV ČR(CZ) KAN200040651; GA MŠk(CZ) LC06035 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : elimination voltammetry * transfer techniques * analysis of oligonucleotides Subject RIV: BO - Biophysics

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

    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...

  10. Sulfur-containing phosphonate monomers for oligonucleotide synthesis

    Kostov, Ondřej; Zborníková, Eva; Buděšínský, Miloš; Novák, Pavel; Rosenberg, Ivan

    Praha : Institute of Organic Chemistry and Biochemistry AS CR, v. v. i, 2014 - (Hocek, M.), s. 308-309 ISBN 978-80-86241-50-0. - (Collection Symposium Series. 14). [Symposium on Chemistry of Nucleic Acid Components /16./. Český Krumlov (CZ), 08.06.2014-13.06.2014] R&D Projects: GA ČR GA13-26526S; GA ČR GA13-24880S Institutional support: RVO:61388963 Keywords : S-MOP * oligonucleotides Subject RIV: CC - Organic Chemistry

  11. Oligonucleotide microarray for subtyping of influenza A viruses

    Influenza is one of the most widespread respiratory viral diseases, infecting humans, horses, pigs, poultry and some other animal populations. Influenza A viruses (IAV) are classified into subtypes on the basis of the surface hemagglutinin (H1 to H16) and neuraminidase (N1 to N9) glycoproteins. The correct determination of IAV subtype is necessary for clinical and epidemiological studies. In this article we propose an oligonucleotide microarray for subtyping of IAV using universal one-step multisegment RT-PCR fluorescent labeling of viral gene segments. It showed to be an advanced approach for fast detection and identification of IAV.

  12. Repair of DNA lesions associated with triplex-forming oligonucleotides.

    Chin, Joanna Y; Glazer, Peter M

    2009-04-01

    Triplex-forming oligonucleotides (TFOs) are gene targeting tools that can bind in the major groove of duplex DNA in a sequence-specific manner. When bound to DNA, TFOs can inhibit gene expression, can position DNA-reactive agents to specific locations in the genome, or can induce targeted mutagenesis and recombination. There is evidence that third strand binding, alone or with an associated cross-link, is recognized and metabolized by DNA repair factors, particularly the nucleotide excision repair pathway. This review examines the evidence for DNA repair of triplex-associated lesions. PMID:19072762

  13. PCR amplification on microarrays of gel immobilized oligonucleotides

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

    2003-11-04

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

  14. A comparison of alternative 60-mer probe designs in an in-situ synthesized oligonucleotide microarray

    Fairbanks Benjamin D

    2006-04-01

    Full Text Available Abstract Background DNA microarrays have proven powerful for functional genomics studies. Several technologies exist for the generation of whole-genome arrays. It is well documented that 25mer probes directed against different regions of the same gene produce variable signal intensity values. However, the extent to which this is true for probes of greater length (60mers is not well characterized. Moreover, this information has not previously been reported for whole-genome arrays designed against bacteria, whose genomes may differ substantially in characteristics directly affecting microarray performance. Results We report here an analysis of alternative 60mer probe designs for an in-situ synthesized oligonucleotide array for the GC rich, β-proteobacterium Burkholderia cenocepacia. Probes were designed using the ArrayOligoSel3.5 software package and whole-genome microarrays synthesized by Agilent, Inc. using their in-situ, ink-jet technology platform. We first validated the quality of the microarrays as demonstrated by an average signal to noise ratio of >1000. Next, we determined that the variance of replicate probes (1178 total probes examined of identical sequence was 3.8% whereas the variance of alternative probes (558 total alternative probes examined designs was 9.5%. We determined that depending upon the definition, about 2.4% of replicate and 7.8% of alternative probes produced outlier conclusions. Finally, we determined none of the probe design subscores (GC content, internal repeat, binding energy and self annealment produced by ArrayOligoSel3.5 were predictive or probes that produced outlier signals. Conclusion Our analysis demonstrated that the use of multiple probes per target sequence is not essential for in-situ synthesized 60mer oligonucleotide arrays designed against bacteria. Although probes producing outlier signals were identified, the use of ratios results in less than 10% of such outlier conclusions. We also determined that

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

    Boyang Cao

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

  16. Creative Commons

    Jensen, Lone

    2006-01-01

    En Creative Commons licens giver en forfatter mulighed for at udbyde sit værk i en alternativ licensløsning, som befinder sig på forskellige trin på en skala mellem yderpunkterne "All rights reserved" og "No rights reserved". Derved opnås licensen "Some rights reserved"......En Creative Commons licens giver en forfatter mulighed for at udbyde sit værk i en alternativ licensløsning, som befinder sig på forskellige trin på en skala mellem yderpunkterne "All rights reserved" og "No rights reserved". Derved opnås licensen "Some rights reserved"...

  17. The use of oligonucleotide probes for meningococcal serotype characterization

    SACCHI Claudio Tavares

    1998-01-01

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

  18. SOMA: a single oligonucleotide mutagenesis and cloning approach.

    Thorsten Pfirrmann

    Full Text Available Modern biology research requires simple techniques for efficient and restriction site-independent modification of genetic material. Classical cloning and mutagenesis strategies are limited by their dependency on restriction sites and the use of complementary primer pairs. Here, we describe the Single Oligonucleotide Mutagenesis and Cloning Approach (SOMA that is independent of restriction sites and only requires a single mutagenic oligonucleotide to modify a plasmid. We demonstrate the broad application spectrum of SOMA with three examples. First, we present a novel plasmid that in a standardized and rapid fashion can be used as a template for SOMA to generate GFP-reporters. We successfully use such a reporter to assess the in vivo knock-down quality of morpholinos in Xenopus laevis embryos. In a second example, we show how to use a SOMA-based protocol for restriction-site independent cloning to generate chimeric proteins by domain swapping between the two human hRMD5a and hRMD5b isoforms. Last, we show that SOMA simplifies the generation of randomized single-site mutagenized gene libraries. As an example we random-mutagenize a single codon affecting the catalytic activity of the yeast Ssy5 endoprotease and identify a spectrum of tolerated and non-tolerated substitutions. Thus, SOMA represents a highly efficient alternative to classical cloning and mutagenesis strategies.

  19. Fast comparison of DNA sequences by oligonucleotide profiling

    Marín Ignacio

    2008-02-01

    Full Text Available Abstract Background The comparison of DNA sequences is a traditional problem in genomics and bioinformatics. Many new opportunities emerge due to the improvement of personal computers, allowing the implementation of novel strategies of analysis. Findings We describe a new program, called UVWORD, which determines the number of times that each DNA word present in a sequence (target is found in a second sequence (source, a procedure that we have called oligonucleotide profiling. On a standard computer, the user may search for words of a size ranging from k = 1 to k = 14 nucleotides. Average counts for groups of contiguous words may also be established. The rate of analysis on standard computers is from 3.4 (k = 14 to 16 millions of words per second (1 ≤ k ≤ 8. This makes feasible the fast screening of even the longest known DNA molecules. Discussion We show that the combination of the ability of analyzing words of relatively long size, which occur very rarely by chance, and the fast speed of the program allows to perform novel types of screenings, complementary to those provided by standard programs such as BLAST. This method can be used to determine oligonucleotide content, to characterize the distribution of repetitive sequences in chromosomes, to determine the evolutionary conservation of sequences in different species, to establish regions of similar DNA among chromosomes or genomes, etc.

  20. In vivo delivery of transcription factors with multifunctional oligonucleotides

    Lee, Kunwoo; Rafi, Mohammad; Wang, Xiaojian; Aran, Kiana; Feng, Xuli; Lo Sterzo, Carlo; Tang, Richard; Lingampalli, Nithya; Kim, Hyun Jin; Murthy, Niren

    2015-07-01

    Therapeutics based on transcription factors have the potential to revolutionize medicine but have had limited clinical success as a consequence of delivery problems. The delivery of transcription factors is challenging because it requires the development of a delivery vehicle that can complex transcription factors, target cells and stimulate endosomal disruption, with minimal toxicity. Here, we present a multifunctional oligonucleotide, termed DARTs (DNA assembled recombinant transcription factors), which can deliver transcription factors with high efficiency in vivo. DARTs are composed of an oligonucleotide that contains a transcription-factor-binding sequence and hydrophobic membrane-disruptive chains that are masked by acid-cleavable galactose residues. DARTs have a unique molecular architecture, which allows them to bind transcription factors, trigger endocytosis in hepatocytes, and stimulate endosomal disruption. The DARTs have enhanced uptake in hepatocytes as a result of their galactose residues and can disrupt endosomes efficiently with minimal toxicity, because unmasking of their hydrophobic domains selectively occurs in the acidic environment of the endosome. We show that DARTs can deliver the transcription factor nuclear erythroid 2-related factor 2 (Nrf2) to the liver, catalyse the transcription of Nrf2 downstream genes, and rescue mice from acetaminophen-induced liver injury.

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

    The goal of the Human Genome Project is to sequence all 3 billion base pairs of the human genome. Progress in this has been rapid; GenBank(regsign) finished 1994 with 286 million bases of sequence and grew by 2470 in the first quarter of 1995. The challenge to the scientific community is to understand the biological relevance of this genetic information. In most cases the sequence being generated for any single region of the genome represents the genotype of a single individual. A complete understanding of the function of specific genes and other regions of the genome and their role in human disease and development will only become apparent when the sequence of many more individuals is known. Access to genetic information is ultimately limited by the ability to screen DNA sequence. Although the pioneering sequencing methods of Sanger et al. (15) and Maxam and Gilbert (11) have become standard in virtually all molecular biology laboratories, the basic protocols remain largely unchanged. The throughput of this sequencing technology is now becoming the rate-limiting step in both large-scale sequencing projects such as the Human Genome Project and the subsequent efforts to understand genetic diversity. This has inspired the development of advanced DNA sequencing technologies (9), Incremental improvements to Sanger sequencing have been made in DNA labeling and detection. High-speed electrophoresis methods using ultrathin gels or capillary arrays are now being more widely employed. However, these methods are throughput-limited by their sequential nature and the speed and resolution of separations. This limitation will become more pronounced as the need to rapidly screen newly discovered genes for biologically relevant polymorphisms increases. An alternative to gel-based sequencing is to use high-density oligonucleotide probe arrays. Oligonucleotide probe arrays display specific oligonucleotide probes at precise locations in a high density, information-rich format (5

  2. Studies on the Syntheses and Properties of 5'-Branched-sugar Isonucleosides and the Related Oligonucleotides

    Tian Xiaobing; Zhang Lihe; Min Jimei

    2001-01-01

    @@ The chemistry of nucleosides and oligonucleotides is an actively investigated field in the search for new drugs. Thesyntheses and the properties of isonucleosides and oligonucleotides have been investigated to improve their stability,antitumor and antiviral activities, and to reduce their toxicity.

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

    Golova, Julia B.; Chernov, Boris K.

    2010-04-27

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

  4. Mass spectrometry of nucleic acids components. Nucleotides, oligonucleotides and application to sequence determination

    The application of the various ionization techniques to the analysis of nucleotides and oligonucleotides is reviewed. The sequence determination of oligonucleotides was chosen to present the growing possibilities of mass spectrometry due to development of new ''soft ionization'' techniques. 119 refs., 6 figs., 2 tabs. (author)

  5. Long-range order of organized oligonucleotide monolayers on Au(111) electrodes

    Wackerbarth, Hainer; Grubb, Mikala; Zhang, Jingdong; Hansen, Allan Glargaard; Ulstrup, Jens

    2004-01-01

    Oligonucleotides modified by a hexamethylene linker group adsorb on gold electrodes via Au-S bond formation. We have obtained novel data for adsorption of thiol-modified (HS) single-strand HS-10A and double-stranded HS-10AT oligonucleotides and for analogous thiol-free 10A (A = adenine) and 10T (T...

  6. A pedagogy of abundance

    Weller, Martin

    2011-01-01

    The digitisation of content combined with a global network for delivery and an open system for sharing has seen radical changes in many industries. The economic model which has underpinned many content based industries has been based on an assumption of scarcity. With a digital, open, networked approach we are witnessing a shift to abundance of content, and subsequently new economic models are being developed which have this as an assumption. In this article the role of scarcity in developing...

  7. Interstellar Atomic Abundances

    Jenkins, E B

    2003-01-01

    A broad array of interstellar absorption features that appear in the ultraviolet spectra of bright sources allows us to measure the abundances and ionization states of many important heavy elements that exist as free atoms in the interstellar medium. By comparing these abundances with reference values in the Sun, we find that some elements have abundances relative to hydrogen that are approximately consistent with their respective solar values, while others are depleted by factors that range from a few up to around 1000. These depletions are caused by the atoms condensing into solid form onto dust grains. Their strengths are governed by the volatility of compounds that are produced, together with the densities and velocities of the gas clouds. We may characterize the depletion trends in terms of a limited set of parameters; ones derived here are based on measurements of 15 elements toward 144 stars with known values of N(H I) and N(H2). In turn, these parameters may be applied to studies of the production, de...

  8. Abundances in galaxies

    Standard (or mildly inhomogeneous) Big Bang nucleosynthesis theory is well confirmed by abundance measurements of light elements up to 7Li and the resulting upper limit to the number of neutrino families confirmed in accelerator experiments. Extreme inhomogeneous models with a closure density in form of baryons seem to be ruled out and there is no evidence for a cosmic 'floor' to 9Be or heavier elements predicted in some versions of those models. Galaxies show a correlation between luminous mass and abundance of carbon and heavier elements, usually attributed to escape of hot gas from shallow potential wells. Uncertainties include the role of dark matter and biparametric behaviour of ellipticals. Spirals have radial gradients which may arise from a variety of causes. In our own Galaxy one can distinguish three stellar populations - disk, halo and bulge - characterised by differing metallicity distribution functions. Differential abundance effects are found among different elements in stars as a function of metallicity and presumably age, notably in the ratio of oxygen and α-particle elements to iron. These may eventually be exploitable to set a time scale for the formation of the halo, bulge and disk. (orig.)

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

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

    2003-01-01

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

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

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

    2003-01-01

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

  11. Report on carbon and nitrogen abundance studies

    Boehm-Vitense, Erika

    1991-01-01

    The aim of the proposal was to determine the nitrogen to carbon abundance ratios from transition layer lines in stars with different T(sub eff) and luminosities. The equations which give the surface emission line fluxes and the measured ratio of the NV to CIV emission line fluxes are presented and explained. The abundance results are compared with those of photospheric abundance studies for stars in common with the photospheric investigations. The results show that the analyses are at least as accurate as the photospheric determinations. These studies can be extended to F and early G stars for which photospheric abundance determinations for giants are hard to do because molecular bands become too weak. The abundance determination in the context of stellar evolution is addressed. The N/C abundance ratio increases steeply at the point of evolution for which the convection zone reaches deepest. Looking at the evolution of the rotation velocities v sin i, a steep decrease in v sin i is related to the increasing depth of the convection zone. It is concluded that the decrease in v sin i for T(sub eff) less than or approximately = 5800 K is most probably due to the rearrangement of the angular momentum in the stars due to deep convective mixing. It appears that the convection zone is rotating with nearly depth independent angular momentum. Other research results and ongoing projects are discussed.

  12. A Data-intensive Assessment of the Species Abundance Distribution

    Baldridge, Elita

    2013-01-01

    The hollow curve species abundance distribution describes the pattern of large numbers of rare species and a small number of common species in a community. The species abundance distribution is one of the most ubiquitous patterns in nature and many models have been proposed to explain the mechanisms that generate this pattern. While there have been numerous comparisons of species abundance distribution models, most of these comparisons only use a small subset of available models, focus on a s...

  13. Application of PCR to Distinguish Common Species of Dermatophytes

    Faggi, Elisabetta; Pini, Gabriella; Campisi, Enza; Bertellini, Chiara; Difonzo, Elisa; Mancianti, Francesca

    2001-01-01

    This report describes the application of PCR fingerprinting for the identification of species and varieties of common dermatophytes and related fungi utilizing as a single primer the simple repetitive oligonucleotide (GACA)4. The primer was able to amplify all the strains, producing species-specific profiles for Microsporum canis, Microsporum gypseum, Trichophyton rubrum, Trichophyton ajelloi, and Epidermophyton floccosum. Intraspecific variability was not observed for these species. Instead,...

  14. [Research progress of probe design software of oligonucleotide microarrays].

    Chen, Xi; Wu, Zaoquan; Liu, Zhengchun

    2014-02-01

    DNA microarray has become an essential medical genetic diagnostic tool for its high-throughput, miniaturization and automation. The design and selection of oligonucleotide probes are critical for preparing gene chips with high quality. Several sets of probe design software have been developed and are available to perform this work now. Every set of the software aims to different target sequences and shows different advantages and limitations. In this article, the research and development of these sets of software are reviewed in line with three main criteria, including specificity, sensitivity and melting temperature (Tm). In addition, based on the experimental results from literatures, these sets of software are classified according to their applications. This review will be helpful for users to choose an appropriate probe-design software. It will also reduce the costs of microarrays, improve the application efficiency of microarrays, and promote both the research and development (R&D) and commercialization of high-performance probe design software. PMID:24804514

  15. Direct microcontact printing of oligonucleotides for biochip applications

    Trévisiol E

    2005-07-01

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

  16. Enzymatic synthesis of modified oligonucleotides by PEAR using Phusion and KOD DNA polymerases.

    Wang, Xuxiang; Zhang, Jianye; Li, Yingjia; Chen, Gang; Wang, Xiaolong

    2015-02-01

    Antisense synthetic oligonucleotides have been developed as potential gene-targeted therapeutics. We previously reported polymerase-endonuclease amplification reaction (PEAR) for amplification of natural and 5'-O-(1-thiotriphosphate) (S)-modified oligonucleotides. Here, we extended the PEAR technique for enzymatic preparation of 2'-deoxy-2'-fluoro-(2'-F) and 2'-F/S double-modified oligonucleotides. The result showed that KOD and Phusion DNA polymerase could synthesize oligonucleotides with one or two modified nucleotides, and KOD DNA polymerase is more suitable than Phusion DNA polymerase for PEAR amplification of 2'-F and 2'-F/S double modified oligonucleotides. The composition of PEAR products were analyzed by electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS) detection and showed that the sequence of the PEAR products are maintained at an extremely high accuracy (>99.9%), and after digestion the area percent of full-length modified oligonucleotides reaches 89.24%. PEAR is suitable for synthesis of modified oligonucleotides efficiently and with high purity. PMID:25517220

  17. The Use of Gel Electrophoresis to Study the Reactions of Activated Amino Acids with Oligonucleotides

    Zieboll, Gerhard; Orgel, Leslie E.

    1994-01-01

    We have used gel electrophoresis to study the primary covalent addition of amino acids to oligonu-cleotides or their analogs and the subsequent addition of further molecules of the amino acids to generate peptides covalently linked to the oligonucleotides. We have surveyed the reactions of a variety of amino acids with the phosphoramidates derived from oligonucleotide 5 inches phosphates and ethylenediamine. We find that arginine and amino acids can interact with oligonucleotidesl through stacking interactions react most efficiently. D- and L-amino acids give indistinguishable families of products.

  18. Structure of the oligonucleotide d(CGTATATACG) as a site-specific complex with nickel ions.

    Abrescia, N. G.; Malinina, L.; Fernandez, L. G.; Huynh-Dinh, T.; Neidle, S; Subirana, J A

    1999-01-01

    In this paper we explore the application of Ni2+to the crystallization of oligonucleotides. We have determined in this way the structure of a fully alternating (Y-R) decanucleotide d(CGTATATACG) by single crystal X-ray diffraction. This is the first oligonucleotide crystal structure with an alternating 5'-(TA)3-3' central part. Alternating oligonucleotides have a particular interest since they often have a unique structure. In this case the general conformation is B-like with an alternating t...

  19. Transfection Efficiency Comparison of Oligonucleotide and Plasmid to the HL-60 Cell Line with Liposomes

    TANG Yi; LIU Wenli; ZHOU Jianfeng; XU Huizhen; LU Wu

    2005-01-01

    The transfection efficiency of oligonucleotide and plasmid to the HL-60 cell line with lipofectaminePLUS was compared through observing the transfection rate and the expression duration of exogenous gene in the target cells. The results showed that the transfection rate of oligonucleotide to the HL-60 was about 90 %-95 % and it had no obvious attenuation within 84 h. However,the plasmid transfection rate was only 5 %-25 % and it was decreased significantly within 60 h. It was suggested that the transfection of oligonucleotide with liposomes was better than that of plasmid.

  20. Modulating anti-MicroRNA-21 activity and specificity using oligonucleotide derivatives and length optimization

    Munoz-Alarcon, Andres; Guterstam, Peter; Romero, Cristian;

    2012-01-01

    MicroRNAs are short, endogenous RNAs that direct posttranscriptional regulation of gene expression vital for many developmental and cellular functions. Implicated in the pathogenesis of several human diseases, this group of RNAs provides interesting targets for therapeutic intervention. Anti......-microRNA oligonucleotides constitute a class of synthetic antisense oligonucleotides used to interfere with microRNAs. In this study, we investigate the effects of chemical modifications and truncations on activity and specificity of anti-microRNA oligonucleotides targeting microRNA-21. We observed an increased activity...

  1. VIP grafted long-circulation liposome for targeted delivery of oligonucleotide to breast cancer cells

    Purpose: To investigate the use of long-circulation liposome (LCL) for the delivery of encapsulated oligonucleotide with or without radioiodine-125 (125I) to MCF-7 breast cancer cells in vitro. Methods: (1) Oligonucleotide was labeled with 125I using thallium chloride tetrahydrate (TICL3) as an oxidant. 125I-oligonucleotide was separated from free oligonucleotide or 125I by column chromatography (Sephadex G-25). The efficiency of labeling and the radiochemistry purity were obtained using chromatography of paper. (2) Oligonucleotide with or without 125I encapsulating LCL were prepared in the means of reverse-phase evaporation. The crude LCL were then repeatedly extruded through 400 nm, 200 nm, 100 nm polycarbonate membranes consecutively. Uncapsulated oligonucleotide with or without 125I was separated from LCL formulations by passing down a sephadex G-50 column in 0.01 M HEPES buffer. Pooled LCL encapsulated oligonucleotide with or without 125I fractions were sterile through 0.22 μm filters prior to use. The method of protamine sulfate precipitation was utilized to gain the efficiency of encapsulation. (3) MCF-7 cells were grown in RPMI1640 media containing 10% heat-inactivated fetal calf serum. (4)Time-dependent uptake of 125I-oligonucleotide was studied by measuring the radioactivity of MCF-7 cells. Results: The efficiency of labeling and radiochemistry purity of bcl-2 antisense-, sense- and nonsense-oligonucleotide were 84.52% and 97.49%, 58.05% and 95.40%, 74.6% and 98.7%, respectively. The efficiency of encapsulation of bcl-2 antisense-, sense- and nonsense-oligonucleotide is 77.58%, 45.98%, 38.2%, respectively. (3) The par cle size of LCL formulations(∼120 nm) was determined by laser scattering techniques. During the period of observation from 20 min to 300 min, the radioactivity of tumor cells was almost as same as the background. Conclusions: LCL can not effectively deliver oligonucleotide with 125I into MCF-7 cells in vitro. To achieve the active

  2. Abundance, Excess, Waste

    Rox De Luca

    2016-02-01

    Her recent work focuses on the concepts of abundance, excess and waste. These concerns translate directly into vibrant and colourful garlands that she constructs from discarded plastics collected on Bondi Beach where she lives. The process of collecting is fastidious, as is the process of sorting and grading the plastics by colour and size. This initial gathering and sorting process is followed by threading the components onto strings of wire. When completed, these assemblages stand in stark contrast to the ease of disposability associated with the materials that arrive on the shoreline as evidence of our collective human neglect and destruction of the environment around us. The contrast is heightened by the fact that the constructed garlands embody the paradoxical beauty of our plastic waste byproducts, while also evoking the ways by which those byproducts similarly accumulate in randomly assorted patterns across the oceans and beaches of the planet.

  3. Carboranyl Nucleosides & Oligonucleotides for Neutron Capture Therapy Final Report

    Schinazi, Raymond F.

    2004-12-01

    This proposal enabled us to synthesize and develop boron-rich nucleosides and oligonucleotide analogues for boron neutron capture therapy (BNCT) and the treatment of various malignancies. First, we determined the relationship between structure, cellular accumulation and tissue distribution of 5-o-carboranyl-2'-deoxyuridine (D-CDU) and its derivatives D-ribo-CU and 5-o-carboranyluracil (CU), to potentially target brain and other solid tumors for neutron capture therapy. Synthesized carborane containing nucleoside derivatives of CDU, D- and L-enantiomers of CDU, D-ribo-CU and CU were used. We measured tissue disposition in xenografted mice bearing 9479 human prostate tumors xenografts and in rats bearing 9L gliosarcoma isografts in their flanks and intracranially. The accumulation of D-CDU, 1-({beta}-L-arabinosyl)-5-o-carboranyluracil, D-ribo-CU, and CU were also studied in LnCap human prostate tumor cells and their retention was measured in male nude mice bearing LnCap and 9479 human prostate tumor xenografts. D-CDU, D-ribo-CU and CU levels were measured after administration in mice bearing 9479 human prostate tumors in their flanks. D-CDU achieved high cellular concentrations in LnCap cells and up to 2.5% of the total cellular compound was recovered in the 5'-monophosphorylated form. D-CDU cellular concentrations were similar in LnCap and 9479 tumor xenografts. Studies in tumor bearing animals indicated that increasing the number of hydroxyl moieties in the sugar constituent of the carboranyl nucleosides lead to increased rate and extent of renal elimination, a decrease in serum half-lives and an increased tissue specificity. Tumor/brain ratios were greatest for CDU and D-ribo-CU, while tumor/prostate ratios were greatest with CU. CDU and D-ribo-CU have potential for BNCT of brain malignancies, while CU may be further developed for prostate cancer. A method was developed for the solid phase synthesis of oligonucleotides containing (ocarboran-1-yl

  4. Carboranyl Oligonucleotides for Neutron Capture Therapy Final Report

    This proposal enabled us to synthesize and develop boron-rich nucleosides and oligonucleotide analogues for boron neutron capture therapy (BNCT) and the treatment of various malignancies. First, we determined the relationship between structure, cellular accumulation and tissue distribution of 5-o-carboranyl-2'-deoxyuridine (D-CDU) and its derivatives D-ribo-CU and 5-o-carboranyluracil (CU), to potentially target brain and other solid tumors for neutron capture therapy. Synthesized carborane containing nucleoside derivatives of CDU, D- and L-enantiomers of CDU, D-ribo-CU and CU were used. We measured tissue disposition in xenografted mice bearing 9479 human prostate tumors xenografts and in rats bearing 9L gliosarcoma isografts in their flanks and intracranially. The accumulation of D-CDU, 1-(β-L-arabinosyl)-5-o-carboranyluracil, D-ribo-CU, and CU were also studied in LnCap human prostate tumor cells and their retention was measured in male nude mice bearing LnCap and 9479 human prostate tumor xenografts. D-CDU, D-ribo-CU and CU levels were measured after administration in mice bearing 9479 human prostate tumors in their flanks. D-CDU achieved high cellular concentrations in LnCap cells and up to 2.5% of the total cellular compound was recovered in the 5'-monophosphorylated form. D-CDU cellular concentrations were similar in LnCap and 9479 tumor xenografts. Studies in tumor bearing animals indicated that increasing the number of hydroxyl moieties in the sugar constituent of the carboranyl nucleosides lead to increased rate and extent of renal elimination, a decrease in serum half-lives and an increased tissue specificity. Tumor/brain ratios were greatest for CDU and D-ribo-CU, while tumor/prostate ratios were greatest with CU. CDU and D-ribo-CU have potential for BNCT of brain malignancies, while CU may be further developed for prostate cancer. A method was developed for the solid phase synthesis of oligonucleotides containing (ocarboran-1-yl

  5. Cleavage of Oligonucleotides Containing a P3’→N5’ Phosphoramidate Linkage Mediated by Single-Stranded Oligonucleotide Templates

    Takeshi Imanishi

    2011-12-01

    Full Text Available Double-stranded DNA (dsDNA templates can hybridize to and accelerate cleavage of oligonucleotides containing a P3’→N5’ phosphoramidate (P-N linkage. This dsDNA-templated cleavage of P-N linkages could be due to conformational strain placed on the linkage upon triplex formation. To determine whether duplex formation also induced conformational strain, we examined the reactivity of the oligonucleotides with a P-N linkage in the presence of single-stranded templates, and compared these reactions to those with dsDNA templates. P-N oligonucleotides that are cleaved upon duplex formation could be used as probes to detect single-stranded nucleic acids.

  6. Correlation test to assess low-level processing of high-density oligonucleotide microarray data

    Bergh Jonas

    2005-03-01

    Full Text Available Abstract Background There are currently a number of competing techniques for low-level processing of oligonucleotide array data. The choice of technique has a profound effect on subsequent statistical analyses, but there is no method to assess whether a particular technique is appropriate for a specific data set, without reference to external data. Results We analyzed coregulation between genes in order to detect insufficient normalization between arrays, where coregulation is measured in terms of statistical correlation. In a large collection of genes, a random pair of genes should have on average zero correlation, hence allowing a correlation test. For all data sets that we evaluated, and the three most commonly used low-level processing procedures including MAS5, RMA and MBEI, the housekeeping-gene normalization failed the test. For a real clinical data set, RMA and MBEI showed significant correlation for absent genes. We also found that a second round of normalization on the probe set level improved normalization significantly throughout. Conclusion Previous evaluation of low-level processing in the literature has been limited to artificial spike-in and mixture data sets. In the absence of a known gold-standard, the correlation criterion allows us to assess the appropriateness of low-level processing of a specific data set and the success of normalization for subsets of genes.

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

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

  8. Performance of a 70-mer oligonucleotide microarray for genotyping of Campylobacter jejuni

    Ljungström Marianne

    2008-05-01

    Full Text Available Abstract Background Campylobacter jejuni is widespread in the environment and is the major cause of bacterial gastroenteritis in humans. In the present study we use microarray-based comparative genomic hybridizations (CGH, pulsed-field gel electrophoresis (PFGE and multilocus sequence typing (MLST to analyze closely related C. jejuni isolates from chicken and human infection. Results With the exception of one isolate, the microarray data clusters the isolates according to the five groups determined by PFGE. In contrast, MLST defines only three genotypes among the isolates, indicating a lower resolution. All methods show that there is no inherit difference between isolates infecting humans and chicken, suggesting a common underlying population of C. jejuni. We further identify regions that frequently differ between isolates, including both previously described and novel regions. Finally, we show that genes that belong to certain functional groups differ between isolates more often than expected by chance. Conclusion In this study we demonstrated the utility of 70-mer oligonucleotide microarrays for genotyping of Campylobacter jejuni isolates, with resolution outperforming MLST.

  9. North Sea Elasmobranchs: distribution, abundance and biodiversity

    Daan, N.; Heessen, H.J.L.; Hofstede, ter, AHM Arthur

    2005-01-01

    Based on data from various international and national surveys, an overview is given of the fine-scale distribution (resolution of 20¿longitude * 10¿ latitude; ¿ 10*10 nm) and trends in abundance of elasmobranch species reported from the North Sea. Presence-absence maps are produced based on 4 surveys, which help to delineate distribution limits of the less common species, while maps in terms of catch rates (International Bottom Trawl Survey data only) are given for the seven most common shark...

  10. Exploiting Protected Maleimides to Modify Oligonucleotides, Peptides and Peptide Nucleic Acids

    Clément Paris

    2015-04-01

    Full Text Available This manuscript reviews the possibilities offered by 2,5-dimethylfuran-protected maleimides. Suitably derivatized building blocks incorporating the exo Diels-Alder cycloadduct can be introduced at any position of oligonucleotides, peptide nucleic acids, peptides and peptoids, making use of standard solid-phase procedures. Maleimide deprotection takes place upon heating, which can be followed by either Michael-type or Diels-Alder click conjugation reactions. However, the one-pot procedure in which maleimide deprotection and conjugation are simultaneously carried out provides the target conjugate more quickly and, more importantly, in better yield. This procedure is compatible with conjugates involving oligonucleotides, peptides and peptide nucleic acids. A variety of cyclic peptides and oligonucleotides can be obtained from peptide and oligonucleotide precursors incorporating protected maleimides and thiols.

  11. Oligonucleotides with rapid turnover of the phosphate groups occur endogenously in eukaryotic cells

    Endogenous oligonucleotides were found in trichloroacetic acid extracts of hamster lung fibroblasts and Tetrahymena cells. Peaks of radioactivity that eluted with retention times similar to oligonucleotide markers (5- to 50-mer) were found by HPLC in cells labeled briefly with 32Pi. Only minute amounts of UV-absorbing material were detected, consistent with a rapid turnover of phosphate groups. The 32P-labeled material also migrated as oligonucleotides on 20% polyacrylamide gels; it was not hydrolyzed by alkaline phosphatase but was digested by snake venom phosphodiesterase, S1 nuclease, and pancreatic RNase and was phosphorylated by T4 polynucleotide kinase. The 32P-labeled material isolated by HPLC was alkali labile and the hydrolyzate ran as nucleotides on paper chromatography. It is concluded that the oligonucleotides are mainly oligoribonucleotides, but it is possible that oligodeoxynucleotides are also present

  12. Fast, copper-free click chemistry: a convenient solid-phase approach to oligonucleotide conjugation

    Singh, Ishwar; Vyle, Joseph S.; Heaney, Frances

    2009-01-01

    Solid-phase oligonucleotide conjugation by nitrile oxide–alkyne click cycloaddition chemistry has been successfully demonstrated; the reaction, compatible with all nucleobases, requires no metal catalyst and proceeds under physiological conditions.

  13. Use of synthetic oligonucleotides for genomic DNA dot hybridization to split the DQw3 haplotype.

    Martell, M; Le Gall, I; Millasseau, P; Dausset, J; Cohen, D

    1988-01-01

    Comparison of two different HLA-DQ beta gene sequences from two DR4 individuals, probably corresponding to DQw3.2 (DQR4) and DQw3.1 (DQR5) specificities, has shown several nucleotide variations. Eight oligonucleotides (24 bases long), derived from these polymorphic areas, have been synthesized. Each oligonucleotide was hybridized to BamHI-digested DNA samples from eight families with HLA-DR4 individuals. Four polymorphic BamHI fragments were detected. Two of eight oligonucleotides gave a single signal (8.9 kilobases) on DQw3.2-positive haplotypes. We used one of these oligonucleotides in a genomic DNA dot hybridization and detected a hybridization signal only in DQw3.2-positive individuals. A very simple test like this allows the screening of a large population sample within a very short period. Images PMID:2895927

  14. On-line coupling of capillary gel electrophoresis with electrospray mass spectrometry for oligonucleotide analysis.

    Freudemann, T; von Brocke, A; Bayer, E

    2001-06-01

    Homooligodeoxyribonucleotides differing one nucleotide in length from 12- to 15-mer and from 17- to 20-mer were separated by size with capillary gel electrophoresis (CGE) using an entangled polymer solution in coated capillaries. The resolved components were analyzed by on-line coupling of CGE with electrospray mass spectrometry (ES-MS), denoted as CGE/ES-MS, in the full-scan negative ion detection mode. Baseline separation was achieved for the 12-15-mer oligonucleotide mixtures. Both synthetic phosphodiester oligonucleotide mixtures as well as their phosphorothioate analogues, serving as model compounds for antisense oligonucleotides, could be analyzed by on-line CGE/ES-MS coupling. Terminally phosphorylated and nonphosphorylated synthetic failure sequences could be electrophoretically separated and mass spectrometically characterized as well. This methodology might be a useful tool for synthesis control of phosphodiester oligonucleotides as well as for analysis of phosphorothioate analogues as they are used in antisense drug development. PMID:11403304

  15. Primordial Deuterium Abundance Measurements

    Levshakov, S A; Takahara, F; Levshakov, Sergei A.; Kegel, Wilhelm H.; Takahara, Fumio

    1997-01-01

    Deuterium abundances measured recently from QSO absorption-line systems lie in the range from 3 10^{-5} to 3 10^{-4}, which shed some questions on standard big bang theory. We show that this discordance may simply be an artifact caused by inadequate analysis ignoring spatial correlations in the velocity field in turbulent media. The generalized procedure (accounting for such correlations) is suggested to reconcile the D/H measurements. An example is presented based on two high-resolution observations of Q1009+2956 (low D/H) [1,2] and Q1718+4807 (high D/H) [8,9]. We show that both observations are compatible with D/H = 4.1 - 4.6 10^{-5}, and thus support SBBN. The estimated mean value = 4.4 10^{-5} corresponds to the baryon-to-photon ratio during SBBN eta = 4.4 10^{-10} which yields the present-day baryon density Omega_b h^2 = 0.015.

  16. P123-T Oligonucleotide Purification Strategies using a New High-Capacity Anion Exchange Resin

    Deetz, M.; Fisher, J. R.; Gehris, A.; Maikner, J.; Kinzey, M.

    2007-01-01

    With the advent of nucleic acid silencing technologies and the need for high purity diagnostic and therapeutic oligonucleotides, there is a need for high-capacity chromatographic supports that can deliver economic purification processes. A new, 30 micron, mono-sized, polymeric resin has been recently developed that provides high resolution and high capacity for synthetic oligonucleotides. Physical properties of this new resin will be described, including particle size uniformity, ion exchange...

  17. Ligation of oligonucleotides to nucleic acids or proteins via disulfide bonds.

    Chu, B C; Orgel, L E

    1988-01-01

    We have developed general methods for joining together, via cleavable disulfide bonds, either two unprotected polynucleotides or a polynucleotide and a peptide or protein. To join two oligonucleotides, each is first converted to an adduct in which cystamine is joined to the 5'-terminal phosphate of the oligonucleotide by a phosphoramidate bond. The adducts are mixed and reduced with dithiothreitol. The dithiothreitol is then removed by dialysis. Oxidation by atmospheric oxygen occurs to yield...

  18. Design and synthesis of polyacrylamide-based oligonucleotide supports for use in nucleic acid diagnostics.

    Fahy, E.; Davis, G R; DiMichele, L J; Ghosh, S. S.

    1993-01-01

    Polyacrylamide supports, in a range of pore sizes, were investigated as nucleic acid affinity matrices for the detection of target DNA or RNA sequences using a sandwich hybridization format. Bromoacetyl and thiol oligonucleotide derivatives were covalently linked to sulfhydryl- and bromoacetyl-polyacrylamide supports with greater than 95% end-attachment efficiencies. These polyacrylamide-oligonucleotide supports were further derivatized with anionic residues to provide multi-functional suppor...

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

    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.

  20. Development, Characterization and Experimental Validation of a Cultivated Sunflower (Helianthus annuus L.) Gene Expression Oligonucleotide Microarray

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

    2012-01-01

    Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed us...

  1. Synthesis and Properties of Oligonucleotides Carrying Isoquinoline Imidazo[1,2-a]azine Fluorescent Units

    Pérez-Rentero, Sonia; Kielland, Nicola; Terrazas, Montserrat; Lavilla, Rodolfo; Eritja Casadellà, Ramón

    2010-01-01

    Oligonucleotides carrying novel fluorescent compounds with a dipolar isoquinoline imidazo[1,2-a]azine core were prepared. Analysis of the melting curves demonstrates that DNA duplexes carrying these fluorescent labels at their ends have a slight increase in DNA duplex stability. The UV absorption and fluorescent properties of the oligonucleotide conjugates were analyzed. The fluorescent label is sensitive to duplex formation, as cooperative melting curves are also observed at 366 nm and fluor...

  2. Improved bioactivity of G-rich triplex-forming oligonucleotides containing modified guanine bases

    Rogers, Faye A.; Lloyd, Janice A; Tiwari, Meetu Kaushik

    2014-01-01

    Triplex structures generated by sequence-specific triplex-forming oligonucleotides (TFOs) have proven to be promising tools for gene targeting strategies. In addition, triplex technology has been highly utilized to study the molecular mechanisms of DNA repair, recombination and mutagenesis. However, triplex formation utilizing guanine-rich oligonucleotides as third strands can be inhibited by potassium-induced self-association resulting in G-quadruplex formation. We report here that guanine-r...

  3. Understanding oligonucleotide-mediated inhibition of gene expression in Xenopus laevis oocytes

    Bailey, Cheryl; Weeks, Daniel L.

    2000-01-01

    Triplex-forming oligonucleotides (TFOs) modified with N,N-diethylethylenediamine can inhibit the expression of a reporter plasmid in Xenopus oocytes if the triplex is preformed prior to injection while unmodified oligonucleotides cannot. Here we show that merely forming a triplex in a reporter plasmid does not disrupt transcription, but when TFOs are targeted to sites within the transcribed region of a reporter gene then gene activity is inhibited. TFO-based inhibition did not lead to large s...

  4. Positively charged oligonucleotides overcome potassium-mediated inhibition of triplex DNA formation.

    Dagle, J M; Weeks, D L

    1996-01-01

    The formation of triplex DNA using unmodified, purine-rich oligonucleotides (ODNs) is inhibited by physiologic levels of potassium. Changing negative phosphodiester bonds in a triplex forming oligonucleotide (TFO) to neutral linkages causes a small increase in triplex formation. When phosphodiester bonds in a TFO are converted to positively-charged linkages the formation of triplex DNA increases dramatically. In the absence of KCl, a 17mer TFO containing 11 positively-charged linkages at a co...

  5. Synthesis and properties of triplex-forming oligonucleotides containing 2'-modified nucleoside analogues

    Lou, Chenguang

    2011-01-01

    Triplex-forming oligonucleotides (TFOs) bind to the major groove of the DNA duplex via the Hoogsteen interactions to generate triple helices. Potential applications of triplex technology are in regulation of gene expression, site-directed gene-knockout, mutation correction and as tools in molecular biotechnology. The presence of 2’-modified nucleosides in therapeutic oligonucleotides inhibits enzymatic degradation in vivo. Therefore such sugar modifications have the potential to improve the b...

  6. Targeted inhibition of transcription elongation in cells mediated by triplex-forming oligonucleotides

    Faria, M.; Wood, C. D.; Perrouault, L; Nelson, J. S.; Winter, A.; White, M. R. H.; Hélène, C; Giovannangeli, C.

    2000-01-01

    Triple-helix-forming oligonucleotides (TFOs) bind in the major groove of double-stranded DNA at oligopyrimidine⋅oligopurine sequences and therefore are candidate molecules for artificial gene regulation, in vitro and in vivo. We recently have described oligonucleotide analogues containing N3′-P5′ phosphoramidate (np) linkages that exhibited efficient inhibition of transcription elongation in vitro. In the present work we provide conclusive evidence that np-modified TFOs targeted to the HIV-1 ...

  7. In vivo stability and kinetics of absorption and disposition of 3' phosphopropyl amine oligonucleotides.

    Zendegui, J G; Vasquez, K M; Tinsley, J H; Kessler, D J; Hogan, M E

    1992-01-01

    Development of oligonucleotide derivatives as therapeutic agents requires an understanding of their pharmacokinetic behavior. The in vivo disposition and stability of a prototype of such compounds are reported here. The compound studied, a relatively G-rich 38 base 3' phosphopropyl amine oligonucleotide (TFO-1), was cleared from the circulation with a half-life of approximately 10 minutes, displaying distribution kinetics consistent with a two compartment model. TFO-1 was also readily absorbe...

  8. Spatial organization of topoisomerase I-mediated DNA cleavage induced by camptothecin–oligonucleotide conjugates

    Arimondo, Paola B.; Angenault, Stéphane; Halby, Ludovic; Boutorine, Alexandre; Schmidt, Frédéric; Monneret, Claude; Garestier, Thérèse; Sun, Jian-Sheng; Bailly, Christian; Hélène, Claude

    2003-01-01

    Triple helix-forming oligonucleotides covalently linked to topoisomerase I inhibitors, in particular the antitumor agent camptothecin, trigger topoisomerase I-mediated DNA cleavage selectively in the proximity of the binding site of the oligonucleotide vector. In the present study, we have performed a systematic analysis of the DNA cleavage efficiency as a function of the positioning of the camptothecin derivative, either on the 3′ or the 5′ side of the triplex, and the location of the cleava...

  9. A New Achiral Linker Reagent for the Incorporation of Multiple Amino Groups Into Oligonucleotides

    1997-01-01

    The present invention relates to a new functionalized achiral linker reagent for incorporating multiple primary amino groups or reporter groups into oligonucleotides following the phosphoramidite methodology. It is possible to substitute any ribodeoxynucleotide, deoxynucleotide, or nucleotide with...... the linker in conventional phosphoamidite or H-phosphonate DNA syntheses. Directly, or via a post modification step, an oligonucleotide is labelled with one or more reporter moieties, e.g. dansyl (5-dimethylamino)-1-naphthalenesulfonyl), biotin, digoxigenin, DOXYL (N-oxyl-4,4-dimethyloxazolidine...

  10. Complexes of carbon nanotubes with oligonucleotides in thin Langmuir-Blodgett films to detect electrochemically hybridization

    Egorov, A. S.; Egorova, V. P.; Krylova, H. V.; Lipnevich, I. V.; Orekhovskaya, T. I.; Veligura, A. A.; Govorov, M. I.; Shulitsky, B. G.

    2014-10-01

    Self-assembled complexes consisting of thin multi-walled carbon nanotubes (MWCNTs) and DNA-oligonucleotides which are able to a cooperative binding to complementary oligonucleotides have been investigated. It was establised a high-performance charge transport in nanostructured Langmuir-Blodgett complexes thin MWCNTs/DNA. A method to electrochemically detect DNA hybridization on the self-organized structures has been proposed.

  11. Fragment-based solid-phase assembly of oligonucleotide conjugates with peptide and polyethylene glycol ligands.

    Dirin, Mehrdad; Urban, Ernst; Noe, Christian R; Winkler, Johannes

    2016-10-01

    Ligand conjugation to oligonucleotides is an attractive strategy for enhancing the therapeutic potential of antisense and siRNA agents by inferring properties such as improved cellular uptake or better pharmacokinetic properties. Disulfide linkages enable dissociation of ligands and oligonucleotides in reducing environments found in endosomal compartments after cellular uptake. Solution-phase fragment coupling procedures for producing oligonucleotide conjugates are often tedious, produce moderate yields and reaction byproducts are frequently difficult to remove. We have developed an improved method for solid-phase coupling of ligands to oligonucleotides via disulfides directly after solid-phase synthesis. A 2'-thiol introduced using a modified nucleotide building block was orthogonally deprotected on the controlled pore glass solid support with N-butylphosphine. Oligolysine peptides and a short monodisperse ethylene glycol chain were successfully coupled to the deprotected thiol. Cleavage from the resin and full removal of oligonucleotide protection groups were achieved using methanolic ammonia. After standard desalting, and without further purification, homogenous conjugates were obtained as demonstrated by HPLC, gel electrophoresis, and mass spectrometry. The attachment of both amphiphilic and cationic ligands proves the versatility of the conjugation procedure. An antisense oligonucleotide conjugate with hexalysine showed pronounced gene silencing in a cell culture tumor model in the absence of a transfection reagent and the corresponding ethylene glycol conjugate resulted in down regulation of the target gene to nearly 50% after naked application. PMID:27236069

  12. Polymorphism in Sahiwal breed of zebu cattle revealed using synthetic oligonucleotide markers

    ''2P using the enzyme polynucleotide kinase by the standard procedure. Hybridization of labelled oligonucleotide probes to genomic DNA on Nylon membranes was carried out at 45 deg. C for probes (GTG)5 and (TCC)5, 43 deg. C for (GT)8 and 65 deg. C for (GT)12. Post-hybridization treatments and autoradiography were carried out and size of each fragment on X-ray film, i.e. DNA fingerprint, was estimated using computer software GelBase (UVP, UK). Number of total bands and shared bands in the fingerprints of each individual were recorded in the range of 2.5 to 23.0 KB. Number of bands, average band sharing rate (BS), mean allelic frequencies (a) and heterozygosity (h) level were calculated. All four probes used produced multilocus fingerprints with differing levels of polymorphism. Means of number of bands per individual, band sharing rate, allele frequencies and heterozygosity was calculated. The probes (GT)8, (GT)12 and (TCC)5 produced fingerprinting patterns of medium to low polymorphism whereas the probe (GTG)5 produced highly polymorphic pattern. The probe (GT)8 probe produced as many as 32 bands in resolvable portion of the gel. However, nearly 40% of the bands were shared by all the individuals hence, the average bands sharing rate was found to be high. High band sharing rate in this study indicate that the animals examined might be genetically more homogeneous with respect to (GT)n sequences. Comparison of average number of bands obtained between different probes reveal that the probe GT8 hybridized to more number of fragments than the other probes. This result indicates that GTn are more abundant in zebu cattle genome compared to other sequences studied. The probe (GT)12 produced a multilocus fingerprints with lower level of polymorphism in comparison with (GT)8 fingerprints. Mean number of bands and polymorphism were low as compared to (GT)8 fingerprints. Variation in the nucleotide constitution of repeat sequences and differences in hybridization and stringency

  13. Chromosome-Specific Painting in Cucumis Species Using Bulked Oligonucleotides.

    Han, Yonghua; Zhang, Tao; Thammapichai, Paradee; Weng, Yiqun; Jiang, Jiming

    2015-07-01

    Chromosome-specific painting is a powerful technique in molecular cytogenetic and genome research. We developed an oligonucleotide (oligo)-based chromosome painting technique in cucumber (Cucumis sativus) that will be applicable in any plant species with a sequenced genome. Oligos specific to a single chromosome of cucumber were identified using a newly developed bioinformatic pipeline and then massively synthesized de novo in parallel. The synthesized oligos were amplified and labeled with biotin or digoxigenin for use in fluorescence in situ hybridization (FISH). We developed three different probes with each containing 23,000-27,000 oligos. These probes spanned 8.3-17 Mb of DNA on targeted cucumber chromosomes and had the densities of 1.5-3.2 oligos per kilobases. These probes produced FISH signals on a single cucumber chromosome and were used to paint homeologous chromosomes in other Cucumis species diverged from cucumber for up to 12 million years. The bulked oligo probes allowed us to track a single chromosome in early stages during meiosis. We were able to precisely map the pairing between cucumber chromosome 7 and chromosome 1 of Cucumis hystrix in a F1 hybrid. These two homeologous chromosomes paired in 71% of prophase I cells but only 25% of metaphase I cells, which may provide an explanation of the higher recombination rates compared to the chiasma frequencies between homeologous chromosomes reported in plant hybrids. PMID:25971668

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

    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.

  15. Characterization of adjacent breast tumors using oligonucleotide microarrays

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

  16. Advancements of antisense oligonucleotides in treatment of breast cancer

    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.

  17. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

    Kim, Jinho; Olsen, Timothy R.; Zhu, Jing; Hilton, John P.; Yang, Kyung-Ae; Pei, Renjun; Stojanovic, Milan N.; Lin, Qiao

    2016-05-01

    We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling highly efficient isolation of aptamers in drastically reduced times and with minimized consumption of biological material. The approach as such also offers broad target applicability by allowing selection of aptamers with respect to targets that are either surface-immobilized or solution-borne, potentially allowing aptamers to be developed as readily available affinity reagents for a wide range of targets. We demonstrate the utility of this approach on two different procedures, respectively for isolating aptamers against a surface-immobilized protein (immunoglobulin E) and a solution-phase small molecule (bisboronic acid in the presence of glucose). In both cases aptamer candidates were isolated in three rounds of SELEX within a total process time of approximately 10 hours.

  18. Nanoexplosive gene therapy using triplex-forming oligonucleotides

    Oh, Eun Jung; Min, Hye Jung; Choe, Jae Gol; Park, Gil Hong; Kim, Meyoung Kon [College of Medicine, Korea Univ., Seoul (Korea, Republic of)

    2001-07-01

    Triplex forming oligonucleotides (TFO) labeled with Auger emitter could be ideal vehicles for delivering radiation energy to specific DNA sequences, and followed by double-stranded DNA breaks and subsequent inactivation of targeted genes. We designed TFOs targeting the selected DNA fragments (i.e., estrogen receptors and N-myc promoter) and labeled with {sup 125}I and {sup 111}In. Various Cancer cells, e.g., MCF-7 (breast adenocarcinoma), MCF-10A (immortalized breast cells), Jurkat (T-cell leukemia), ARO (thyroid cancer), SNU-449 (Colon Caner), and HL-60 (polymyelocytic leukemia), were prepared and treated with radiolabeled TFO for 24 h. After the incubation, subcellular fractions (i.e., cell nucleus, cytoplasm and cultured medium) were collected and measured radioactivity by a gamma scintillation counter, respectively. The mean value of % injected dose for each fraction was ranged as follows: nucleus, 4.4-20%; cytoplasm, 8.2-29%; and medium, 64-87%. Therefore, we speculated that TFO labeled with Auger emitter could be a next-generation therapeutic tool in nanoexplosive gene therapy.

  19. Triplex formation on DNA targets: how to choose the oligonucleotide.

    Vekhoff, Pierre; Ceccaldi, Alexandre; Polverari, David; Pylouster, Jean; Pisano, Claudio; Arimondo, Paola B

    2008-11-25

    Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA binders. TFOs provide a tool for controlling gene expression or, when attached to an appropriate chemical reagent, for directing DNA damage. Here, we report a set of rules for predicting the best out of five different triple-helical binding motifs (TM, UM, GA, GT, and GU, where M is 5-methyldeoxycytidine and U is deoxyuridine) by taking into consideration the sequence composition of the underlying duplex target. We tested 11 different triplex targets present in genes having an oncogenic role. The rules have predictive power and are very useful in the design of TFOs for antigene applications. Briefly, we retained motifs GU and TM, and when they do form a triplex, TFOs containing G and U are preferred over those containing T and M. In the case of the G-rich TFOs, triplex formation is principally dependent on the percentage of G and the length of the TFO. In the case of the pyrimidine motif, replacement of T with U is destabilizing; triplex formation is dependent on the percentage of T and destabilized by the presence of several contiguous M residues. An equation to choose between a GU and TM motif is given. PMID:18954091

  20. Nanoexplosive gene therapy using triplex-forming oligonucleotides

    Triplex forming oligonucleotides (TFO) labeled with Auger emitter could be ideal vehicles for delivering radiation energy to specific DNA sequences, and followed by double-stranded DNA breaks and subsequent inactivation of targeted genes. We designed TFOs targeting the selected DNA fragments (i.e., estrogen receptors and N-myc promoter) and labeled with 125I and 111In. Various Cancer cells, e.g., MCF-7 (breast adenocarcinoma), MCF-10A (immortalized breast cells), Jurkat (T-cell leukemia), ARO (thyroid cancer), SNU-449 (Colon Caner), and HL-60 (polymyelocytic leukemia), were prepared and treated with radiolabeled TFO for 24 h. After the incubation, subcellular fractions (i.e., cell nucleus, cytoplasm and cultured medium) were collected and measured radioactivity by a gamma scintillation counter, respectively. The mean value of % injected dose for each fraction was ranged as follows: nucleus, 4.4-20%; cytoplasm, 8.2-29%; and medium, 64-87%. Therefore, we speculated that TFO labeled with Auger emitter could be a next-generation therapeutic tool in nanoexplosive gene therapy

  1. Preparation and detection of nonradioactive nucleic acid and oligonucleotide

    There is increasing interest worldwide in the development of nucleic acid probes which are detected by nonradioactive means. In the research laboratory, the use of 32P for detection is undoubtedly the method of choice and is likely to remain so for the forseeable future, in spite of the half life of only 14 days for 32P. In the diagnostic laboratory on the other hand, the use of nonradioactive probes has many potential advantages. Perhaps the major one is that nonradioactive probes are stable for at least 6 to 12 months, and probably much longer if properly stored, thus leading to a substantial reduction in cost by obviating the need to prepare them every 2 to 3 weeks. In addition, there is no radiation exposure from routine daily use and there are no storage and disposal problems. Numerous methods are described in this chapter for the preparation by enzymatic and chemical techniques of nonradioactive nucleic acid and oligonucleotide probes. In many cases, the resulting probes have yet to be fully tested under hybridization conditions. In others, initial results look very promising since some nonradioactive probes can provide a sensitivity of detection of target sequences similar to that provided by 32P-labeled probes

  2. Quality assurance of radiolabeled proteins, peptides and antisense oligonucleotides

    Radiopharmaceuticals (RP) labeled with nonmetallic (I-123, C-11, F-18) and metallic radionuclides (Tc-99m, Ga-67, In-111) are used for diagnosis and therapy; they could be classified as blood flow markers, metabolic substrates, receptor ligands, peptide/proteins and antisense oligonucleotide analogs (I-123, In-111). For safety and efficacy of the test using these tracers, quality assurance (QA) of RP (Chemical, radionuclidic, radiochemical impurities, enantiomers, immunoreactivity, sterility, apyrogenicity, cell-viability) is required. This test is more critical for the RP under clinical investigations. FDA allows a maximum permissible limit of 10% of the injected radionuclide as impurity. Quality assurance of RP is carried out by thin-layer, size-exclusion and high pressure liquid chromatography. For therapeutic RP labeled with I-131 (β,γ), Re-186 (β,γ), Re-188 (β), Y-90 (β), Y-90 (β), At-211(α) and Bi-212 (α), etc., the level of chemical alterations/degradations, directly by energetic particles or indirectly by free-radicals, is higher for the α-,β- than γ-emitting RP and chemical alterations are time-dependent processes. Considering the adverse reactions (marrow-suppression), unnecessary radiation due to unbound tracers and impurities, QA of RP should be performed and impurities eliminated before RP administration

  3. Antineoplastic Effect of Decoy Oligonucleotide Derived from MGMT Enhancer

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

    2014-01-01

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

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

    Tamar Canello

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

  5. Dicationic Surfactants with Glycine Counter Ions for Oligonucleotide Transportation.

    Pietralik, Zuzanna; Skrzypczak, Andrzej; Kozak, Maciej

    2016-08-01

    Gemini surfactants are good candidates to bind, protect, and deliver nucleic acids. Herein, the concept of amino acids (namely glycine) as counter ions of gemini surfactants for gene therapy application was explored. This study was conducted on DNA and RNA oligomers and two quaternary bis-imidazolium salts, having 2,5-dioxahexane and 2,8-dioxanonane spacer groups. The toxicity level of surfactants was assessed by an MTT assay, and their ability to bind nucleic acids was tested through electrophoresis. The nucleic acid conformation was established based on circular dichroism and infrared spectroscopic analyses. The structures of the formed complexes were characterized by small-angle scattering of synchrotron radiation. Both studied surfactants appear to be suitable for gene therapy; however, although they vary by only three methylene groups in the spacer, they differ in binding ability and toxicity. The tested oligonucleotides maintained their native conformations upon surfactant addition and the studied lipoplexes formed a variety of structures. In systems based on a 2,5-dioxahexane spacer, a hexagonal phase was observed for DNA-surfactant complexes and a micellar phase was dominant with RNA. For the surfactant with a 2,8-dioxanonane spacer group, the primitive cubic phase prevailed. PMID:27214208

  6. Novel Cationic Carotenoid Lipids as Delivery Vectors of Antisense Oligonucleotides for Exon Skipping in Duchenne Muscular Dystrophy

    Vassilia Partali

    2012-01-01

    Full Text Available Duchenne Muscular Dystrophy (DMD is a common, inherited, incurable, fatal muscle wasting disease caused by deletions that disrupt the reading frame of the DMD gene such that no functional dystrophin protein is produced. Antisense oligonucleotide (AO-directed exon skipping restores the reading frame of the DMD gene, and truncated, yet functional dystrophin protein is expressed. The aim of this study was to assess the efficiency of two novel rigid, cationic carotenoid lipids, C30-20 and C20-20, in the delivery of a phosphorodiamidate morpholino (PMO AO, specifically designed for the targeted skipping of exon 45 of DMD mRNA in normal human skeletal muscle primary cells (hSkMCs. The cationic carotenoid lipid/PMO-AO lipoplexes yielded significant exon 45 skipping relative to a known commercial lipid, 1,2-dimyristoyl-sn-glycero-3-ethylphosphocholine (EPC.

  7. Polycationic ligands of different chemical classes stimulate DNA strand displacement between short oligonucleotides in a protein-free system.

    Volodin, Alexander A; Bocharova, Tatiana N; Smirnova, Elena A

    2016-09-01

    The ability of polycationic ligands to stimulate DNA strand displacement between short oligonucleotides in a protein-free system is demonstrated. We show that two ligands, tetracationic aliphatic amine (spermine) and a dicationic intercalating drug (chloroquine), promote strand displacement in a concentration-dependent manner. At low concentrations both ligands decelerate spontaneous strand displacement because of their impact on the stability of the DNA duplex. At elevated concentrations they accelerate strand displacement via formation of intermediate structures containing three DNA strands. The rate of the last process does not correlate with the thermal dissociation rate of the entire DNA duplex. It indicates that, possibly, the action of these agents cannot be explained by their influence on the stability of the DNA duplex. In general, our results suggest that the ability to stimulate DNA strand displacement appears to be a common feature of polycations of different chemical and structural classes. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 633-641, 2016. PMID:27106951

  8. Down-regulation of Survivin by Antisense Oligonucleotides Increases Apoptosis, Inhibits Cytokinesis and Anchorage-Independent Growth

    Jun Chen

    2000-05-01

    Full Text Available Survivin, a member of the inhibitor of apoptosis protein (IAP family, is detected in most common human cancers but not in adjacent normal cells. Previous studies suggest that survivin associates with the mitotic spindle and directly inhibits caspase activity. To further investigate the function of survivin, we used a survivin antisense (AS oligonucleotide to downregulate survivin expression in normal and cancer cells. We found that inhibition of survivin expression increased apoptosis and polyploidy while decreasing colony formation in soft agar. Immunohistochemistry showed that cells without survivin can initiate the cleavage furrow and contractile ring, but cannot complete cytokinesis, thus resulting in multinucleated cells. These findings indicate that survivin plays important roles in a late stage of cytokinesis, as well as in apoptosis.

  9. Assessing differential gene expression with small sample sizes in oligonucleotide arrays using a mean-variance model.

    Hu, Jianhua; Wright, Fred A

    2007-03-01

    The identification of the genes that are differentially expressed in two-sample microarray experiments remains a difficult problem when the number of arrays is very small. We discuss the implications of using ordinary t-statistics and examine other commonly used variants. For oligonucleotide arrays with multiple probes per gene, we introduce a simple model relating the mean and variance of expression, possibly with gene-specific random effects. Parameter estimates from the model have natural shrinkage properties that guard against inappropriately small variance estimates, and the model is used to obtain a differential expression statistic. A limiting value to the positive false discovery rate (pFDR) for ordinary t-tests provides motivation for our use of the data structure to improve variance estimates. Our approach performs well compared to other proposed approaches in terms of the false discovery rate. PMID:17447928

  10. The Galactic Thick Disk Stellar Abundances

    Prochaska, J X; Carney, B W; McWilliam, A; Wolfe, A M; Prochaska, Jason X.; Naumov, Sergei O.; Carney, Bruce W.; William, Andrew Mc; Wolfe, Arthur M.

    2000-01-01

    We present first results from a program to measure the chemical abundances of a large (N>30) sample of thick disk stars with the principal goal of investigating the formation history of the Galactic thick disk. Our analysis confirms previous studies of O and Mg in the thick disk stars which reported enhancements in excess of the thin disk population. Furthermore, the observations of Si, Ca, Ti, Mn, Co, V, Zn, Al, and Eu all argue that the thick disk population has a distinct chemical history from the thin disk. With the exception of V and Co, the thick disk abundance patterns match or tend towards the values observed for halo stars with [Fe/H]~-1. This suggests that the thick disk stars had a chemical enrichment history similar to the metal-rich halo stars. With the possible exception of Si, the thick disk abundance patterns are in excellent agreement with the chemical abundances observed in the metal-poor bulge stars suggesting the two populations formed from the same gas reservoir at a common epoch. We disc...

  11. Diffusion of Oligonucleotides from within Iron-Crosslinked Polyelectrolyte-Modified Alginate Beads: A Model System for Drug Release

    Privman, Vladimir; Luz, Roberto A S; Guz, Nataliia; Glasser, M Lawrence; Katz, Evgeny

    2016-01-01

    We developed and experimentally verified an analytical model to describe diffusion of oligonucleotides from stable hydrogel beads. The synthesized alginate beads are Fe3+-cross-linked as well as polyelectrolyte-doped for uniformity and stability at physiological pH. Data on diffusion of oligonucleotides from inside the beads provide physical insights into the volume nature of the immobilization of a fraction of oligonucleotides due to polyelectrolyte cross-linking, i.e., the absence of the surface-layer barrier in this case. Furthermore, our results suggest a new simple approach to measuring the diffusion coefficient of the mobile oligonucleotide molecules inside hydrogel. The considered alginate beads provide a model for a well-defined component in drug release systems and for the oligonucleotide-release transduction steps in drug-delivering and biocomputing applications. This is illustrated by destabilizing the beads with citrate that induces full oligonucleotide release with non-diffusional kinetics.

  12. Diffusion of Oligonucleotides from within Iron-Cross-Linked, Polyelectrolyte-Modified Alginate Beads: A Model System for Drug Release.

    Privman, Vladimir; Domanskyi, Sergii; Luz, Roberto A S; Guz, Nataliia; Glasser, M Lawrence; Katz, Evgeny

    2016-04-01

    An analytical model to describe diffusion of oligonucleotides from stable hydrogel beads is developed and experimentally verified. The synthesized alginate beads are Fe(3+) -cross-linked and polyelectrolyte-doped for uniformity and stability at physiological pH. Data on diffusion of oligonucleotides from inside the beads provide physical insights into the volume nature of the immobilization of a fraction of oligonucleotides due to polyelectrolyte cross-linking, that is, the absence of a surface-layer barrier in this case. Furthermore, the results suggest a new simple approach to measuring the diffusion coefficient of mobile oligonucleotide molecules inside hydrogels. The considered alginate beads provide a model for a well-defined component in drug-release systems and for the oligonucleotide-release transduction steps in drug-delivering and biocomputing applications. This is illustrated by destabilizing the beads with citrate, which induces full oligonucleotide release with nondiffusional kinetics. PMID:26762598

  13. Intercalator conjugates of pyrimidine locked nucleic acid-modified triplex-forming oligonucleotides: improving DNA binding properties and reaching cellular activities

    Brunet, Erika; Corgnali, Maddalena; Perrouault, Loïc; Roig, Victoria; Asseline, Ulysse; Sørensen, Mads D.; Babu, B. Ravindra; Wengel, Jesper; Giovannangeli, Carine

    2005-01-01

    Triplex-forming oligonucleotides (TFOs) are powerful tools to interfere sequence-specifically with DNA-associated biological functions. (A/T,G)-containing TFOs are more commonly used in cells than (T,C)-containing TFOs, especially C-rich sequences; indeed the low intracellular stability of the non-covalent pyrimidine triplexes make the latter less active. In this work we studied the possibility to enhance DNA binding of (T,C)-containing TFOs, aiming to reach cellular activities; to this end, ...

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

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

  15. Modified oligonucleotides for triple helix studies and for the obtention of structures with biomedical and technological interest

    Alvira Torre, Margarita

    2010-01-01

    [eng] Oligonucleotides are short fragments of DNA (10-100nt) which are of great interest because their applications in molecular biology, biomedicine and nanotechnology. As a result of their ability to base pairing, oligonucleotides can be used as primers, hybridization probes in biosensors, agents for controlling gene expression, structural material in nanotechnology or as substrates for a variety of biochemical and biophysical studies. Chemical modification of oligonucleotides as well as co...

  16. Inhibition of transcription by platinated triplex-forming oligonucleotides.

    Graham, Mindy K; Miller, Paul S

    2012-12-01

    Platinated triplex-forming oligonucleotides (TFOs) consisting of 2'-methoxythymidine and 2'-methoxy-5-methylcytidine and an N-7 platinated deoxyguanosine ((Pt)G) at the 5'-((Pt)G-TFO), 3'-(TFO-G(Pt)), or 3'- and 5'-((Pt)G-TFO-G(Pt)) ends of the TFO form mono-((Pt)G-TFO and TFO-G(Pt)) and interstrand ((Pt)G-TFO-G(Pt)) cross-links with target DNA as a result of reaction of the (Pt)G with guanines adjacent to the homopurine TFO binding site in the target. The extent of cross-linking is greatest when the (Pt)G is located on the 3' end of the TFO and the target guanine is on the same strand as the TFO binding site. Multiple, contiguous deoxyguanosines in the TFO binding site or a cytosine adjacent to the G(Pt) of the TFO significantly reduce cross-linking. DNA reporter plasmids in which platinated TFOs were cross-linked at a site in the transcribed region between a CMV promoter and a luciferase reporter gene were transfected into Chinese hamster ovary cells, and luciferase expression was compared with that for the corresponding non-cross-linked plasmid. Luciferase expression was inhibited 95 % when TFO-G(Pt) was bound and cross-linked to the transcribed strand, demonstrating that the cross-linked TFO was able to block transcription elongation. Further inhibition (99 %) was observed in nucleotide excision repair (NER) deficient cells, suggesting that NER may repair this lesion. The 3'-G(Pt) group of TFO-G(Pt) protects the TFO from degradation by exonucleases found in mammalian serum. Taken together, these results suggest that platinated TFOs of the type TFO-G(Pt) may find applications as agents for suppressing DNA transcription and consequently inhibiting gene expression in mammalian cells. PMID:22965663

  17. Solar System Abundances of the Elements

    Lodders, Katharina

    2010-01-01

    Representative abundances of the chemical elements for use as a solar abundance standard in astronomical and planetary studies are summarized. Updated abundance tables for solar system abundances based on meteorites and photospheric measurements are presented.

  18. Attenuation of species abundance distributions by sampling.

    Shimadzu, Hideyasu; Darnell, Ross

    2015-04-01

    Quantifying biodiversity aspects such as species presence/ absence, richness and abundance is an important challenge to answer scientific and resource management questions. In practice, biodiversity can only be assessed from biological material taken by surveys, a difficult task given limited time and resources. A type of random sampling, or often called sub-sampling, is a commonly used technique to reduce the amount of time and effort for investigating large quantities of biological samples. However, it is not immediately clear how (sub-)sampling affects the estimate of biodiversity aspects from a quantitative perspective. This paper specifies the effect of (sub-)sampling as attenuation of the species abundance distribution (SAD), and articulates how the sampling bias is induced to the SAD by random sampling. The framework presented also reveals some confusion in previous theoretical studies. PMID:26064626

  19. 2'-O-[2-(guanidinium)ethyl]-modified oligonucleotides: stabilizing effect on duplex and triplex structures

    Prakash, T.P.; Puschl, A.; Lesnik, E.; Mohan, V.; Tereshko, V.; Egli, M.; Manoharan, M. (Vanderbilt); (UC)

    2010-03-08

    Oligonucleotides with a novel 2'-O-[2-(guanidinium)ethyl] (2'-O-GE) modification have been synthesized using a novel protecting group strategy for the guanidinium group. This modification enhances the binding affinity of oligonucleotides to RNA as well as duplex DNA ({Delta}T{sub m} 3.2 C per modification). The 2'-O-GE modified oligonucleotides exhibited exceptional resistance to nuclease degradation. The crystal structure of a palindromic duplex formed by a DNA oligonucleotide with a single 2'-O-GE modification was solved at 1.16 {angstrom} resolution.

  20. Experimental analysis of oligonucleotide microarray design criteria to detect deletions by comparative genomic hybridization

    Moerman Donald G

    2008-10-01

    Full Text Available Abstract Background Microarray comparative genomic hybridization (CGH is currently one of the most powerful techniques to measure DNA copy number in large genomes. In humans, microarray CGH is widely used to assess copy number variants in healthy individuals and copy number aberrations associated with various diseases, syndromes and disease susceptibility. In model organisms such as Caenorhabditis elegans (C. elegans the technique has been applied to detect mutations, primarily deletions, in strains of interest. Although various constraints on oligonucleotide properties have been suggested to minimize non-specific hybridization and improve the data quality, there have been few experimental validations for CGH experiments. For genomic regions where strict design filters would limit the coverage it would also be useful to quantify the expected loss in data quality associated with relaxed design criteria. Results We have quantified the effects of filtering various oligonucleotide properties by measuring the resolving power for detecting deletions in the human and C. elegans genomes using NimbleGen microarrays. Approximately twice as many oligonucleotides are typically required to be affected by a deletion in human DNA samples in order to achieve the same statistical confidence as one would observe for a deletion in C. elegans. Surprisingly, the ability to detect deletions strongly depends on the oligonucleotide 15-mer count, which is defined as the sum of the genomic frequency of all the constituent 15-mers within the oligonucleotide. A similarity level above 80% to non-target sequences over the length of the probe produces significant cross-hybridization. We recommend the use of a fairly large melting temperature window of up to 10°C, the elimination of repeat sequences, the elimination of homopolymers longer than 5 nucleotides, and a threshold of -1 kcal/mol on the oligonucleotide self-folding energy. We observed very little difference in data

  1. Non-additive effects of genotypic diversity increase floral abundance and abundance of floral visitors.

    Mark A Genung

    Full Text Available BACKGROUND: In the emerging field of community and ecosystem genetics, genetic variation and diversity in dominant plant species have been shown to play fundamental roles in maintaining biodiversity and ecosystem function. However, the importance of intraspecific genetic variation and diversity to floral abundance and pollinator visitation has received little attention. METHODOLOGY/PRINCIPAL FINDINGS: Using an experimental common garden that manipulated genotypic diversity (the number of distinct genotypes per plot of Solidago altissima, we document that genotypic diversity of a dominant plant can indirectly influence flower visitor abundance. Across two years, we found that 1 plant genotype explained 45% and 92% of the variation in flower visitor abundance in 2007 and 2008, respectively; and 2 plant genotypic diversity had a positive and non-additive effect on floral abundance and the abundance of flower visitors, as plots established with multiple genotypes produced 25% more flowers and received 45% more flower visits than would be expected under an additive model. CONCLUSIONS/SIGNIFICANCE: These results provide evidence that declines in genotypic diversity may be an important but little considered factor for understanding plant-pollinator dynamics, with implications for the global decline in pollinators due to reduced plant diversity in both agricultural and natural ecosystems.

  2. Conjugates of Phthalocyanines With Oligonucleotides as Reagents for Sensitized or Catalytic DNA Modification

    2006-01-01

    Full Text Available Several conjugates of metallophthalocyanines with deoxyribooligonucleotides were synthesized to investigate sequence-specific modification of DNA by them. Oligonucleotide parts of these conjugates were responsible for the recognition of selected complementary sequences on the DNA target. Metallophthalocyanines were able to induce the DNA modification: phthalocyanines of Zn(II and Al(III were active as photosensitizers in the generation of singlet oxygen 1 O 2 , while phthalocyanine of Co(II promoted DNA oxidation by molecular oxygen through the catalysis of formation of reactive oxygen species ( ⋅ O 2 − , O 2 H 2 , OH. Irradiation of the reaction mixture containing either Zn(II- or Al(III-tetracarboxyphthalocyanine conjugates of oligonucleotide pd(TCTTCCCA with light of > 340 nm wavelength (Hg lamp or He/Ne laser resulted in the modification of the 22-nucleotide target d(TGAATGGGAAGAGGGTCAGGTT. A conjugate of Co(II-tetracarboxyphthalocyanine with the oligonucleotide was found to modify the DNA target in the presence of O 2 and 2-mercaptoethanol or in the presence of O 2 H 2 . Under both sensitized and catalyzed conditions, the nucleotides G 13 – G 15 were mainly modified, providing evidence that the reaction proceeded in the double-stranded oligonucleotide. These results suggest the possible use of phthalocyanine-oligonucleotide conjugates as novel artificial regulators of gene expression and therapeutic agents for treatment of cancer.

  3. Liposome-encapsulated polyethylenimine/oligonucleotide polyplexes prepared by reverse-phase evaporation technique.

    Ko, Young Tag; Bickel, Ulrich

    2012-06-01

    Liposome-encapsulated polyplex system represents a promising delivery system for oligonucleotide-based therapeutics such as siRNA and asODN. Here, we report a novel method to prepare liposome-encapsulated cationic polymer/oligonucleotide polyplexes based on the reverse-phase evaporation following organic extraction of the polyplexes. The polyplexes of polyethylenimine and oligonucleotide were first formed in aqueous buffer at an N/P ratio of 6. The overall positively charged polyplexes were then mixed with the anionic phospholipids in overall organic media. The overall organic environment and electrostatic interaction between anionic phospholipids and positively charged polyplexes resulted in inverted micelle-like particles with the polyplexes in the core. After phase separation, the hydrophobic particles were recovered in organic phase. Reverse-phase evaporation of the organic solvent in the presence of hydrophilic polymer-grafted lipids resulted in a stable aqueous dispersion of hydrophilic lipid-coated particles with the polyplex in the core. Transmission electron microscopy visualization revealed spherical structures with heavily stained polyplex cores surrounded by lightly stained lipid coats. The lipid-coated polyplex particles showed colloidal stability, complete protection of the loaded oligonucleotide molecules from enzymatic degradation, and high loading efficiency of more than 80%. Thus, this technique represents an alternative method to prepare lipid-coated polyplex particles as a delivery system of oligonucleotide therapeutics. PMID:22328240

  4. Oligonucleotide delivery with cell surface binding and cell penetrating Peptide amphiphile nanospheres.

    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

  5. LNA-modified isothermal oligonucleotide microarray for differentiating bacilli of similar origin

    Jing Yan; Ying Yuan; Runqing Mu; Hong Shang; Yifu Guan

    2014-12-01

    Oligonucleotide microarray has been one of the most powerful tools in the ‘Post-Genome Era’ for its high sensitivity, high throughput and parallel processing capability. To achieve high detection specificity, we fabricated an isothermal microarray using locked nucleic acid (LNA)-modified oligonucleotide probes, since LNA has demonstrated the advanced ability to enhance the binding affinity toward their complementary nucleotides. After designing the nucleotide sequences of these oligonucleotide probes for gram-positive bacilli of similar origin (Bacillus subtilis, Bacillus licheniformis, Bacillus pumilus, Bacillus megaterium and Bacillus circulans), we unified the melting temperatures of these oligonucleotide probes by modifying some nucleotides using LNA. Furthermore, we optimized the experimental procedures of hydrating microarray slides, blocking side surface as well as labelling the PCR products. Experimental results revealed that KOD Dash DNA polymerase could efficiently incorporate Cy3-dCTP into the PCR products, and the LNA-isothermal oligonucleotide microarray were able to distinguish the bacilli of similar origin with a high degree of accuracy and specificity under the optimized experimental condition.

  6. Identification and distribution of three serologically undetected alleles of HLA-DR by oligonucleotide x DNA typing analysis

    Recent progress in the molecular biology of human major histocompatibility complex class II genes (HLA-DP, -DQ, -DR) have shown that the genetic complexity and allelic polymorphism are greater than expected. In the case of HLA-DR, three DR β-chain loci have been identified and linked, two of which (DR βI and DR βIII, now assigned names HLA-DR1B and HLA-DR3B) are functional. The authors have shown that the HLA micropolymorphism detected at the DNA sequence level can easily be analyzed by hybridization with allele-specific oligonucleotides (HLA oligotyping). In the case of the HLA DRw52 supertypic specificity, which includes the DR3, DR5, DRw6, and DRw8 haplotypes, three alleles, referred to as DRw52a, DRw52b, and DRw52c, have recently been identified at the HLA-DR3B locus by DNA sequencing. Hybridization with locus- and allele-specific oligonucleotide probes (designated 52a, 52b, and 52c) has been performed on DNA from normal individuals forming a panel of 82 haplotypes to establish the distribution of these three alleles. Individuals of the DR3 haplotype had either the DRw52a or DRw52b allele, and individuals of extended haplotype HLA-A1,B8,DR3 had only the DRw52a allele. DR5 individuals all had the DRw52b allele, while individuals of DRw6 haplotype had the DRw52a, -52b, or -52c allele. None of these three alleles are found in DRw8 individuals. Analysis of this micropolymorphism, undetectable by common typing procedures, is therefore now operational for more accurate HLA matching for transplantation and for improving correlations between HLA and disease susceptibility

  7. Herbivory: effects on plant abundance, distribution and population growth

    Maron, John L.; Crone, Elizabeth

    2006-01-01

    Plants are attacked by many different consumers. A critical question is how often, and under what conditions, common reductions in growth, fecundity or even survival that occur due to herbivory translate to meaningful impacts on abundance, distribution or dynamics of plant populations. Here, we review population-level studies of the effects of consumers on plant dynamics and evaluate: (i) whether particular consumers have predictably more or less influence on plant abundance, (ii) whether par...

  8. Zooplankton composition and abundance in Mida Creek, Kenya

    Osore, M.K.W.; Mwaluma, J.M.; FIERS, F; Daro, M.H.

    2004-01-01

    In order to determine the resident assemblages of zooplankton in Mida Creek, Kenya, a survey was conducted from May 1996 to Apr. 1997 for which we studied their seasonal composition, abundance, and distribution. Twenty-seven major zooplankton taxa were identified. The order Copepoda was the most abundant taxon dominated mainly by the genera Acartia, Paracalanus, Labidocera, Temora, Centropages, and Calanopia. Other common zooplankton taxa included the Medusae, Ctenophora, Brachyura larvae, an...

  9. Non-Additive Effects of Genotypic Diversity Increase Floral Abundance and Abundance of Floral Visitors

    Mark A Genung; Jean-Philippe Lessard; Claire B Brown; Bunn, Windy A.; Cregger, Melissa A.; W M Nicholas Reynolds; Emmi Felker-Quinn; Stevenson, Mary L.; Hartley, Amanda S.; Gregory M. Crutsinger; Schweitzer, Jennifer A.; Bailey, Joseph K.

    2010-01-01

    BACKGROUND: In the emerging field of community and ecosystem genetics, genetic variation and diversity in dominant plant species have been shown to play fundamental roles in maintaining biodiversity and ecosystem function. However, the importance of intraspecific genetic variation and diversity to floral abundance and pollinator visitation has received little attention. METHODOLOGY/PRINCIPAL FINDINGS: Using an experimental common garden that manipulated genotypic diversity (the number of dist...

  10. Selective Neuromuscular Denervation in Taiwanese Severe SMA Mouse Can Be Reversed by Morpholino Antisense Oligonucleotides

    Lin, Te-Lin; Chen, Tai-Heng; Hsu, Ya-Yun; Cheng, Yu-Hua; Juang, Bi-Tzen; Jong, Yuh-Jyh

    2016-01-01

    Spinal muscular atrophy (SMA) is an autosomal recessive motor neuron disease caused by deficiency of the survival of motor neuron (SMN) protein, which leads to synaptic defects and spinal motor neuron death. Neuromuscular junction (NMJ) abnormalities have been found to be involved in SMA pathogenesis in the SMNΔ7 SMA mouse model. However, whether similar NMJ pathological findings present in another commonly used mouse model, the Taiwanese SMA mouse, has not been fully investigated. To examine the NMJs of the Taiwanese severe SMA mouse model (Smn-/-; SMN2tg/0), which is characterized by severe phenotype and death before postnatal day (P) 9, we investigated 25 axial and appendicular muscles from P1 to P9. We labelled the muscles with anti-neurofilament and anti-synaptophysin antibodies for nerve terminals and α-bungarotoxin for acetylcholine receptors (AChRs). We found that severe NMJ denervation (<50% fully innervated endplates) selectively occurred in the flexor digitorum brevis 2 and 3 (FDB-2/3) muscles from P5, and an increased percentage of fully denervated endplates correlated with SMA progression. Furthermore, synaptophysin signals were absent at the endplate compared to control littermate mice, suggesting that vesicle transport might only be affected at the end stage. Subsequently, we treated the Taiwanese severe SMA mice with morpholino (MO) antisense oligonucleotides (80 μg/g) via subcutaneous injection at P0. We found that MO significantly reversed the NMJ denervation in FDB-2/3 muscles and extended the survival of Taiwanese severe SMA mice. We conclude that early NMJ denervation in the FDB-2/3 muscles of Taiwanese severe SMA mice can be reversed by MO treatment. The FDB-2/3 muscles of Taiwanese severe SMA mice provide a very sensitive platform for assessing the effectiveness of drug treatments in SMA preclinical studies. PMID:27124114

  11. Antisense oligonucleotide induced exon skipping and the dystrophin gene transcript: cocktails and chemistries

    Fletcher Sue

    2007-07-01

    Full Text Available Abstract Background Antisense oligonucleotides (AOs can interfere with exon recognition and intron removal during pre-mRNA processing, and induce excision of a targeted exon from the mature gene transcript. AOs have been used in vitro and in vivo to redirect dystrophin pre-mRNA processing in human and animal cells. Targeted exon skipping of selected exons in the dystrophin gene transcript can remove nonsense or frame-shifting mutations that would otherwise have lead to Duchenne Muscular Dystrophy, the most common childhood form of muscle wasting. Results Although many dystrophin exons can be excised using a single AO, several exons require two motifs to be masked for efficient or specific exon skipping. Some AOs were inactive when applied individually, yet pronounced exon excision was induced in transfected cells when the AOs were used in select combinations, clearly indicating synergistic rather than cumulative effects on splicing. The necessity for AO cocktails to induce efficient exon removal was observed with 2 different chemistries, 2'-O-methyl modified bases on a phosphorothioate backbone and phosphorodiamidate morpholino oligomers. Similarly, other trends in exon skipping, as a consequence of 2'-O-methyl AO action, such as removal of additional flanking exons or variations in exon skipping efficiency with overlapping AOs, were also seen when the corresponding sequences were prepared as phosphorodiamidate morpholino oligomers. Conclusion The combination of 2 AOs, directed at appropriate motifs in target exons was found to induce very efficient targeted exon skipping during processing of the dystrophin pre-mRNA. This combinatorial effect is clearly synergistic and is not influenced by the chemistry of the AOs used to induce exon excision. A hierarchy in exon skipping efficiency, observed with overlapping AOs composed of 2'-O-methyl modified bases, was also observed when these same sequences were evaluated as phosphorodiamidate morpholino

  12. Multi-exon Skipping Using Cocktail Antisense Oligonucleotides in the Canine X-linked Muscular Dystrophy.

    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

  13. A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

    Chen Feng

    2010-10-01

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

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

    Zhang Wei

    2009-09-01

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

  15. Comparison of gene coverage of mouse oligonucleotide microarray platforms

    Medrano Juan F

    2006-03-01

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

  16. Estimating Animal Abundance: Review III

    Schwarz, Carl J; Seber, George A. F.

    1999-01-01

    The literature describing methods for estimating animal abundance and related parameters continues to grow. This paper reviews recent developments in the subject over the past seven years and updates two previous reviews.

  17. Polymorphism identification and quantitative detection of genomic DNA by invasive cleavage of oligonucleotide probes

    Lyamichev, V.; Mast, A.L.; Hall, J.G. [Third Wave Technologies, Madison, WI (United States)] [and others

    1999-03-01

    Flap endonucleases (FENs) isolated from archaea are shown to recognize and cleave a structure formed when two overlapping oligonucleotides hybridize to a target DNA strand. The downstream oligonucleotide probe is cleaved, and the precise site of cleavage is dependent on the amount of overlap with the upstream oligonucleotide. The authors have demonstrated that use of thermostable archaeal FENs allows the reaction to be performed at temperatures that promote probe turnover without the need for temperature cycling. The resulting amplification of the cleavage signal enables the detection of specific DNA targets at sub-attomole levels within complex mixtures. Moreover, the authors provide evidence that this cleavage is sufficiently specific to enable discrimination of single-base differences and can differentiate homozygotes from heterozygotes in single-copy genes in genomic DNA.

  18. Study of the simultaneous preparation of oligopeptides and oligonucleotides under prebiotic conditions

    Caroon, J.M.

    1975-08-01

    The effect of imidazole in promoting the formation of biopolymers under chemical evolutionary conditions was investigated. At the onset of this work, it was known that short-chain oligonucleotides formed in aqueous solutions of ATP, imidazole, and MgCl/sub 2/; in addition, short peptides had been shown to form in aqueous solutions of aminoacylimidazolides and MgCl/sub 2/. The products formed in an aqueous solution of ATP, imidazole, alanine, and MgCl/sub 2/ was investigated and it was found that, under plausible prebiotic conditions, the formation of representatives of both peptides and oligonucleotides could be demonstrated. Furthermore, it was shown that the simultaneous formation of peptides increased the formation of oligonucleotides. A preliminary study was made on the possibility of inducing asymmetry in the clay catalyzed polymerization of peptides, but the results were discouraging. (auth)

  19. Study of HIV-2 primer-template initiation complex using antisense oligonucleotides

    Boulmé, F; Freund, F; Gryaznov, S;

    2000-01-01

    HIV-2 reverse transcription is initiated by the retroviral DNA polymerase (reverse transcriptase) from a cellular tRNALys3 partially annealed to the primer binding site in the 5'-region of viral RNA. The HIV-2 genome has two A-rich regions upstream of the primer binding site. In contrast to HIV-1...... approach, first validated in our in vitro HIV-1 reverse transcription system. Annealing of the antisense oligonucleotides to the pre-primer binding site (the upstream region contiguous to the HIV-2 primer binding site) was determined in the presence of native tRNALys3 or synthetic primers. Using natural...... and chemically modified antisense oligonucleotides we found that interactions between the anticodon of tRNALys3 and an A-rich loop of viral RNA led to an important destabilization of the pre-primer binding site; this region became accessible to anti-pre-primer binding site oligonucleotides in a...

  20. Hydrolysis of microporous polyamide-6 membranes as substrate for in situ synthesis of oligonucleotides

    Tang, Jianxin; He, Nongyue; Nie, Libo; Xiao, Pengfeng; Chen, Hong

    2004-02-01

    This article provides a novel method of preparing substrate for in situ synthesis of oligonucleotide by hydrolyzing microporous polyamide-6 membranes in a 0.01 mol/l/NaOH/(H 2O-CH 3OH) mixture medium with refluxing about 36 h. Attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) demonstrated the emergence of amines (NH 2) on the surface. Optimum hydrolyzing conditions were determined through the ultra-violet (UV) spectra. A pH value of 12 and a hydrolysis time of 36 h are the preferred conditions for the modification. The treated membrane can be applied to in situ synthesis of oligonucleotide and, for example, the oligonucleotide probes of 5 '-AAC CAC CAA ACA CAC-3 ' were successfully synthesized on the hydrolyzed membrane. The single step coupling efficiency determined by ultraviolet (UV) spectra is above 98%.

  1. Potent triple helix stabilization by 5',3'-modified triplex-forming oligonucleotides.

    Ben Gaied, Nouha; Zhao, Zhengyun; Gerrard, Simon R; Fox, Keith R; Brown, Tom

    2009-07-20

    Anthraquinone and pyrene analogues attached to the 3' and/or 5' termini of triplex-forming oligonucleotides (TFOs) by various linkers increased the stability of parallel triple helices. The modifications are simple to synthesize and can be introduced during standard solid-phase oligonucleotide synthesis. Potent triplex stability was achieved by using doubly modified TFOs, which in the most favourable cases gave an increase in melting temperature of 30 degrees C over the unmodified counterparts and maintained their selectivity for the correct target duplex. Such TFOs can produce triplexes with melting temperatures of 40 degrees C at pH 7 even though they do not contain any triplex-stabilizing base analogues. These studies have implications for the design of triplex-forming oligonucleotides for use in biology and nanotechnology. PMID:19554592

  2. Smart polymeric micelles as nanocarriers for oligonucleotides and siRNA delivery.

    Kataoka, Kazunori; Itaka, Keiji; Nishiyama, Nobuhiro; Yamasaki, Yuichi; Oishi, Motoi; Nagasaki, Yukio

    2005-01-01

    The development of in vivo delivery systems for oligonucleotides and siRNA is strongly desired to achieve their clinical applications. Recently, polyplex micelles, which are formed through an electrostatic interaction between nucleic acid compounds (DNA and RNA) and poly(ethylene glycol) (PEG)-polycation block copolymers, have received much attention due to their nanometric-scaled size and excellent biocompatibility. Here, three types of newly engineered block copolymers were developed to construct polyplex micelles useful for oligonucleotides and siRNA delivery: (1) PEG-polycation diblock copolymers possessing diamine side-chain with distinctive pKa for siRNA encapsulation into polyplex micelles with high endosomal escaping ability, (2) Lactosylated PEG-(oligonucleotide or siRNA) conjugate through acid-labile beta-thiopropionate linkage to construct pH-sensitive PIC micelles, and (3) PEG-poly(methacrylic acid) block copolymer for the construction of organic/inorganic hybrid nanoparticles encapsulating siRNA. PMID:17150611

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

    The dynamics of the human oligonucleotide AG3(T2AG3)3 has been investigated by incoherent neutron scattering in the sub-nanosecond timescale. A hydration-dependent dynamical activation of thermal fluctuations in weakly hydrated samples was found, similar to that of protein powders. The amplitudes of such thermal fluctuations were evaluated in two different exchanged wave-vector ranges, so as to single out the different contributions from intra- and inter-nucleotide dynamics. The activation energy was calculated from the temperature-dependent characteristic times of the corresponding dynamical processes. The trends of both amplitudes and activation energies support a picture where oligonucleotides possess a larger conformational flexibility than long DNA sequences. This additional flexibility, which likely results from a significant relative chain-end contribution to the average chain dynamics, could be related to the strong structural polymorphism of the investigated oligonucleotides

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

    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.

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

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

    2015-07-07

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

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

    Nåbo, Lina J.; Madsen, Charlotte Stahl; Jensen, Knud Jørgen;

    2015-01-01

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

  7. Chlorine Abundances in Cool Stars

    Maas, Z G; Hinkle, K

    2016-01-01

    Chlorine abundances are reported in 15 evolved giants and one M dwarf in the solar neighborhood. The Cl abundance was measured using the vibration-rotation 1-0 P8 line of H$^{35}$Cl at 3.69851 $\\mu$m. The high resolution L-band spectra were observed using the Phoenix infrared spectrometer on the Kitt Peak Mayall 4m telescope. The average [$^{35}$Cl/Fe] abundance in stars with --0.72$<$[Fe/H]$<$0.20 is [$^{35}$Cl/Fe]=(--0.10$\\pm$0.15) dex. The mean difference between the [$^{35}$Cl/Fe] ratios measured in our stars and chemical evolution model values is (0.16$\\pm$0.15) dex. The [$^{35}$Cl/Ca] ratio has an offset of $\\sim$0.35 dex above model predictions suggesting chemical evolution models are under producing Cl at the high metallicity range. Abundances of C, N, O, Si, and Ca were also measured in our spectral region and are consistent with F and G dwarfs. The Cl versus O abundances from our sample match Cl abundances measured in planetary nebula and \\ion{H}{2} regions. In one star where both H$^{35}$Cl a...

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

    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

  9. The shape of terrestrial abundance distributions.

    Alroy, John

    2015-09-01

    Ecologists widely accept that the distribution of abundances in most communities is fairly flat but heavily dominated by a few species. The reason for this is that species abundances are thought to follow certain theoretical distributions that predict such a pattern. However, previous studies have focused on either a few theoretical distributions or a few empirical distributions. I illustrate abundance patterns in 1055 samples of trees, bats, small terrestrial mammals, birds, lizards, frogs, ants, dung beetles, butterflies, and odonates. Five existing theoretical distributions make inaccurate predictions about the frequencies of the most common species and of the average species, and most of them fit the overall patterns poorly, according to the maximum likelihood-related Kullback-Leibler divergence statistic. Instead, the data support a low-dominance distribution here called the "double geometric." Depending on the value of its two governing parameters, it may resemble either the geometric series distribution or the lognormal series distribution. However, unlike any other model, it assumes both that richness is finite and that species compete unequally for resources in a two-dimensional niche landscape, which implies that niche breadths are variable and that trait distributions are neither arrayed along a single dimension nor randomly associated. The hypothesis that niche space is multidimensional helps to explain how numerous species can coexist despite interacting strongly. PMID:26601249

  10. Silica nanoparticles surface-modified with thiacalixarenes selectively adsorb oligonucleotides and proteins

    Yuskova, Elena A. [Kazan (Volga Region) Federal University, Department of Chemistry, A. M. Butlerov Chemical Institute (Russian Federation); Ignacio-de Leon, Patricia Anne A.; Khabibullin, Amir [University of Utah, Department of Chemistry (United States); Stoikov, Ivan I., E-mail: ivan.stoikov@mail.ru [Kazan (Volga Region) Federal University, Department of Chemistry, A. M. Butlerov Chemical Institute (Russian Federation); Zharov, Ilya, E-mail: i.zharov@utah.edu [University of Utah, Department of Chemistry (United States)

    2013-10-15

    We prepared silica nanospheres 360 nm in diameter surface-modified with p-tert-butylthiacalix[4]arenes containing amine, carboxyl, and guanidinium groups. We found that these silica nanoparticles selectively adsorb model oligonucleotides and proteins. The particles modified with the macrocycle containing guanidinium fragments selectively adsorbed long-chain oligonucleotides and those modified with the macrocycle containing amine groups adsorbed BSA and hemoglobin with pH-dependent selectivity. We compared this behavior with that of silica nanoparticles carrying amine and carboxyl groups, and concluded that both electrostatic interactions and specific binding are responsible for the observed selectivity.

  11. Effects of HSP70 Antisense Oligonucleotide on the Proliferation and Apoptosis of Human Hepatocellular Carcinoma Cells

    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...

  12. Oligonucleotide Fingerprinting of rRNA Genes for Analysis of Fungal Community Composition

    Valinsky, Lea; Della Vedova, Gianluca; Jiang, Tao; Borneman, James

    2002-01-01

    Thorough assessments of fungal diversity are currently hindered by technological limitations. Here we describe a new method for identifying fungi, oligonucleotide fingerprinting of rRNA genes (OFRG). ORFG sorts arrayed rRNA gene (ribosomal DNA [rDNA]) clones into taxonomic clusters through a series of hybridization experiments, each using a single oligonucleotide probe. A simulated annealing algorithm was used to design an OFRG probe set for fungal rDNA. Analysis of 1,536 fungal rDNA clones d...

  13. An Oligonucleotide Microarray for High-Throughput Sequencing of the Mitochondrial Genome

    Zhou, Shaoyu; Kassauei, Keyaunoosh; Cutler, David J.; Kennedy, Giulia C; Sidransky, David; Maitra, Anirban; Califano, Joseph

    2006-01-01

    Previously we developed an oligonucleotide sequencing microarray (MitoChip) as an array-based sequencing platform for rapid and high-throughput analysis of mitochondrial DNA. The first generation MitoChip, however, was not tiled with probes for the noncoding D-loop region, a site frequently mutated in human cancers. Here we report the development of a second-generation MitoChip (v2.0) with oligonucleotide probes to sequence the entire mitochondrial genome. In addition, the MitoChip v2.0 conta...

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

    Andersson, A M; Gaardsvoll, H; Giladi, E; Dahl, B; Bock, Elisabeth Marianne

    1990-01-01

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

  15. Sterilization of sterlet Acipenser ruthenus by using knockdown agent, antisense morpholino oligonucleotide, against dead end gene.

    Linhartová, Zuzana; Saito, Taiju; Kašpar, Vojtěch; Rodina, Marek; Prášková, Eva; Hagihara, Seishi; Pšenička, Martin

    2015-10-15

    Sturgeons (chondrostean, acipenseridae) are ancient fish species, widely known for their caviar. Nowadays, most of them are critically endangered. The sterlet (Acipenser ruthenus) is a common Eurasian sturgeon species with a small body size and the fastest reproductive cycle among sturgeons. Such species can be used as a host for surrogate production; application is of value for recovery of critically endangered and huge sturgeon species with an extremely long reproductive cycle. One prerequisite for production of the donor's gametes only is to have a sterile host. Commonly used sterilization techniques in fishes such as triploidization or hybridization do not guarantee sterility in sturgeon. Alternatively, sterilization can be achieved by using a temporary germ cell exclusion-specific gene by a knockdown agent, the antisense morpholino oligonucleotide (MO). The targeted gene for the MO is the dead end gene (dnd) which is a vertebrate-specific gene encoding a RNA-binding protein which is crucial for migration and survival of primordial germ cells (PGCs). For this purpose, a dnd homologue of Russian sturgeon (Agdnd), resulting in the same sequence in the start codon region with isolated fragments of sterlet dnd (Ardnd), was used. Reverse transcription polymerase chain reaction confirmed tissue-specific expression of Ardnd only in the gonads of both sexes. Dnd-MO for depletion of PGCs together with fluorescein isothiocyanate (FITC)-biotin-dextran for PGCs labeling was injected into the vegetal region of one- to four-cell-stage sterlet embryos. In the control groups, only FITC was injected to validate the injection method and labeling of PGCs. After optimization of MO concentration together with volume injection, 250-μM MO was applied for sterilization of sturgeon embryos. Primordial germ cells were detected under a fluorescent stereomicroscope in the genital ridge of the FITC-labeled control group only, whereas no PGCs were present in the body cavities of morphants

  16. Solar and Stellar Photospheric Abundances

    Prieto, Carlos Allende

    2016-01-01

    The determination of photospheric abundances in late-type stars from spectroscopic observations is a well-established field, built on solid theoretical foundations. Improving those foundations to refine the accuracy of the inferred abundances has proven challenging, but progress has been made. In parallel, developments on instrumentation, chiefly regarding multi-object spectroscopy, have been spectacular, and a number of projects are collecting large numbers of observations for stars across the Milky Way and nearby galaxies, promising important advances in our understanding of galaxy formation and evolution. After providing a brief description of the basic physics and input data involved in the analysis of stellar spectra, a review is made of the analysis steps, and the available tools to cope with large observational efforts. The paper closes with a quick overview of relevant ongoing and planned spectroscopic surveys, and highlights of recent research on photospheric abundances.

  17. Solar and stellar photospheric abundances

    Allende Prieto, Carlos

    2016-07-01

    The determination of photospheric abundances in late-type stars from spectroscopic observations is a well-established field, built on solid theoretical foundations. Improving those foundations to refine the accuracy of the inferred abundances has proven challenging, but progress has been made. In parallel, developments on instrumentation, chiefly regarding multi-object spectroscopy, have been spectacular, and a number of projects are collecting large numbers of observations for stars across the Milky Way and nearby galaxies, promising important advances in our understanding of galaxy formation and evolution. After providing a brief description of the basic physics and input data involved in the analysis of stellar spectra, a review is made of the analysis steps, and the available tools to cope with large observational efforts. The paper closes with a quick overview of relevant ongoing and planned spectroscopic surveys, and highlights of recent research on photospheric abundances.

  18. Mapping of HIV-1 integrase preferences for target site selection with various oligonucleotides

    Snášel, Jan; Rosenberg, Ivan; Pačes, Ondřej; Pichová, Iva

    2009-01-01

    Roč. 488, č. 2 (2009), s. 153-162. ISSN 0003-9861 R&D Projects: GA MŠk 1M0508; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z40550506 Keywords : HIV integrase * strand transfer reaction * oligonucleotide substrates Subject RIV: CE - Biochemistry Impact factor: 3.046, year: 2009

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

    Stolc, Viktor; Li, Lei; Wang, Xiangfeng; Li, Xueyong; Su, Ning; Tongprasit, Waraporn; Han, Bin; Xue, Yongbiao; Li, Jiayang; Snyder, Michael; Gerstein, Mark; Wang, Jun; Deng, Xing Wang

    2005-01-01

    As the international efforts to sequence the rice genome are completed, an immediate challenge and opportunity is to comprehensively and accurately define all transcription units in the rice genome. Here we describe a strategy of using high-density oligonucleotide tiling-path microarrays to map t...

  20. nuID: a universal naming scheme of oligonucleotides for Illumina, Affymetrix, and other microarrays

    Kibbe Warren A

    2007-05-01

    Full Text Available Abstract Background Oligonucleotide probes that are sequence identical may have different identifiers between manufacturers and even between different versions of the same company's microarray; and sometimes the same identifier is reused and represents a completely different oligonucleotide, resulting in ambiguity and potentially mis-identification of the genes hybridizing to that probe. Results We have devised a unique, non-degenerate encoding scheme that can be used as a universal representation to identify an oligonucleotide across manufacturers. We have named the encoded representation 'nuID', for nucleotide universal identifier. Inspired by the fact that the raw sequence of the oligonucleotide is the true definition of identity for a probe, the encoding algorithm uniquely and non-degenerately transforms the sequence itself into a compact identifier (a lossless compression. In addition, we added a redundancy check (checksum to validate the integrity of the identifier. These two steps, encoding plus checksum, result in an nuID, which is a unique, non-degenerate, permanent, robust and efficient representation of the probe sequence. For commercial applications that require the sequence identity to be confidential, we have an encryption schema for nuID. We demonstrate the utility of nuIDs for the annotation of Illumina microarrays, and we believe it has universal applicability as a source-independent naming convention for oligomers. Reviewers This article was reviewed by Itai Yanai, Rong Chen (nominated by Mark Gerstein, and Gregory Schuler (nominated by David Lipman.

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

    Behrens, Carsten; Petersen, Kenneth H.; Egholm, Michael; Nielsen, John; Buchard, Ole; Dahl, Otto

    1995-01-01

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

  2. Efficient large-scale preparation and purification of short single-stranded RNA oligonucleotides.

    Zlobina, Maria; Sedo, Ondrej; Chou, Ming-Yuan; Slepankova, Lucia; Lukavsky, Peter J

    2016-01-01

    Sequence-specific RNA recognition by RNA-binding proteins plays a crucial role in the post-translational regulation of gene expression. Biophysical and biochemical studies help to unravel the principles of sequence-specific RNA recognition, but the methods used require large amounts of single-stranded RNA (ssRNA). Here we present a fast and robust method for large-scale preparation and purification of short ssRNA oligonucleotides for biochemical, biophysical, and structural studies. We designed an efficiently folding, self-cleaving hammerhead (HH) ribozyme to prepare ssRNA oligonucleotides. Hammerhead ribozyme RNAs self-cleave with over 95% efficiency during in vitro transcription as a function of magnesium concentration to produce high yields of the desired ssRNA products. The resulting ssRNAs can be purified from crude transcription reactions by denaturing anion-exchange chromatography and then desalted by weak anion-exchange chromatography using volatile ammonium bicarbonate buffer solutions. The ssRNA oligonucleotides produced this way are homogenous, as judged by mass spectrometry (MS), and are suitable for biochemical and biophysical studies. Moreover, for high-resolution NMR structure determination of RNA-protein complexes, our protocol enables efficient preparation of ssRNA oligonucleotides with various isotope-labeling schemes which are not commercially available. PMID:26842352

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

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

    2013-01-01

    Myotonic dystrophy type 1 (DM1) is caused by DM protein kinase (DMPK) transcripts containing an expanded (CUG)n repeat. Antisense oligonucleotide (AON)-mediated suppression of these mutant RNAs is considered a promising therapeutic strategy for this severe disorder. Earlier, we identified a 2'-O-met

  4. Oligo switches: photoresponsive oligonucleotide conjugates by solid-supported click chemistry

    Freeman, Colin; Vyle, Joseph S; Heaney, Frances

    2013-01-01

    Photoresponsive oligonucleotides (ONs) incorporating isoxazole-linked azobenzene (AB) moieties were prepared by resin-supported nitrile oxide-alkyne cycloaddition (NOAC) chemistry. The thermal and photochromic properties of the modified ONs were significantly influenced by the extent of π-conjugation between the isoxazole and the AB modules

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

    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

  6. A triple-helix forming oligonucleotide targeting genomic DNA fails to induce mutation.

    Reshat, Reshat; Priestley, Catherine C; Gooderham, Nigel J

    2012-11-01

    Purine tracts in duplex DNA can bind oligonucleotide strands in a sequence specific manner to form triple-helix structures. Triple-helix forming oligonucleotides (TFOs) targeting supFG1 constructs have previously been shown to be mutagenic raising safety concerns for oligonucleotide-based pharmaceuticals. We have engineered a TFO, TFO27, to target the genomic Hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus to define the mutagenic potential of such structures at genomic DNA. We report that TFO27 was resistant to nuclease degradation and readily binds to its target motif in a cell free system. Contrary to previous studies using the supFG1 reporter construct, TFO27 failed to induce mutation within the genomic HPRT locus. We suggest that it is possible that previous reports of triplex-mediated mutation using the supFG1 reporter construct could be confounded by DNA quadruplex formation. Although the present study indicates that a TFO targeting a genomic locus lacks mutagenic activity, it is unclear if this finding can be generalised to all TFOs and their targets. For the present, we suggest that it is prudent to avoid large purine stretches in oligonucleotide pharmaceutical design to minimise concern regarding off-target genotoxicity. PMID:22914677

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

    Eriksen, K W; Nielsen, M; Kaltoft, K; Svejgaard, A; Nissen, Mogens Holst; Röpke, C; Ødum, N

    2001-01-01

    -stranded oligonucleotide probes containing a STAT6-binding gene-sequence from the promotor of the immunoglobulin heavy chain germline epsilon transcript to study the IL-4-induced DNA binding of STAT6. Using these probes, we show that repeated adjacent STAT6-binding sites result in enhanced STAT6-DNA binding. Moreover, the...

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

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

    2012-01-01

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

  9. Long-range order of organized oligonucleotide monolayers on Au(111) electrodes

    Wackerbarth, Hainer; Grubb, Mikala; Zhang, Jingdong;

    2004-01-01

    -S bond. The coverage is high and in keeping with dense monolayers of adsorbed HS-10A and HS-10AT in an upright or tilted orientation, with the oligonucleotide backbone repelled from the strongly negatively charged electrode surface. Adsorbed thiol-free 10A only gives aAu(111)-reconstruction peak, while...

  10. TmPrime: fast, flexible oligonucleotide design software for gene synthesis

    Bode, Marcus; Khor, Samuel; Ye, Hongye; Li, Mo-Huang; Ying, Jackie Y.

    2009-01-01

    Herein we present TmPrime, a computer program to design oligonucleotide sets for gene assembly by both ligase chain reaction (LCR) and polymerase chain reaction (PCR). TmPrime offers much flexibility with no constraints on the gene and oligonucleotide lengths. The program divides the long input DNA sequence based on the input desired melting temperature, and dynamically optimizes the length of oligonucleotides to achieve homologous melting temperatures. The output reports the melting temperatures, oligonucleotide sequences and potential formation of secondary structures. Our program also provides functions on sequence pooling to separate long genes into smaller pieces for multi-pool assembly and codon optimization for expression. The software has been successfully used in the design and synthesis of green fluorescent protein fragment (GFPuv) (760 bp), human protein kinase B-2 (PKB2) (1446 bp) and the promoter of human calcium-binding protein A4 (S100A4) (752 bp) using real-time PCR assembly with LCGreen I, which offers a novel approach to compare the efficiency of gene synthesis. The purity of assembled products is successfully estimated with the use of melting curve analysis, which would potentially eliminate the necessity for agarose gel electrophoresis. This program is freely available at http://prime.ibn.a-star.edu.sg. PMID:19515937

  11. MOLECULAR-MODELLING AND BIOPHYSICAL STUDIES ON MODIFIED AND UNMODIFIED OLIGONUCLEOTIDES

    GUNNING, J.; CUTBUSH, S.; Kuroda, R; Neidle, S

    1984-01-01

    Computer graphics modelling and energy calculations have been carried out on a series of modified and unmodified oligonucleotides to investigate the effects of base deletion, base substitution and base methylation, particularly 06-methylguanine and 5-methylcytosine, as well as the substitution of bases by analogues.

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

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

    2015-01-01

    Roč. 13, č. 15 (2015), s. 4449-4458. ISSN 1477-0520 R&D Projects: GA ČR GAP207/11/0108; GA ČR GA13-26526S Institutional support: RVO:61388963 Keywords : acyclic nucleoside phosphonates * oligonucleotides * solid phase synthesis Subject RIV: CC - Organic Chemistry Impact factor: 3.562, year: 2014

  13. Prophylactic vaccines mimic synthetic CpG oligonucleotides in their ability to modulate immune responses

    Vries, I.J.M. de; Tel, J.; Benitez-Ribas, D.; Torensma, R.; Figdor, C.G.

    2011-01-01

    Synthetic oligonucleotide ligands that bind to toll-like receptors are known to modulate the immune response via the activation of antigen presenting cells, and were therefore proposed as a novel form of vaccine adjuvant. Clinical-grade they are, however, not readily available. Here, we show that co

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

    Lindholm, Marie W; Elmén, Joacim; Fisker, Niels; Hansen, Henrik; Persson, Hans Egon Robert; Møller, Dorte Marianne; Rosenbohm, Christoph; Ørum, Henrik; Straarup, Ellen Marie; Koch, Troels

    2012-01-01

    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...

  15. A highly sensitive and selective viral protein detection method based on RNA oligonucleotide nanoparticle

    Changhyun Roh

    2010-04-01

    Full Text Available Changhyun Roh1, Ho-Young Lee2, Sang-Eun Kim2, Sung-Kee Jo11Radiation Research Division for Biotechnology, Advanced Radiation Technology Institute (ARTI, Korea Atomic Energy Research Institute (KAERI, Sinjeong-dong, Jeongeup, Jeonbuk, South Korea; 2Department of Nuclear Medicine, College of Medicine, Seoul National University, South KoreaAbstract: Globally, approximately 170 million people (representing approximately 3% of the population worldwide, are infected with hepatitis C virus (HCV and at risk of serious liver disease, including chronic hepatitis. We propose a new quantum dots (QDs-supported RNA oligonucleotide approach for the specific and sensitive detection of viral protein using a biochip. This method was developed by immobilizing a HCV nonstructural protein 5B (NS5B on the surface of a glass chip via the formation of a covalent bond between an amine protein group and a ProLinkerTM glass chip. The QDs-supported RNA oligonucleotide was conjugated via an amide formation reaction from coupling of a 5′-end-amine-modified RNA oligonucleotide on the surface of QDs displaying carboxyl groups via standard EDC coupling. The QDs-conjugated RNA oligonucleotide was interacted to immobilized viral protein NS5B on the biochip. The detection is based on the variation of signal of QDs-supported RNA oligonucleotide bound on an immobilized biochip. It was demonstrated that the value of the signal has a linear relationship with concentrations of the HCV NS5B viral protein in the 1 μg mL-1 to 1 ng mL-1 range with a detection limit of 1 ng mL-1. The major advantages of this RNA-oligonucleotide nanoparticle assay are its good specificity, ease of performance, and ability to perform one-spot monitoring. The proposed method could be used as a general method of HCV detection and is expected to be applicable to other types of diseases as well.Keywords: hepatitis C virus, viral protein, RNA oligonucleotide, quantum dots, biochip

  16. Abundance Inequality in Freshwater Communities Has an Ecological Origin.

    Passy, Sophia I

    2016-04-01

    The hollow-shaped species abundance distribution (SAD) and its allied rank abundance distribution (RAD)-showing that abundance is unevenly distributed among species-are some of the most studied patterns in ecology. To explain the nature of abundance inequality, I developed a novel framework identifying environmental favorability, which controls the balance between reproduction and immigration, as the ultimate source and species stress tolerance as a proximate factor. Thus, under harsh conditions, only a few tolerant species can reproduce, while some sensitive species can be present in low numbers due to chance immigration. This would lead to high abundance inequality between the two groups of species. Under benign conditions, both groups can reproduce and give rise to higher abundance equality. To test these ideas, I examined the variability in the parameters of a Poisson lognormal fit of the SAD and a square root fit of the RAD in diatom and fish communities across US streams. Indeed, as environmental favorability increased, more sensitive forms were able to establish large populations, diminishing the abundance disparity between locally common and rare species. Finally, it was demonstrated that in diatoms, the RAD belonged to the same family of relationships as those of population density with body size and regional distribution. PMID:27028078

  17. Seasonal Abundance of Aphids and Aphidophagous Insects in Pecan

    Ghulam Abbas

    2012-12-01

    Full Text Available Seasonal occurrence of aphids and aphidophagous insects was monitored for six years (2006–2011 from full leaf expansion in May to leaf fall in October in “Desirable” variety pecan trees that were not treated with insecticides. Aphid outbreaks occurred two times per season, once in the spring and again in the late summer. Yellow pecan and blackmargined aphids exceeded the recommended treatment thresholds one time and black pecan aphids exceeded the recommended treatment levels three times over the six seasons. Increases in aphidophagous insect abundance coincided with aphid outbreaks in five of the six seasons. Among aphidophagous insects Harmonia axyridis and Olla v-nigrum were frequently collected in both the tree canopy and at the ground level, whereas, Coccinella septempunctata, Hippodamia convergens were rarely found in the tree canopy and commonly found at the ground level. Green lacewing abundance was higher in the ground level than in the tree canopy. Brown lacewings were more abundant in the tree canopy than at the ground level. Dolichopodid and syrphid fly abundance, at the ground level increased during peak aphid abundance in the tree canopy. Application of an aqueous solution of fermenting molasses to the pecan foliage during an aphid outbreak significantly increased the abundance of ladybeetles and lacewings and significantly reduced the abundance of yellow pecan, blackmargined and black pecan aphids.

  18. Relative abundance of desert tortoises on the Nevada Test Site

    Seven hundred fifty-nine transects having a total length of 1,191 km were walked during 1981--1986 to determine the distribution and relative abundance of desert tortoises (Gopherus agassizii) on the Nevada Test Site (NTS). The abundance of tortoises on NTS was low to very low relative to other populations in the Mojave Desert. Sign of tortoises was found from 880 to 1,570 m elevation and was more abundant above 1,200 m than has been reported previously for Nevada. Tortoises were more abundant on NTS on the upper alluvial fans and slopes of mountains than in valley bottoms. They also were more common on or near limestone and dolomite mountains than on mountains of volcanic origin

  19. Steelhead Abundance - Point Features [ds184

    California Department of Resources — The CalFish Abundance Database contains a comprehensive collection of anadromous fisheries abundance information. Beginning in 1998, the Pacific States Marine...

  20. Coho Abundance - Point Features [ds182

    California Department of Resources — The CalFish Abundance Database contains a comprehensive collection of anadromous fisheries abundance information. Beginning in 1998, the Pacific States Marine...

  1. Coho Abundance - Linear Features [ds183

    California Department of Resources — The CalFish Abundance Database contains a comprehensive collection of anadromous fisheries abundance information. Beginning in 1998, the Pacific States Marine...

  2. Chinook Abundance - Point Features [ds180

    California Department of Resources — The CalFish Abundance Database contains a comprehensive collection of anadromous fisheries abundance information. Beginning in 1998, the Pacific States Marine...

  3. Steelhead Abundance - Linear Features [ds185

    California Department of Resources — The CalFish Abundance Database contains a comprehensive collection of anadromous fisheries abundance information. Beginning in 1998, the Pacific States Marine...

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

    Parekh-Olmedo Hetal

    2006-10-01

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

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

    Muyrers Joep PP

    2003-01-01

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

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

    Elela Sherif

    2006-01-01

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

  7. Development, Characterization and Experimental Validation of a Cultivated Sunflower (Helianthus annuus L.) Gene Expression Oligonucleotide Microarray

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

    2012-01-01

    Oligonucleotide-based microarrays with accurate gene coverage represent a key strategy for transcriptional studies in orphan species such as sunflower, H. annuus L., which lacks full genome sequences. The goal of this study was the development and functional annotation of a comprehensive sunflower unigene collection and the design and validation of a custom sunflower oligonucleotide-based microarray. A large scale EST (>130,000 ESTs) curation, assembly and sequence annotation was performed using Blast2GO (www.blast2go.de). The EST assembly comprises 41,013 putative transcripts (12,924 contigs and 28,089 singletons). The resulting Sunflower Unigen Resource (SUR version 1.0) was used to design an oligonucleotide-based Agilent microarray for cultivated sunflower. This microarray includes a total of 42,326 features: 1,417 Agilent controls, 74 control probes for sunflower replicated 10 times (740 controls) and 40,169 different non-control probes. Microarray performance was validated using a model experiment examining the induction of senescence by water deficit. Pre-processing and differential expression analysis of Agilent microarrays was performed using the Bioconductor limma package. The analyses based on p-values calculated by eBayes (p<0.01) allowed the detection of 558 differentially expressed genes between water stress and control conditions; from these, ten genes were further validated by qPCR. Over-represented ontologies were identified using FatiScan in the Babelomics suite. This work generated a curated and trustable sunflower unigene collection, and a custom, validated sunflower oligonucleotide-based microarray using Agilent technology. Both the curated unigene collection and the validated oligonucleotide microarray provide key resources for sunflower genome analysis, transcriptional studies, and molecular breeding for crop improvement. PMID:23110046

  8. Coupling strategies for the synthesis of Peptide-oligonucleotide conjugates for patterned synthetic biomineralization.

    Carter, Joshua D; Labean, Thomas H

    2011-01-01

    This work describes preparation strategies for peptide-oligonucleotide conjugates that combine the self-assembling behavior of DNA oligonucleotides with the molecular recognition capabilities of peptides. The syntheses include a solution-phase fragment coupling reaction and a solid-phase fragment coupling strategy where the oligonucleotide has been immobilized on DEAE Sepharose. The yield of four coupling reagents is evaluated, two reagents in water, EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) and DMTMM (4-(4,6-dimethoxy[1,3,5]triazin-2-yl)-4-methyl-morpholinium chloride), and two in dimethylformamide (DMF), PyBOP ((Benzotriazol-1-yloxy) tripyrrolidinophosphonium hexafluorophosphate) and HBTU (O-benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate), while the oligonucleotide fragment is either in solution or immobilized on DEAE. These coupling strategies rely on an unprotected 5' amino linker on the oligonucleotide reacting with the peptide C-terminus. The peptide, selected from a combinatorial library for its gold-binding behavior, was 12 amino acids long with an N-terminus acetyl cap. Formation of the conjugates was confirmed by gel electrophoresis and mass spectrometry while molecular recognition functionality of the peptide portion was verified using atomic force microscopy. Solution-phase yields were superior to their solid-phase counterparts. EDC resulted in the highest yield for both solution-phase (95%) and solid-phase strategies (24%), while the DMF-based reagents, PyBOP and HBTU, resulted in low yields with reduced recovery. All recoverable conjugates demonstrated gold nanoparticle templating capability. PMID:22007290

  9. Coupling Strategies for the Synthesis of Peptide-Oligonucleotide Conjugates for Patterned Synthetic Biomineralization

    Joshua D. Carter

    2011-01-01

    Full Text Available This work describes preparation strategies for peptide-oligonucleotide conjugates that combine the self-assembling behavior of DNA oligonucleotides with the molecular recognition capabilities of peptides. The syntheses include a solution-phase fragment coupling reaction and a solid-phase fragment coupling strategy where the oligonucleotide has been immobilized on DEAE Sepharose. The yield of four coupling reagents is evaluated, two reagents in water, EDC (1-ethyl-3-(3-dimethylaminopropyl carbodiimide hydrochloride and DMTMM (4-(4,6-dimethoxy[1,3,5]triazin-2-yl-4-methyl-morpholinium chloride, and two in dimethylformamide (DMF, PyBOP ((Benzotriazol-1-yloxy tripyrrolidinophosphonium hexafluorophosphate and HBTU (O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate, while the oligonucleotide fragment is either in solution or immobilized on DEAE. These coupling strategies rely on an unprotected 5′ amino linker on the oligonucleotide reacting with the peptide C-terminus. The peptide, selected from a combinatorial library for its gold-binding behavior, was 12 amino acids long with an N-terminus acetyl cap. Formation of the conjugates was confirmed by gel electrophoresis and mass spectrometry while molecular recognition functionality of the peptide portion was verified using atomic force microscopy. Solution-phase yields were superior to their solid-phase counterparts. EDC resulted in the highest yield for both solution-phase (95% and solid-phase strategies (24%, while the DMF-based reagents, PyBOP and HBTU, resulted in low yields with reduced recovery. All recoverable conjugates demonstrated gold nanoparticle templating capability.

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

    Paula Fernandez

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

  11. Gd Transition Probabilities and Abundances

    Den Hartog, E A; Sneden, C; Cowan, J J

    2006-01-01

    Radiative lifetimes, accurate to +/- 5%, have been measured for 49 even-parity and 14 odd-parity levels of Gd II using laser-induced fluorescence. The lifetimes are combined with branching fractions measured using Fourier transform spectrometry to determine transition probabilities for 611 lines of Gd II. This work is the largest-scale laboratory study to date of Gd II transition probabilities and the first using a high performance Fourier transform spectrometer. This improved data set has been used to determine a new solar photospheric Gd abundance, log epsilon = 1.11 +/- 0.03. Revised Gd abundances have also been derived for the r-process-rich metal-poor giant stars CS 22892-052, BD+17 3248, and HD 115444. The resulting Gd/Eu abundance ratios are in very good agreement with the solar-system r-process ratio. We have employed the increasingly accurate stellar abundance determinations, resulting in large part from the more precise laboratory atomic data, to predict directly the Solar System r-process elemental...

  12. Abundance estimation and Conservation Biology

    Nichols, J. D.

    2004-06-01

    Full Text Available Abundance is the state variable of interest in most population–level ecological research and in most programs involving management and conservation of animal populations. Abundance is the single parameter of interest in capture–recapture models for closed populations (e.g., Darroch, 1958; Otis et al., 1978; Chao, 2001. The initial capture–recapture models developed for partially (Darroch, 1959 and completely (Jolly, 1965; Seber, 1965 open populations represented efforts to relax the restrictive assumption of population closure for the purpose of estimating abundance. Subsequent emphases in capture–recapture work were on survival rate estimation in the 1970’s and 1980’s (e.g., Burnham et al., 1987; Lebreton et al.,1992, and on movement estimation in the 1990’s (Brownie et al., 1993; Schwarz et al., 1993. However, from the mid–1990’s until the present time, capture–recapture investigators have expressed a renewed interest in abundance and related parameters (Pradel, 1996; Schwarz & Arnason, 1996; Schwarz, 2001. The focus of this session was abundance, and presentations covered topics ranging from estimation of abundance and rate of change in abundance, to inferences about the demographic processes underlying changes in abundance, to occupancy as a surrogate of abundance. The plenary paper by Link & Barker (2004 is provocative and very interesting, and it contains a number of important messages and suggestions. Link & Barker (2004 emphasize that the increasing complexity of capture–recapture models has resulted in large numbers of parameters and that a challenge to ecologists is to extract ecological signals from this complexity. They offer hierarchical models as a natural approach to inference in which traditional parameters are viewed as realizations of stochastic processes. These processes are governed by hyperparameters, and the inferential approach focuses on these hyperparameters. Link & Barker (2004 also suggest that

  13. Effect of CD44 Suppression by Antisense Oligonucleotide on Attachment of Human Trabecular Meshwork Cells to HA

    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.

  14. Simple Method To Prepare Oligonucleotide-Conjugated Antibodies and Its Application in Multiplex Protein Detection in Single Cells.

    Gong, Haibiao; Holcomb, Ilona; Ooi, Aik; Wang, Xiaohui; Majonis, Daniel; Unger, Marc A; Ramakrishnan, Ramesh

    2016-01-20

    The diversity of nucleic acid sequences enables genomics studies in a highly multiplexed format. Since multiplex protein detection is still a challenge, it would be useful to use genomics tools for this purpose. This can be accomplished by conjugating specific oligonucleotides to antibodies. Upon binding of the oligonucleotide-conjugated antibodies to their targets, the protein levels can be converted to oligonucleotide levels. In this report we describe a simple method for preparing oligonucleotide-conjugated antibodies and discuss this method's application in oligonucleotide extension reaction (OER) for multiplex protein detection. Conjugation is based on strain-promoted alkyne-azide cycloaddition (the Cu-free click reaction), in which the antibody is activated with a dibenzocyclooctyne (DBCO) moiety and subsequently linked covalently with an azide-modified oligonucleotide. In the functional test, the reaction conditions and purification processes were optimized to achieve maximum yield and best performance. The OER assay employs a pair of antibody binders (two antibodies, each conjugated with its own oligonucleotide) developed for each protein target. The two oligonucleotides contain unique six-base complementary regions at their 3' prime ends to allow annealing and extension by DNA synthesis enzymes to form a DNA template. Following preamplification, the DNA template is detected by qPCR. Distinct oligonucleotide sequences are assigned to different antibody binders to enable multiplex protein detection. When tested using recombinant proteins, some antibody binders, such as those specific to CSTB, MET, EpCAM, and CASP3, had dynamic ranges of 5-6 logs. The antibody binders were also used in a multiplexed format in OER assays, and the binders successfully detected their protein targets in cell lysates, and in single cells in combination with the C1 system. This click reaction-based antibody conjugation procedure is cost-effective, needs minimal hands-on time, and

  15. Error, reproducibility and sensitivity: a pipeline for data processing of Agilent oligonucleotide expression arrays

    Posch Wilfried

    2010-06-01

    Full Text Available Abstract Background Expression microarrays are increasingly used to obtain large scale transcriptomic information on a wide range of biological samples. Nevertheless, there is still much debate on the best ways to process data, to design experiments and analyse the output. Furthermore, many of the more sophisticated mathematical approaches to data analysis in the literature remain inaccessible to much of the biological research community. In this study we examine ways of extracting and analysing a large data set obtained using the Agilent long oligonucleotide transcriptomics platform, applied to a set of human macrophage and dendritic cell samples. Results We describe and validate a series of data extraction, transformation and normalisation steps which are implemented via a new R function. Analysis of replicate normalised reference data demonstrate that intrarray variability is small (only around 2% of the mean log signal, while interarray variability from replicate array measurements has a standard deviation (SD of around 0.5 log2 units ( 6% of mean. The common practise of working with ratios of Cy5/Cy3 signal offers little further improvement in terms of reducing error. Comparison to expression data obtained using Arabidopsis samples demonstrates that the large number of genes in each sample showing a low level of transcription reflect the real complexity of the cellular transcriptome. Multidimensional scaling is used to show that the processed data identifies an underlying structure which reflect some of the key biological variables which define the data set. This structure is robust, allowing reliable comparison of samples collected over a number of years and collected by a variety of operators. Conclusions This study outlines a robust and easily implemented pipeline for extracting, transforming normalising and visualising transcriptomic array data from Agilent expression platform. The analysis is used to obtain quantitative estimates of

  16. Sequence diversity within the HA-1 gene as detected by melting temperature assay without oligonucleotide probes

    Mattiuz Pier

    2005-10-01

    Full Text Available Abstract Background The minor histocompatibility antigens (mHags are self-peptides derived from common cellular proteins and presented by MHC class I and II molecules. Disparities in mHags are a potential risk for the development of graft-versus-host disease (GvHD in the recipients of bone marrow from HLA-identical donors. Two alleles have been identified in the mHag HA-1. The correlation between mismatches of the mHag HA-1 and GvHD has been suggested and methods to facilitate large-scale testing were afterwards developed. Methods We used sequence specific primer (SSP PCR and direct sequencing to detect HA-1 gene polymorphisms in a sample of 131 unrelated Italian subjects. We then set up a novel melting temperature (Tm assay that may help identification of HA-1 alleles without oligonucleotide probes. Results We report the frequencies of HA-1 alleles in the Italian population and the presence of an intronic 5 base-pair deletion associated with the immunogeneic allele HA-1H. We also detected novel variable sites with respect to the consensus sequence of HA-1 locus. Even though recombination/gene conversion events are documented, there is considerable linkage disequilibrium in the data. The gametic associations between HA-1R/H alleles and the intronic 5-bp ins/del polymorphism prompted us to try the Tm analysis with SYBR® Green I. We show that the addition of dimethylsulfoxide (DMSO during the assay yields distinct patterns when amplicons from HA-1H homozygotes, HA-1R homozygotes, and heterozygotes are analysed. Conclusion The possibility to use SYBR® Green I to detect Tm differences between allelic variants is attractive but requires great caution. We succeeded in allele discrimination of the HA-1 locus using a relatively short (101 bp amplicon, only in the presence of DMSO. We believe that, at least in certain assets, Tm assays may benefit by the addition of DMSO or other agents affecting DNA strand conformation and stability.

  17. Abundance anomalies in tidal disruption events

    Kochanek, C. S.

    2016-05-01

    The ˜10 per cent of tidal disruption events (TDEs) due to stars more massive than M* ≳ M⊙ should show abundance anomalies due to stellar evolution in helium, carbon and nitrogen, but not oxygen. Helium is always enhanced, but only by up to ˜25 per cent on average because it becomes inaccessible once it is sequestered in the high-density core as the star leaves the main sequence. However, portions of the debris associated with the disrupted core of a main-sequence star can be enhanced in helium by factors of 2-3 for debris at a common orbital period. These helium abundance variations may be a contributor to the observed diversity of hydrogen and helium line strengths in TDEs. A still more striking anomaly is the rapid enhancement of nitrogen and the depletion of carbon due to the CNO cycle - stars with M* ≳ M⊙ quickly show an increase in their average N/C ratio by factors of 3-10. Because low-mass stars evolve slowly and high-mass stars are rare, TDEs showing high N/C will almost all be due to ˜1-2 M⊙ stars disrupted on the main sequence. Like helium, portions of the debris will show still larger changes in C and N, and the anomalies decline as the star leaves the main sequence. The enhanced [N/C] abundance ratio of these TDEs provides the first natural explanation for the rare, nitrogen-rich quasars and may also explain the strong nitrogen emission seen in ultraviolet spectra of ASASSN-14li.

  18. Amino acids attached to 2'-amino-LNA: Synthesis of DNA mixmer oligonucleotides with increased duplex stability

    Johannsen, Marie Willaing; Wengel, Jesper; Wamberg, Michael Chr.;

    2010-01-01

    The synthesis of 2'-amino-LNA (locked nucleic acid) opens up exciting possibilities for modification of nucleic acids by conjugation to the 2'-nitrogen. Incorporation of unmodified and N-functionalized 2'-amino-LNA nucleotides improve duplex stability compared to unmodified DNA. 2'-Amino......-LNA nucleosides derivatized with amino acids have been synthesized and incorporated into DNA oligonucleotides. Following oligonucleotide synthesis, peptides have been added using solid phase peptide coupling chem. Modification of oligonucleotides with pos. charged residues greatly improves thermal stability....

  19. Abundance Anomalies In Tidal Disruption Events

    Kochanek, C S

    2015-01-01

    The ~10% of tidal disruption events (TDEs) due to stars more massive than the Sun should show abundance anomalies due to stellar evolution in helium, carbon and nitrogen, but not oxygen. Helium is always enhanced, but only by up to ~25% on average because it becomes inaccessible once it is sequestered in the high density core as the star leaves the main sequence. However, portions of the debris associated with the disrupted core of a main sequence star can be enhanced in helium by factors of 2-3 for debris at a common orbital period. These helium abundance variations may be a contributor to the observed diversity of hydrogen and helium line strengths in TDEs. A still more striking anomaly is the rapid enhancement of nitrogen and the depletion of carbon due to the CNO cycle -- stars more massive than the Sun quickly show an increase in their average N/C ratio by factors of 3-10. Because low mass stars evolve slowly and high mass stars are rare, TDEs showing high N/C will almost all be due to 1-2Msun stars disr...

  20. Sm Transition Probabilities and Abundances

    Lawler, J E; Sneden, C; Cowan, J J

    2005-01-01

    Radiative lifetimes, accurate to +/- 5%, have been measured for 212 odd-parity levels of Sm II using laser-induced fluorescence. The lifetimes are combined with branching fractions measured using Fourier-transform spectrometry to determine transition probabilities for more than 900 lines of Sm II. This work is the largest-scale laboratory study to date of Sm II transition probabilities using modern methods. This improved data set has been used to determine a new solar photospheric Sm abundance, log epsilon = 1.00 +/- 0.03, from 26 lines. The spectra of three very metal-poor, neutron-capture-rich stars also have been analyzed, employing between 55 and 72 Sm II lines per star. The abundance ratios of Sm relative to other rare earth elements in these stars are in agreement, and are consistent with ratios expected from rapid neutron-capture nucleosynthesis (the r-process).

  1. TCP1 complex proteins interact with phosphorothioate oligonucleotides and can co-localize in oligonucleotide-induced nuclear bodies in mammalian cells

    Liang, Xue-hai; Shen, Wen; Sun, Hong; Prakash, Thazha P.; Crooke, Stanley T.

    2014-01-01

    Phosphorothioate (PS) antisense oligonucleotides (ASOs) have been successfully developed as drugs to reduce the expression of disease-causing genes. PS-ASOs can be designed to induce degradation of complementary RNAs via the RNase H pathway and much is understood about that process. However, interactions of PS-ASOs with other cellular proteins are not well characterized. Here we report that in cells transfected with PS-ASOs, the chaperonin T-complex 1 (TCP1) proteins interact with PS-ASOs and...

  2. Abundances in stars with exoplanets

    Israelian, Garik

    2003-01-01

    Extensive spectroscopic studies of stars with and without planets have concluded that stars hosting planets are significantly more metal-rich than those without planets. More subtle trends of different chemical elements begin to appear as the number of detected extrasolar planetary systems continues to grow. I review our current knowledge concerning the observed abundance trends of various chemical elements in stars with exoplanets and their possible implications.

  3. Forms and genesis of species abundance distributions

    Evans O. Ochiaga

    2015-12-01

    Full Text Available Species abundance distribution (SAD is one of the most important metrics in community ecology. SAD curves take a hollow or hyperbolic shape in a histogram plot with many rare species and only a few common species. In general, the shape of SAD is largely log-normally distributed, although the mechanism behind this particular SAD shape still remains elusive. Here, we aim to review four major parametric forms of SAD and three contending mechanisms that could potentially explain this highly skewed form of SAD. The parametric forms reviewed here include log series, negative binomial, lognormal and geometric distributions. The mechanisms reviewed here include the maximum entropy theory of ecology, neutral theory and the theory of proportionate effect.

  4. Oxygen Gas Phase Abundance Revisited

    André, M K; Howk, J C; Ferlet, R; Désert, J M; Hébrard, G; Lacour, S; Lecavelier-des-Etangs, A; Vidal-Madjar, A; Moos, H W

    2003-01-01

    We present new measurements of the interstellar gas-phase oxygen abundance along the sight lines towards 19 early-type galactic stars at an average distance of 2.6 kpc. We derive O {\\small I} column densities from {\\it HST}/STIS observations of the weak 1355 \\AA intersystem transition. We derive total hydrogen column densities [N(H {\\small I})+2N(H$_2$)] using {\\it HST}/STIS observations of \\lya and {\\it FUSE} observations of molecular hydrogen. The molecular hydrogen content of these sight lines ranges from f(H$_2$) = 2N(H$_2$)/[N(H {\\small I})+2N(H$_2$)] = 0.03 to 0.47. The average $$ of 6.3$\\times10^{21}$ cm$^{-2}$ mag$^{-1}$ with a standard deviation of 15% is consistent with previous surveys. The mean oxygen abundance along these sight lines, which probe a wide range of galactic environments in the distant ISM, is 10$^6$ \\oh = $408 \\pm 13$ (1 $\\sigma$ in the mean). %$({\\rm O/H})_{gas} = 408 \\pm 14$(1 $\\sigma$). We see no evidence for decreasing gas-phase oxygen abundance with increasing molecular hydroge...

  5. Planetary nebulae abundances and stellar evolution

    Pottasch, S. R.; Bernard-Salas, J.

    2006-01-01

    A summary is given of planetary nebulae abundances from ISO measurements. It is shown that these nebulae show abundance gradients (with galactocentric distance), which in the case of neon, argon, sulfur and oxygen (with four exceptions) are the same as HII regions and early type star abundance gradients. The abundance of these elements predicted from these gradients at the distance of the Sun from the center are exactly the solar abundance. Sulfur is the exception to this; the reason for this...

  6. Common Privacy Myths

    ... home > privacy + phrs > common privacy myths Common Privacy Myths With the new federal laws protecting the privacy ... are the truths to some of the common myths: Health information cannot be faxed – FALSE Your information ...

  7. Migraine and Common Morbidities

    ... headaches . Home > Migraine and Common Morbidities Print Email Migraine and Common Morbidities ACHE Newsletter Sign up for ... newsletter by entering your e-mail address below. Migraine and Common Morbidities For many patients, migraine is ...

  8. Development and experimental validation of a 20K Atlantic cod (Gadus morhua) oligonucleotide microarray based on a collection of over 150,000 ESTs.

    Booman, Marije; Borza, Tudor; Feng, Charles Y; Hori, Tiago S; Higgins, Brent; Culf, Adrian; Léger, Daniel; Chute, Ian C; Belkaid, Anissa; Rise, Marlies; Gamperl, A Kurt; Hubert, Sophie; Kimball, Jennifer; Ouellette, Rodney J; Johnson, Stewart C; Bowman, Sharen; Rise, Matthew L

    2011-08-01

    The collapse of Atlantic cod (Gadus morhua) wild populations strongly impacted the Atlantic cod fishery and led to the development of cod aquaculture. In order to improve aquaculture and broodstock quality, we need to gain knowledge of genes and pathways involved in Atlantic cod responses to pathogens and other stressors. The Atlantic Cod Genomics and Broodstock Development Project has generated over 150,000 expressed sequence tags from 42 cDNA libraries representing various tissues, developmental stages, and stimuli. We used this resource to develop an Atlantic cod oligonucleotide microarray containing 20,000 unique probes. Selection of sequences from the full range of cDNA libraries enables application of the microarray for a broad spectrum of Atlantic cod functional genomics studies. We included sequences that were highly abundant in suppression subtractive hybridization (SSH) libraries, which were enriched for transcripts responsive to pathogens or other stressors. These sequences represent genes that potentially play an important role in stress and/or immune responses, making the microarray particularly useful for studies of Atlantic cod gene expression responses to immune stimuli and other stressors. To demonstrate its value, we used the microarray to analyze the Atlantic cod spleen response to stimulation with formalin-killed, atypical Aeromonas salmonicida, resulting in a gene expression profile that indicates a strong innate immune response. These results were further validated by quantitative PCR analysis and comparison to results from previous analysis of an SSH library. This study shows that the Atlantic cod 20K oligonucleotide microarray is a valuable new tool for Atlantic cod functional genomics research. PMID:21127932

  9. Histopathological Defects in Intestine in Severe Spinal Muscular Atrophy Mice Are Improved by Systemic Antisense Oligonucleotide Treatment.

    Palittiya Sintusek

    Full Text Available Gastrointestinal (GI defects, including gastroesophageal reflux, constipation and delayed gastric emptying, are common in patients with spinal muscular atrophy (SMA. Similar GI dysmotility has been identified in mouse models with survival of motor neuron (SMN protein deficiency. We previously described vascular defects in skeletal muscle and spinal cord of SMA mice and we hypothesized that similar defects could be involved in the GI pathology observed in these mice. We therefore investigated the gross anatomical structure, enteric vasculature and neurons in the small intestine in a severe mouse model of SMA. We also assessed the therapeutic response of GI histopathology to systemic administration of morpholino antisense oligonucleotide (AON designed to increase SMN protein expression. Significant anatomical and histopathological abnormalities, with striking reduction of vascular density, overabundance of enteric neurons and increased macrophage infiltration, were detected in the small intestine in SMA mice. After systemic AON treatment in neonatal mice, all the abnormalities observed were significantly restored to near-normal levels. We conclude that the observed GI histopathological phenotypes and functional defects observed in these SMA mice are strongly linked to SMN deficiency which can be rescued by systemic administration of AON. This study on the histopathological changes in the gastrointestinal system in severe SMA mice provides further indication of the complex role that SMN plays in multiple tissues and suggests that at least in SMA mice restoration of SMN production in peripheral tissues is essential for optimal outcome.

  10. Conversion of adenine to 5-amino-4-pyrimidinylimidazole caused by acetyl capping during solid phase oligonucleotide synthesis.

    Rodriguez, Andrew A; Cedillo, Isaiah; McPherson, Andrew K

    2016-08-01

    The acetyl capping reaction used throughout solid phase oligonucleotide synthesis is meant to minimize n-1 deletionmer impurities by terminating sequences that fail to couple to a phosphoramidite. However, the reaction is also responsible for the formation of a number of impurities. One capping-related impurity has an additional mass of 98amu from the parent oligonucleotide. The n+98 amu impurity was found to result from modification of an adenine nucleobase. The structure of the impurity was determined by preparation of an oligonucleotide enriched in n+98 amu, enzymatic digestion to individual nucleosides, isolation of the pure nucleoside+98 amu species, crystallization, and X-ray crystallographic analysis. The n+98 amu impurity is an oligonucleotide in which one adenine residue has been converted to 5-amino-4-pyrimidinylimidazole. The mechanism of formation of the impurity was investigated, and a mechanism is proposed. PMID:27353533

  11. Lead abundance in the uranium star CS 31082-001

    Plez, B.; Hill, V.; Cayrel, R.; Spite, M.; Barbuy, B.; Beers, T.C.; Bonifacio, P.; Primas, F.; Nordström, B.

    2004-01-01

    stars:abundances- physical data and processes: nuclear reactions, nucleosynthesis, abundances- atomic data......stars:abundances- physical data and processes: nuclear reactions, nucleosynthesis, abundances- atomic data...

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

    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

  13. Ultrahigh molecular recognition specificity of competing DNA oligonucleotide strands in thermal equilibrium

    Schenkelberger, Marc; Mai, Timo; Ott, Albrecht

    2016-01-01

    The specificity of molecular recognition is important to molecular self-organization. A prominent example is the biological cell where, within a highly crowded molecular environment, a myriad of different molecular receptor pairs recognize their binding partner with astonishing accuracy. In thermal equilibrium it is usually admitted that the affinity of recognizer pairs only depends on the nature of the two binding molecules. Accordingly, Boltzmann factors of binding energy differences relate the molecular affinities among different target molecules that compete for the same probe. Here, we consider the molecular recognition of short DNA oligonucleotide single strands. We show that a better matching oligonucleotide strand can prevail against a disproportionally more concentrated competitor that exhibits reduced affinity due to a mismatch. The magnitude of deviation from the simple picture above may reach several orders of magnitude. In our experiments the effective molecular affinity of a given strand remains...

  14. Site-Specific Oligonucleotide Binding Represses Transcription of the Human c-myc Gene in vitro

    Cooney, Michael; Czernuszewicz, Graznya; Postel, Edith H.; Flint, S. Jane; Hogan, Michael E.

    1988-07-01

    A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.

  15. P122-M The Effect of Ion-Pairing Reagents on the Reversed Phase Purification of DMT-off Oligonucleotides

    Fisher, J. R.; Deetz, M.; Gehris, A.; Maikner, J.; Kinzey, M.

    2007-01-01

    The use of ion-pairing reagents for reversed phase peptide separations has been investigated in numerous publications. Reversed phase oligonucleotide separations typically employ TEAA as an ion-pairing reagent. However, other ion-pair reagents may provide better separation results. We investigated several different ion-pairing reagents using a ten micron, mono-sized, reversed phase polymeric resin, Amberchrom HPR10. DMT-off purifications of two synthetic 12mer DNA oligonucleotides (AAA CCT GA...

  16. Family- and Genus-Level 16S rRNA-Targeted Oligonucleotide Probes for Ecological Studies of Methanotrophic Bacteria

    J. Gulledge; Ahmad, A; Steudler, P. A.; Pomerantz, W. J.; Cavanaugh, Colleen Marie

    2001-01-01

    Methanotrophic bacteria play a major role in the global carbon cycle, degrade xenobiotic pollutants, and have the potential for a variety of biotechnological applications. To facilitate ecological studies of these important organisms, we developed a suite of oligonucleotide probes for quantitative analysis of methanotroph-specific 16S rRNA from environmental samples. Two probes target methanotrophs in the family Methylocystaceae (type II methanotrophs) as a group. No oligonucleotide signature...

  17. In vivo transcription of a progesterone-responsive gene is specifically inhibited by a triplex-forming oligonucleotide.

    Ing, N H; Beekman, J M; Kessler, D J; Murphy, M.; Jayaraman, K; Zendegui, J G; Hogan, M E; O'Malley, B W; Tsai, M J

    1993-01-01

    Oligonucleotides provide novel reagents for inhibition of gene expression because of their high affinity binding to specific nucleotide sequences. We describe a 38 base, single-stranded DNA that forms a triple helix or 'triplex' on progesterone response elements of a target gene. This triplex-forming oligonucleotide binds with a Kd = 100 nM at 37 degrees C and physiological pH, and blocks binding of progesterone receptors to the target. Furthermore, it completely inhibited progesterone recept...

  18. Accessibility of nuclear DNA to triplex-forming oligonucleotides: The integrated HIV-1 provirus as a target

    Giovannangeli, Carine; Diviacco, Silvia; Labrousse, Valérie; Gryaznov, Sergei; Charneau, Pierre; Helene, Claude

    1997-01-01

    The control of gene transcription by antigene oligonucleotides rests upon the specific recognition of double-helical DNA by triplex-forming oligonucleotides. The development of the antigene strategy requires access to the targeted DNA sequence within the chromatin structure of the cell nucleus. In this sudy we have used HIV-1 chronically infected cells containing the HIV provirus as endogenous genes to demonstrate that the integrated HIV-1 proviral genome is accessible to triplex-forming olig...

  19. Inhibition of certain strains of HIV-1 by cell surface polyanions in the form of cholesterol-labeled oligonucleotides

    Cholesterol-labeled oligonucleotides were found several years ago to inhibit HIV-1 in tissue culture at nanomolar concentrations. We present evidence that this is mainly due to an electrostatic interaction between polyanionic oligonucleotide concentrated at the cell surface and a positively charged region in the V3 loop of the HIV-1 envelope protein. When added to tissue culture, cholesterol-labeled oligonucleotides became concentrated at the plasma membrane and potently inhibited virus entry and cell fusion mediated by the envelope protein of some X4 strains of HIV-1, but had little effect on fusion mediated by R5 strains of HIV-1, amphotropic MLV envelope protein, or VSV-G protein. Noncholesterol-labeled oligonucleotides did not bind to the cell surface or inhibit fusion. The pattern of susceptibility to cholesterol-labeled oligonucleotides among HIV-1 strains was the same as reported for nonmembrane-associating polyanions such as dextran sulfate, but the cholesterol-labeled oligonucleotides were effective at lower concentrations. Substitution of a basic 33 amino acid V3 loop sequence from the envelope protein of a resistant strain into a susceptible strain made the envelope protein resistant to inhibition. Inhibition by cholesterol-labeled oligonucleotides was abrogated by the polycation DEAE-dextran. Cholesterol-labeled oligonucleotides bound to nonraft regions of the plasma membrane and did not inhibit HIV virus binding to cells. Many infectious agents first associate with target cells via relatively nonspecific charge interactions; our data suggest that molecules that combine a membrane-targeting motif with multiple negative charges might be useful to modify these interactions

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

    Magness Charles L; Thomas Matthew J; Proll Sean C; Paeper Bryan; Korth Marcus J; Wallace James C; Iadonato Shawn P; Nelson Charles; Katze Michael G

    2007-01-01

    Abstract Background Until recently, few genomic reagents specific for non-human primate research have been available. To address this need, we have constructed a macaque-specific high-density oligonucleotide microarray by using highly fragmented low-pass sequence contigs from the rhesus genome project together with the detailed sequence and exon structure of the human genome. Using this method, we designed oligonucleotide probes to over 17,000 distinct rhesus/human gene orthologs and increase...

  1. Investigating Host Star Abundances as Signatures of Terrestrial Planets

    Teske, J.; Schuler, S.; Cunha, K.; Smith, V.

    2014-03-01

    Kepler has fundamentally changed our view of exoplanets, revealing that Jupiter and Saturn-sized planets are not the most common. Of the current ~3600 Kepler planet candidates, ~65% are ≤2.5 REarth and nearly 300 orbit in/near their host stars' habitable zones (180Kanalog, host star abundances are indicative of the precursor materials available in the protoplanetary disk for incorporation into planets. Our own Sun is deficient by ~20% in refractory elements (Tc=900 K) relative to volatile elements when compared to most (~85%) solar-type stars [3,4,5,6]. This has been proposed as a signature of terrestrial planet formation, with the "missing" refractory elements incorporated into rocky planets [3,7,8]. The amount of missing material in our Sun amounts to that needed to form terrestrial planets [3,7,8], and the abundance patterns in meteorites mirror this solar abundance anomaly [9,10]. However, subsequent studies of stars with/without planets indicate that their abundance patterns may not be so different, or indistinguishable from Galactic chemical evolution [11,12,13]. Here we use traditional stellar abundance analysis (non-automated) to independently re-analyze the Keck/HIRES data presented in Melendez et al. (2012) [6] of one of the best solar twins known to date - though not yet known to host any planets - to investigate their finding of refractory elemental abundance depletion similar to the Sun. We compare our results to similar studies implementing the same type differential abundance analysis to search for a Sun-like abundance pattern using Keck/HIRES spectra of stars discovered by Kepler to host small (terrestrial) planets.

  2. Clinical Expert Panel on Monitoring Potential Lung Toxicity of Inhaled Oligonucleotides: Consensus Points and Recommendations

    Alton, Eric W.; Boushey, Homer A.; Garn, Holger; Green, Francis H.; Hodges, Michael; Richard J. Martin; Murdoch, Robert D.; Renz, Harald; Shrewsbury, Stephen B; Seguin, Rosanne; Johnson, Graham; Parry, Joel D.; Tepper, Jeff; Renzi, Paolo; Cavagnaro, Joy

    2012-01-01

    Oligonucleotides (ONs) are an emerging class of drugs being developed for the treatment of a wide variety of diseases including the treatment of respiratory diseases by the inhalation route. As a class, their toxicity on human lungs has not been fully characterized, and predictive toxicity biomarkers have not been identified. To that end, identification of sensitive methods and biomarkers that can detect toxicity in humans before any long term and/or irreversible side effects occur would be h...

  3. Application of custom-designed oligonucleotide array CGH in 145 patients with autistic spectrum disorders

    Wiśniowiecka-Kowalnik, Barbara; Kastory-Bronowska, Monika; Bartnik, Magdalena; Derwińska, Katarzyna; Dymczak-Domini, Wanda; Szumbarska, Dorota; Ziemka, Ewa; Szczałuba, Krzysztof; Sykulski, Maciej; Gambin, Tomasz; Gambin, Anna; Shaw, Chad A.; Mazurczak, Tadeusz; Obersztyn, Ewa; Bocian, Ewa

    2012-01-01

    Autism spectrum disorders (ASDs) are a heterogeneous group of neurodevelopmental disorders, including childhood autism, atypical autism, and Asperger syndrome, with an estimated prevalence of 1.0–2.5% in the general population. ASDs have a complex multifactorial etiology, with genetic causes being recognized in only 10–20% of cases. Recently, copy-number variants (CNVs) have been shown to contribute to over 10% of ASD cases. We have applied a custom-designed oligonucleotide array comparative ...

  4. A new linker for solid-phase synthesis of oligonucleotides with terminal phosphate group

    Pačes, Ondřej; Točík, Zdeněk; Rosenberg, Ivan

    2008-01-01

    Roč. 73, č. 1 (2008), s. 32-43. ISSN 0010-0765 R&D Projects: GA ČR GA203/05/0827; GA ČR GA202/05/0628; GA MŠk LC512; GA MŠk(CZ) LC06077 Institutional research plan: CEZ:AV0Z40550506 Keywords : solid-phase synthesis * modified LCAA-CPG * oligonucleotide 3'-phosphates Subject RIV: CC - Organic Chemistry Impact factor: 0.784, year: 2008

  5. Synthesis and hybridization of oligonucleotides modified at AMP sites with adenine pyrrolidine phosphonate nucleotides

    Rejman, Dominik; Kočalka, Petr; Pohl, Radek; Točík, Zdeněk; Rosenberg, Ivan

    2009-01-01

    Roč. 74, č. 6 (2009), s. 935-955. ISSN 0010-0765 R&D Projects: GA MŠk 2B06065; GA MŠk(CZ) LC06077; GA MŠk(CZ) LC06061; GA AV ČR KAN200520801 Institutional research plan: CEZ:AV0Z40550506 Keywords : oligonucleotide * pyrrolidine * phosphonate Subject RIV: CC - Organic Chemistry Impact factor: 0.856, year: 2009

  6. Simulation and visualization of flow pattern in microarrays for liquid phase oligonucleotide and peptide synthesis.

    O-Charoen, Sirimon; Srivannavit, Onnop; Gulari, Erdogan

    2007-01-01

    Microfluidic microarrays have been developed for economical and rapid parallel synthesis of oligonucleotide and peptide libraries. For a synthesis system to be reproducible and uniform, it is crucial to have a uniform reagent delivery throughout the system. Computational fluid dynamics (CFD) is used to model and simulate the microfluidic microarrays to study geometrical effects on flow patterns. By proper design geometry, flow uniformity could be obtained in every microreactor in the microarrays. PMID:17480053

  7. SIMULATION AND VISUALIZATION OF FLOW PATTERN IN MICROARRAYS FOR LIQUID PHASE OLIGONUCLEOTIDE AND PEPTIDE SYNTHESIS

    O-Charoen, Sirimon; Srivannavit, Onnop; Gulari, Erdogan

    2007-01-01

    Microfluidic microarrays have been developed for economical and rapid parallel synthesis of oligonucleotide and peptide libraries. For a synthesis system to be reproducible and uniform, it is crucial to have a uniform reagent delivery throughout the system. Computational fluid dynamics (CFD) is used to model and simulate the microfluidic microarrays to study geometrical effects on flow patterns. By proper design geometry, flow uniformity could be obtained in every microreactor in the microarr...

  8. Simultaneous Detection of Four Human Pathogenic Microsporidian Species from Clinical Samples by Oligonucleotide Microarray

    Wang, Zheng; Orlandi, Palmer A.; David A Stenger

    2005-01-01

    Microsporidian species have been rapidly emerging as human enteric pathogens in immunocompromised and immunocompetent individuals in recent years. Routine diagnostic techniques for microsporidia in clinical laboratories are laborious and insensitive and tend to underestimate their presence. In most instances, they are unable to differentiate species of spores due to their small sizes and similar morphologies. In this study, we report the development of another protozoan oligonucleotide microa...

  9. Immobilization of nucleic acids at solid surfaces: effect of oligonucleotide length on layer assembly.

    Steel, A B; Levicky, R L; Herne, T M; Tarlov, M J

    2000-01-01

    This report investigates the effect of DNA length and the presence of an anchoring group on the assembly of presynthesized oligonucleotides at a gold surface. The work seeks to advance fundamental insight into issues that impact the structure and behavior of surface-immobilized DNA layers, as in, for instance, DNA microarray and biosensor devices. The present study contrasts immobilization of single-stranded DNA (ssDNA) containing a terminal, 5' hexanethiol anchoring group with that of unfunc...

  10. Distinguishing Colonization From Infection With Staphylococcus aureus in Diabetic Foot Ulcers With Miniaturized Oligonucleotide Arrays

    Sotto, Albert; Richard, Jean-Louis; Messad, Nourredine; Molinari, Nicolas; Jourdan, Nathalie; Schuldiner, Sophie; Sultan, Ariane; Carrière, Christian; Canivet, Bertrand; Landraud, Luce; Lina, Gérard; Lavigne, Jean-Philippe; ,

    2012-01-01

    OBJECTIVE To extend our previous work on evaluating the use of oligonucleotide arrays to discriminate colonization from infection owing to Staphylococcus aureus in diabetic foot ulcers (DFUs). RESEARCH DESIGN AND METHODS Patients admitted to 14 French diabetic foot departments for a DFU were screened for entry into the study. At admission, ulcers were classified based on clinical examination according to the Infectious Diseases Society of America system. Only patients with monomicrobial cultu...

  11. Double-tailed lipid modification as a promising candidate for oligonucleotide delivery in mammalian cells

    Ugarte-Uribe, Begoña; Grijalvo, Santiago; Busto, Jon V.; Martín, César; Eritja Casadellà, Ramón; Goñi, Félix Lix M; Alkorta, Itziar

    2013-01-01

    Background The potential use of nucleic acids as therapeutic drugs has triggered the quest for oligonucleotide conjugates with enhanced cellular permeability. To this end, the biophysical aspects of previously reported potential lipid oligodeoxyribonucleotide conjugates were studied including its membrane-binding properties and cellular uptake. Methods These conjugates were fully characterized by MALDI-TOF mass spectrometry and HPLC chromatography. Their ability to insert into lipid model mem...

  12. Application of Oligonucleotide Microarrays for Bacterial Source Tracking of Environmental Enterococcus sp. Isolates

    Furey, John S.; Kelley Betts; Indest, Karl J.

    2005-01-01

    In an effort towards adapting new and defensible methods for assessing and managing the risk posed by microbial pollution, we evaluated the utility of oligonucleotide microarrays for bacterial source tracking (BST) of environmental Enterococcus sp. isolates derived from various host sources. Current bacterial source tracking approaches rely on various phenotypic and genotypic methods to identify sources of bacterial contamination resulting from point or non-point pollution. For this study Ent...

  13. Development of Multiexon Skipping Antisense Oligonucleotide Therapy for Duchenne Muscular Dystrophy

    Yoshitsugu Aoki; Toshifumi Yokota; Wood, Matthew J. A.

    2013-01-01

    Duchenne muscular dystrophy (DMD) is an incurable, X-linked progressive muscle degenerative disorder that results from the absence of dystrophin protein and leads to premature death in affected individuals due to respiratory and/or cardiac failure typically by age of 30. Very recently the exciting prospect of an effective oligonucleotide therapy has emerged which restores dystrophin protein expression to affected tissues in DMD patients with highly promising data from a series of clinical tri...

  14. An activated triple bond linker enables 'click' attachment of peptides to oligonucleotides on solid support

    Wenska, Malgorzata; Alvira, Margarita; Steunenberg, Peter; Stenberg, Åsa; Murtola, Merita; Strömberg, Roger

    2011-01-01

    A general procedure, based on a new activated alkyne linker, for the preparation of peptide–oligonucleotide conjugates (POCs) on solid support has been developed. With this linker, conjugation is effective at room temperature (RT) in millimolar concentration and submicromolar amounts. This is made possible since the use of a readily attachable activated triple bond linker enhances the Cu(I) catalyzed 1,3-dipolar cycloaddition (‘click’ reaction). The preferred scheme for conjugate preparation ...

  15. Modular construction of plasmids through ligation-free assembly of vector components with oligonucleotide linkers.

    Vroom, Jonathan A; Wang, Clifford L

    2008-06-01

    We have developed a modular method of plasmid construction that can join multiple DNA components in a single reaction. A nicking enzyme is used to create 5' and 3' overhangs on PCR-generated DNA components. Without the use of ligase or restriction enzymes, components are joined using oligonucleotide linkers that recognize the overhangs. By specifying the sequences of the linkers, desired components can be assembled in any combination and order to generate different plasmid vectors. PMID:18533903

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

    Chen, Fuqiang; Pruett-Miller, Shondra M; Huang, Yuping; Gjoka, Monika; Duda, Katarzyna; Taunton, Jack; Collingwood, Trevor N; Frödin, Morten; Davis, Gregory D

    2011-01-01

    Zinc-finger nucleases (ZFNs) have enabled highly efficient gene targeting in multiple cell types and organisms. Here we describe methods for using simple ssDNA oligonucleotides in tandem with ZFNs to efficiently produce human cell lines with three distinct genetic outcomes: (i) targeted point mut...... mutation, (ii) targeted genomic deletion of up to 100 kb and (iii) targeted insertion of small genetic elements concomitant with large genomic deletions....

  17. Genome dynamics of short oligonucleotides: the example of bacterial DNA uptake enhancing sequences.

    Mohammed Bakkali

    Full Text Available Among the many bacteria naturally competent for transformation by DNA uptake-a phenomenon with significant clinical and financial implications- Pasteurellaceae and Neisseriaceae species preferentially take up DNA containing specific short sequences. The genomic overrepresentation of these DNA uptake enhancing sequences (DUES causes preferential uptake of conspecific DNA, but the function(s behind this overrepresentation and its evolution are still a matter for discovery. Here I analyze DUES genome dynamics and evolution and test the validity of the results to other selectively constrained oligonucleotides. I use statistical methods and computer simulations to examine DUESs accumulation in Haemophilus influenzae and Neisseria gonorrhoeae genomes. I analyze DUESs sequence and nucleotide frequencies, as well as those of all their mismatched forms, and prove the dependence of DUESs genomic overrepresentation on their preferential uptake by quantifying and correlating both characteristics. I then argue that mutation, uptake bias, and weak selection against DUESs in less constrained parts of the genome combined are sufficient enough to cause DUESs accumulation in susceptible parts of the genome with no need for other DUES function. The distribution of overrepresentation values across sequences with different mismatch loads compared to the DUES suggests a gradual yet not linear molecular drive of DNA sequences depending on their similarity to the DUES. Other genomically overrepresented sequences, both pro- and eukaryotic, show similar distribution of frequencies suggesting that the molecular drive reported above applies to other frequent oligonucleotides. Rare oligonucleotides, however, seem to be gradually drawn to genomic underrepresentation, thus, suggesting a molecular drag. To my knowledge this work provides the first clear evidence of the gradual evolution of selectively constrained oligonucleotides, including repeated, palindromic and protein

  18. Targeting duplex DNA with chimeric α,β-triplex-forming oligonucleotides

    Kolganova, N. A.; Shchyolkina, A K; Chudinov, A. V.; Zasedatelev, A S; Florentiev, V L; Timofeev, E. N.

    2012-01-01

    Triplex-directed DNA recognition is strictly limited by polypurine sequences. In an attempt to address this problem with synthetic biology tools, we designed a panel of short chimeric α,β-triplex-forming oligonucleotides (TFOs) and studied their interaction with fluorescently labelled duplex hairpins using various techniques. The hybridization of hairpin with an array of chimeric probes suggests that recognition of double-stranded DNA follows complicated rules combining reversed Hoogsteen and...

  19. Using Triplex-Forming Oligonucleotide Probes for the Reagentless, Electrochemical Detection of Double-Stranded DNA

    Patterson, Adriana; Caprio, Felice; Vallée-Bélisle, Alexis; Moscone, Danila; Plaxco, Kevin W.; Palleschi, Giuseppe; Ricci, Francesco

    2010-01-01

    We report a reagentless, electrochemical sensor for the detection of double-stranded DNA targets that employs triplex-forming oligonucleotides (TFOs) as its recognition element. These sensors are based on redox-tagged TFO probes strongly chemisorbed onto an interrogating gold electrode. Upon the addition of the relevant double-stranded DNA target, the probe forms a rigid triplex structure via reverse Hoogsteen base pairing in the major groove. The formation of the triplex impedes contact betw...

  20. Triple helix-forming oligonucleotides target psoralen adducts to specific chromosomal sequences in human cells.

    Oh, D H; Hanawalt, P C

    1999-01-01

    The ability to target photochemical adducts to specific genomic DNA sequences in cells is useful for studying DNA repair and mutagenesis in intact cells, and also as a potential mode of gene-specific therapy. Triple helix-forming DNA oligonucleotides linked to psoralen (psoTFOs) were designed to deliver UVA-induced psoralen photoadducts to two distinct sequences within the human interstitial collagenase gene. A primer extension assay demonstrated that the appropriate psoTFO selectively damage...

  1. High-frequency intrachromosomal gene conversion induced by triplex-forming oligonucleotides microinjected into mouse cells

    Luo, Zhongjun; Macris, Margaret A.; Faruqi, A. Fawad; Glazer, Peter M.

    2000-01-01

    To test the ability of triple helix-forming oligonucleotides (TFOs) to promote recombination within chromosomal sites in mammalian cells, a mouse LTK− cell line was established carrying two mutant copies of the herpes simplex virus thymidine kinase (TK) gene as direct repeats in a single chromosomal locus. Recombination between these repeats can produce a functional TK gene and occurs at a spontaneous frequency of 4 × 10−6 under standard culture conditions. When cells were microinjected with ...

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

    Oh, Dennis H.; Rajagopal Krishnan

    2010-01-01

    Triplex-forming oligonucleotides (TFOs) with both DNA and 2′-O-methyl RNA backbones can direct psoralen photoadducts to specific DNA sequences. However, the functional consequences of these differing structures on psoralen photoreactivity are unknown. We designed TFO sequences with DNA and 2′-O-methyl RNA backbones conjugated to psoralen by 2-carbon linkers and examined their ability to bind and target damage to model DNA duplexes corresponding to sequences within the human HPRT gene. While T...

  3. Site-specific mutagenesis by triple helix-forming oligonucleotides containing a reactive nucleoside analog

    Nagatsugi, Fumi; Sasaki, Shigeki; Miller, Paul S.; Seidman, Michael M.

    2003-01-01

    The specific recognition of homopurine–homo pyrimidine regions in duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Alkylation of nucleobases with functionalized TFOs would have the potential for site-directed mutagenesis. Recently, we demonstrated that a TFO bearing 2-amino-6-vinylpurine derivative, 1, achieves triplex-mediated reaction with high selectivity toward the cytosine of the G-C target site. In this report, we have inves...

  4. Recognition of RNA duplexes by chemically modified triplex-forming oligonucleotides

    Zhou, Yuan; Kierzek, Elzbieta; Loo, Zi Ping; Antonio, Meraldo; Yau, Yin Hoe; Chuah, York Wieo; Geifman-Shochat, Susana; Kierzek, Ryszard; Chen, Gang

    2013-01-01

    Triplex is emerging as an important RNA tertiary structure motif, in which consecutive non-canonical base pairs form between a duplex and a third strand. RNA duplex region is also often functionally important site for protein binding. Thus, triplex-forming oligonucleotides (TFOs) may be developed to regulate various biological functions involving RNA, such as viral ribosomal frameshifting and reverse transcription. How chemical modification in TFOs affects RNA triplex stability, however, is n...

  5. Peptide conjugates for chromosomal gene targeting by triplex-forming oligonucleotides

    Rogers, Faye A.; Manoharan, Muthiah; Rabinovitch, Peter; Ward, David C.; Glazer, Peter M.

    2004-01-01

    Triplex-forming oligonucleotides (TFOs) are DNA-binding molecules, which offer the potential to selectively modulate gene expression. However, the biological activity of TFOs as potential antigene compounds has been limited by cellular uptake. Here, we investigate the effect of cell-penetrating peptides on the biological activity of TFOs as measured in an assay for gene-targeted mutagenesis. Using the transport peptide derived from the third helix of the homeodomain of antennapedia (Antp), we...

  6. Optimal design of parallel triplex forming oligonucleotides containing Twisted Intercalating Nucleic Acids—TINA

    Schneider, Uffe V.; Mikkelsen, Nikolaj D.; Jøhnk, Nina; Okkels, Limei M.; Westh, Henrik; Lisby, Gorm

    2010-01-01

    Twisted intercalating nucleic acid (TINA) is a novel intercalator and stabilizer of Hoogsteen type parallel triplex formations (PT). Specific design rules for position of TINA in triplex forming oligonucleotides (TFOs) have not previously been presented. We describe a complete collection of easy and robust design rules based upon more than 2500 melting points (T m) determined by FRET. To increase the sensitivity of PT, multiple TINAs should be placed with at least 3 nt in-between or preferabl...

  7. Covalent crosslinks introduced via a triple helix-forming oligonucleotide coupled to psoralen are inefficiently repaired.

    Barre, F X; Giovannangeli, C.; Hélène, C; Harel-Bellan, A

    1999-01-01

    Triple helix-forming oligonucleotides (TFOs) represent potentially powerful tools to artificially modulate gene activity. In particular, they can be used to specifically introduce a lesion into a selected target sequence: interstrand crosslinks and monoadducts can be introduced via TFOs coupled to psoralen. The efficiency of these strategies depends on the cell ability to repair these lesions, an issue which is still controversial. Here we show, using psoralen-coupled TFOs and the yeast as a ...

  8. Gene activation by triplex-forming oligonucleotide coupled to the activating domain of protein VP16.

    Kuznetsova, S.; Ait-Si-Ali, S; Nagibneva, I; Troalen, F; Le Villain, J P; Harel-Bellan, A; Svinarchuk, F

    1999-01-01

    Triplex-forming oligonucleotides (TFOs) are generally designed to inhibit transcription or DNA replication but can be used for more diverse purposes. Here we have designed a chimera peptide-TFO able to activate transcription from a target gene. The designed hybrid molecule contains a triplex-forming sequence, linked through a phosphoroamidate bond to several minimal transcriptional activation domains derived from Herpes simplex virus protein 16 (VP16). We show here that this TFO-peptide chime...

  9. HPMA Polymer-based Site-specific Delivery of Oligonucleotides to Hepatic Stellate Cells

    Yang, Ningning; Ye, Zhaoyang; Li, Feng; Mahato, Ram I.

    2009-01-01

    The objective was to determine whether bioconjugation of type I collagen specific triplex forming oligonucleotide (TFO) to N-(2-hydroxypropyl) methacrylamide (HPMA) containing tetrapeptide Gly-Phe-Leu-Gly (GFLG) and mannose 6-phosphate (M6P) can provide their targeted delivery to hepatic stellate cells (HSCs). Following bioconjugation, M6P-GFLG-HPMA-GFLG-32P-TFO was characterized by PAGE, HPLC and GPC, and then its biodistribution was determined. TFO was dissociated from the conjugate when in...

  10. Exploring the Cellular Activity of Camptothecin-Triple-Helix-Forming Oligonucleotide Conjugates†

    Arimondo, Paola B.; Thomas, Craig J.; Oussedik, Kahina; Baldeyrou, Brigitte; Mahieu, Christine; Halby, Ludovic; Guianvarc'h, Dominique; Lansiaux, Amélie; Hecht, Sidney M.; Bailly, Christian; Giovannangeli, Carine

    2006-01-01

    Topoisomerase I is a ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins (CPTs). These drugs stimulate DNA cleavage by topoisomerase I but exhibit little sequence preference, inducing toxicity and side effects. A convenient strategy to confer sequence specificity consists of the linkage of topoisomerase poisons to DNA sequence recognition elements. In this context, triple-helix-forming oligonucleotides (TFOs) covalently linked to CPTs we...

  11. Priming DNA Replication from Triple Helix Oligonucleotides: Possible Threestranded DNA in DNA Polymerases

    Lestienne, Patrick P.

    2011-01-01

    Triplex associate with a duplex DNA presenting the same polypurine or polypyrimidine-rich sequence in an antiparallel orientation. So far, triplex forming oligonucleotides (TFOs) are known to inhibit transcription, replication, and to induce mutations. A new property of TFO is reviewed here upon analysis of DNA breakpoint yielding DNA rearrangements; the synthesized sequence of the first direct repeat displays a skewed polypurine- rich sequence. This synthesized sequence can bind the second h...

  12. Detection of hepatitis B virus DNA in serum using synthetic non-radioactive oligonucleotides.

    Manzin, A; Pauri, P.; Bagnarelli, P; F. Brecciaroli; Varaldo, P E; Colloca, S; Clementi, M.

    1989-01-01

    A rapid and simplified technique for detecting hepatitis B virus (HBV) DNA by spot hybridisation in the sera of patients with different clinical forms of HBV infection was investigated using enzyme conjugated synthetic oligodeoxyribonucleotides as probes. These are able to hybridize to the S and C regions of the HBV L(-) DNA strand. When compared with a complete 32P-labelled HBV DNA probe, the synthetic oligonucleotides provided a sensitive and quick method for the routine survey of HBV infec...

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

    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.

  14. Refinement of Light-Responsive Transcript Lists Using Rice Oligonucleotide Arrays: Evaluation of Gene-Redundancy

    Jung, Ki-Hong; Dardick, Christopher; Bartley, Laura E.; Cao, Peijian; Phetsom, Jirapa; Canlas, Patrick; Seo, Young-Su; Shultz, Michael; Ouyang, Shu; Yuan, Qiaoping; Frank, Bryan C; Ly, Eugene; Zheng, Li; Jia, Yi; Hsia, An-Ping

    2008-01-01

    Studies of gene function are often hampered by gene-redundancy, especially in organisms with large genomes such as rice (Oryza sativa). We present an approach for using transcriptomics data to focus functional studies and address redundancy. To this end, we have constructed and validated an inexpensive and publicly available rice oligonucleotide near-whole genome array, called the rice NSF45K array. We generated expression profiles for light- vs. dark-grown rice leaf tissue and validated the ...

  15. Use of locked nucleic acid oligonucleotides to add functionality to plasmid DNA

    Hertoghs, Kirsten M. L.; Ellis, Jonathan H.; Catchpole, Ian R.

    2003-01-01

    The available reagents for the attachment of functional moieties to plasmid DNA are limiting. Most reagents bind plasmid DNA in a non-sequence- specific manner, with undefined stoichiometry, and affect DNA charge and delivery properties or involve chemical modifications that abolish gene expression. The design and ability of oligonucleotides (ODNs) containing locked nucleic acids (LNAs) to bind supercoiled, double-stranded plasmid DNA in a sequence-specific manner are described for the first ...

  16. Complete genome sequence of Treponema pallidum ssp. pallidum strain SS14 determined with oligonucleotide arrays

    Sodergren Erica; Petrosino Joseph F; Palzkill Timothy; Norris Steven J; Šmajs David; Strouhal Michal; Matějková Petra; Norton Jason E; Singh Jaz; Richmond Todd A; Molla Michael N; Albert Thomas J; Weinstock George M

    2008-01-01

    Abstract Background Syphilis spirochete Treponema pallidum ssp. pallidum remains the enigmatic pathogen, since no virulence factors have been identified and the pathogenesis of the disease is poorly understood. Increasing rates of new syphilis cases per year have been observed recently. Results The genome of the SS14 strain was sequenced to high accuracy by an oligonucleotide array strategy requiring hybridization to only three arrays (Comparative Genome Sequencing, CGS). Gaps in the resultin...

  17. Kinetics and mechanisms of steps in anti-sense oligonucleotide synthesis

    Russell, Mark A.

    2007-01-01

    Mechanistic studies are reported for the detritylation, coupling and sulphurisation reactions involved in oligonucleotide synthesis by the phosphoramidite method. Detritylation is the acid catalysed removal of a 4,4-dimethoxytrityl protecting group from the 5' protected nucleotide to give the 5' deprotected nucleotide and the 4,4- dimethoxytrityl carbocation. In the absence of water and at high acid concentrations the equilibrium favours carbocation formation. Equilibrium profi...

  18. Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers.

    Jolly, Pawan; Estrela, Pedro; Ladomery, Michael

    2016-06-30

    There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications. PMID:27365033

  19. Efficient inhibition of human telomerase activity by antisense oligonucleotides sensitizes cancer cells to radiotherapy

    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.

  20. Rapid, high-resolution in situ hybridization histochemistry with radioiodinated synthetic oligonucleotides

    In situ hybridization histochemistry is a valuable technique for localizing specific messenger RNA (mRNA) and detecting changes in gene expression. Generally, the mRNA of interest has been detected by probes obtained from cloned DNA and labelled to high specific activity by nick translation. Such probes have a number of disadvantages which can be circumvented by the use of short synthetic oligonucleotides designed to be complementary to a known mRNA sequence. We report here that synthetic oligonucleotides complementary to part of the mRNA coding for rat arginine-vasopressin (AVP) can be labelled to high specific activity with [125I], using either the primer extension method with the Klenow fragment of DNA polymerase I or the 3'-tailing method with terminal deoxynucleotidyl transferase. Both AVP probes hybridized well to the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. A strong autoradiographic signal was present by 2 days, with grains largely confined to the perikaryon. These results compare favorably to those obtained with [32P]- or [3H]-labelled probes. Given the ease of the 3'-tailing method, [125I]-labelled oligonucleotides appear to be especially useful probes for in situ hybridization histochemistry

  1. Evaluation of Therapeutic Oligonucleotides for Familial Amyloid Polyneuropathy in Patient-Derived Hepatocyte-Like Cells.

    Niemietz, Christoph J; Sauer, Vanessa; Stella, Jacqueline; Fleischhauer, Lutz; Chandhok, Gursimran; Guttmann, Sarah; Avsar, Yesim; Guo, Shuling; Ackermann, Elizabeth J; Gollob, Jared; Monia, Brett P; Zibert, Andree; Schmidt, Hartmut H-J

    2016-01-01

    Familial amyloid polyneuropathy (FAP) is caused by mutations of the transthyretin (TTR) gene, predominantly expressed in the liver. Two compounds that knockdown TTR, comprising a small interfering RNA (siRNA; ALN-TTR-02) and an antisense oligonucleotide (ASO; IONIS-TTRRx), are currently being evaluated in clinical trials. Since primary hepatocytes from FAP patients are rarely available for molecular analysis and commercial tissue culture cells or animal models lack the patient-specific genetic background, this study uses primary cells derived from urine of FAP patients. Urine-derived cells were reprogrammed to induced pluripotent stem cells (iPSCs) with high efficiency. Hepatocyte-like cells (HLCs) showing typical hepatic marker expression were obtained from iPSCs of the FAP patients. TTR mRNA expression of FAP HLCs almost reached levels measured in human hepatocytes. To assess TTR knockdown, siTTR1 and TTR-ASO were introduced to HLCs. A significant downregulation (>80%) of TTR mRNA was induced in the HLCs by both oligonucleotides. TTR protein present in the cell culture supernatant of HLCs was similarly downregulated. Gene expression of other hepatic markers was not affected by the therapeutic oligonucleotides. Our data indicate that urine cells (UCs) after reprogramming and hepatic differentiation represent excellent primary human target cells to assess the efficacy and specificity of novel compounds. PMID:27584576

  2. Synthesis of a duplex oligonucleotide containing a nitrogen mustard interstrand DNA-DNA cross-link.

    Ojwang, J O; Grueneberg, D A; Loechler, E L

    1989-12-01

    Many cancer chemotherapeutic agents react with DNA and give adducts that block DNA replication, which is thought to result in cytotoxicity, especially in rapidly proliferating cells such as cancer cells. One class of these agents is bifunctionally reactive (e.g., the nitrogen mustards) and forms DNA-DNA cross-links. It is unknown whether inter- or intrastrand cross-links are more effective at blocking DNA replication. To evaluate this, a DNA shuttle vector is being constructed with an interstrand cross-link at a unique site. In the first step of this project, a duplex oligonucleotide containing an interstrand cross-link is isolated by denaturing polyacrylamide gel electrophoresis from the reaction of nitrogen mustard with two partially complementary oligodeoxynucleotides. The purified oligonucleotide product is characterized and shown to be cross-linked in a 5'-GAC-3' 3'-CTG-5' sequence by a nitrogen mustard moiety that is bound at the N(7)-position of the guanines in the opposing strands; the glycosylic bonds of these guanine adducts are stabilized in their corresponding imidazole ring-opened form. Nitrogen mustard is shown to react with a variety of oligonucleotides and, based upon these results, its preferred targets for interstrand cross-linking are 5'-GXC-3' sequences, where X can be any of the four deoxyribonucleotide bases. PMID:2819709

  3. Targeting DNA with triplex-forming oligonucleotides to modify gene sequence.

    Simon, Philippe; Cannata, Fabio; Concordet, Jean-Paul; Giovannangeli, Carine

    2008-08-01

    Molecules that interact with DNA in a sequence-specific manner are attractive tools for manipulating gene sequence and expression. For example, triplex-forming oligonucleotides (TFOs), which bind to oligopyrimidine.oligopurine sequences via Hoogsteen hydrogen bonds, have been used to inhibit gene expression at the DNA level as well as to induce targeted mutagenesis in model systems. Recent advances in using oligonucleotides and analogs to target DNA in a sequence-specific manner will be discussed. In particular, chemical modification of TFOs has been used to improve binding to chromosomal target sequences in living cells. Various oligonucleotide analogs have also been found to expand the range of sequences amenable to manipulation, including so-called "Zorro" locked nucleic acids (LNAs) and pseudo-complementary peptide nucleic acids (pcPNAs). Finally, we will examine the potential of TFOs for directing targeted gene sequence modification and propose that synthetic nucleases, based on conjugation of sequence-specific DNA ligands to DNA damaging molecules, are a promising alternative to protein-based endonucleases for targeted gene sequence modification. PMID:18460344

  4. Electron stimulated desorption of anions from native and brominated single stranded oligonucleotide trimers

    Polska, Katarzyna; Rak, Janusz [Department of Chemistry, University of Gdansk, Sobieskiego 18, 80-952 Gdansk (Poland); Bass, Andrew D.; Cloutier, Pierre; Sanche, Leon [Research Group in the Radiation Sciences, Faculty of Medicine, Universite de Sherbrooke, Sherbrooke, Quebec J1H 5N4 (Canada)

    2012-02-21

    We measured the low energy electron stimulated desorption (ESD) of anions from thin films of native (TXT) and bromine monosubstituted (TBrXT) oligonucleotide trimers deposited on a gold surface (T = thymidine, X = T, deoxycytidine (C), deoxyadenosine (A) or deoxyguanosine (G), Br = bromine). The desorption of H{sup -}, CH{sub 3}{sup -}/NH{sup -}, O{sup -}/NH{sub 2}{sup -}, OH{sup -}, CN{sup -}, and Br{sup -} was induced by 0 to 20 eV electrons. Dissociative electron attachment, below 12 eV, and dipolar dissociation, above 12 eV, are responsible for the formation of these anions. The comparison of the results obtained for the native and brominated trimers suggests that the main pathways of TBrXT degradation correspond to the release of the hydride and bromide anions. Significantly, the presence of bromine in oligonucleotide trimers blocks the electron-induced degradation of nuclobases as evidenced by a dramatic decrease in CN{sup -} desorption. An increase in the yields of OH{sup -} is also observed. The debromination yield of particular oligonucleotides diminishes in the following order: BrdU > BrdA > BrdG > BrdC. Based on these results, 5-bromo-2{sup '}-deoxyuridine appears to be the best radiosensitizer among the studied bromonucleosides.

  5. Phosphorylation or Mutation of the ERK2 Activation Loop Alters Oligonucleotide Binding.

    McReynolds, Andrea C; Karra, Aroon S; Li, Yan; Lopez, Elias Daniel; Turjanski, Adrian G; Dioum, Elhadji; Lorenz, Kristina; Zaganjor, Elma; Stippec, Steve; McGlynn, Kathleen; Earnest, Svetlana; Cobb, Melanie H

    2016-03-29

    The mitogen-activated protein kinase ERK2 is able to elicit a wide range of context-specific responses to distinct stimuli, but the mechanisms underlying this versatility remain in question. Some cellular functions of ERK2 are mediated through regulation of gene expression. In addition to phosphorylating numerous transcriptional regulators, ERK2 is known to associate with chromatin and has been shown to bind oligonucleotides directly. ERK2 is activated by the upstream kinases MEK1/2, which phosphorylate both tyrosine 185 and threonine 183. ERK2 requires phosphorylation on both sites to be fully active. Some additional ERK2 phosphorylation sites have also been reported, including threonine 188. It has been suggested that this phospho form has distinct properties. We detected some ERK2 phosphorylated on T188 in bacterial preparations of ERK2 by mass spectrometry and further demonstrate that phosphomimetic substitution of this ERK2 residue impairs its kinase activity toward well-defined substrates and also affects its DNA binding. We used electrophoretic mobility shift assays with oligonucleotides derived from the insulin gene promoter and other regions to examine effects of phosphorylation and mutations on the binding of ERK2 to DNA. We show that ERK2 can bind oligonucleotides directly. Phosphorylation and mutations alter DNA binding and support the idea that signaling functions may be influenced through an alternate phosphorylation site. PMID:26950759

  6. Application of Oligonucleotide Microarrays for Bacterial Source Tracking of Environmental Enterococcus sp. Isolates

    John S. Furey

    2005-04-01

    Full Text Available In an effort towards adapting new and defensible methods for assessing and managing the risk posed by microbial pollution, we evaluated the utility of oligonucleotide microarrays for bacterial source tracking (BST of environmental Enterococcus sp. isolates derived from various host sources. Current bacterial source tracking approaches rely on various phenotypic and genotypic methods to identify sources of bacterial contamination resulting from point or non-point pollution. For this study Enterococcus sp. isolates originating from deer, bovine, gull, and human sources were examined using microarrays. Isolates were subjected to Box PCR amplification and the resulting amplification products labeled with Cy5. Fluorescent-labeled templates were hybridized to in-house constructed nonamer oligonucleotide microarrays consisting of 198 probes. Microarray hybridization profiles were obtained using the ArrayPro image analysis software. Principal Components Analysis (PCA and Hierarchical Cluster Analysis (HCA were compared for their ability to visually cluster microarray hybridization profiles based on the environmental source from which the Enterococcus sp. isolates originated. The PCA was visually superior at separating origin-specific clusters, even for as few as 3 factors. A Soft Independent Modeling (SIM classification confirmed the PCA, resulting in zero misclassifications using 5 factors for each class. The implication of these results for the application of random oligonucleotide microarrays for BST is that, given the reproducibility issues, factor-based variable selection such as in PCA and SIM greatly outperforms dendrogram-based similarity measures such as in HCA and K-Nearest Neighbor KNN.

  7. Specific inhibition of hepatitis B virus gene expression by an antisense oligonucleotide in vitro

    It was previously shown that a number of antisense oligonucleotides against hepatitis B virus (HBV) mRNAa were highly effective in inhibition of HBV gene expression. Here, using radioisotope techniques, we report a specific inhibition of HBV surface antigen (HBsAg) production in vitro by 2.2.15 cells (Hep-G2 cells transfected with HBV genome) by the antisense oligonucleotide 15-S-asON, a 15-mer phosphorothioate analogue complementary to the cap site of the SPII promoter of HBV mRNA, ar a concentration of 2 - 5 :m:mol/l. After 24 and 48 hours of incubation of cells with 15-S-asON, the intracellular concentration of the latter rose to 69.4 and 75.8 nmol/l, respectively, and the HBsAg level assayed by ELISA was reduced by 50.0% and 70.6%, respectively. The results were checked by use of the radio-immunoprecipitation method: 2.2.15 cells exposed to 15-S-asON and labelled with [35S]-methionine for 48 hours showed a decrease of the HBsAg level by 81.26% but almost none of the total proteins. No cytotoxicity of the 15-S-asON was observed with regard to the cell morphology and growth. These results indicate that the tested antisense oligonucleotide specifically inhibits the HBV gene expression. (author)

  8. Pressure-Mediated Oligonucleotide Transfection of Rat and Human Cardiovascular Tissues

    Mann, Michael J.; Gibbons, Gary H.; Hutchinson, Howard; Poston, Robert S.; Hoyt, E. Grant; Robbins, Robert C.; Dzau, Victor J.

    1999-05-01

    The application of gene therapy to human disease is currently restricted by the relatively low efficiency and potential hazards of methods of oligonucleotide or gene delivery. Antisense or transcription factor decoy oligonucleotides have been shown to be effective at altering gene expression in cell culture expreriments, but their in vivo application is limited by the efficiency of cellular delivery, the intracellular stability of the compounds, and their duration of activity. We report herein the development of a highly efficient method for naked oligodeoxynucleotide (ODN) transfection into cardiovascular tissues by using controlled, nondistending pressure without the use of viral vectors, lipid formulations, or exposure to other adjunctive, potentially hazardous substances. In this study, we have documented the ability of ex vivo, pressure-mediated transfection to achieve nuclear localization of fluorescent (FITC)-labeled ODN in approximately 90% and 50% of cells in intact human saphenous vein and rat myocardium, respectively. We have further documented that pressure-mediated delivery of antisense ODN can functionally inhibited target gene expression in both of these tissues in a sequence-specific manner at the mRNA and protein levels. This oligonucleotide transfection system may represent a safe means of achieving the intraoperative genetic engineering of failure-resistant human bypass grafts and may provide an avenue for the genetic manipulation of cardiac allograft rejection, allograft vasculopathy, or other transplant diseases.

  9. Methicillin-Resistant Bacteria Inhabiting Surface Waters Monitored by mecA-Targeted Oligonucleotide Probes.

    Seyedmonir, Elnaz; Yilmaz, Fadime; Icgen, Bulent

    2016-08-01

    Part of a 20-60 kb staphylococcal chromosome cassette called mecA encodes low-affinity penicillin-binding protein PBP2a and causes methicillin resistance. Among all methicillin-resistant bacteria, methicillin-resistant Staphylococcus aureus is a major pathogen and main concern worldwide. Although the origin of the mecA is not very well-defined, mecA homologues are also ubiquitous in methicillin-resistant non-staphylococcal bacteria. Due to the dissemination of methicillin resistance through the transmission of mecA gene among staphylococcal and non-staphylococcal bacteria inhabiting surface waters, there is a need to monitor mecA gene in these waters for public health safety. Therefore, this study aimed at monitoring mecA harboring bacteria inhabiting surface waters by using fluorescently labelled mecA-targeted oligonucleotide probes. Under the hybridization conditions of 55 % formamide and 0.020 M NaCl at 46°C, the oligonucleotide probe used in the study showed high hybridization stringency to the mecA gene targeted. The strong linear relationships observed between the signal intensity and the target gene were used to assess the population dynamics of mecA harboring isolates over a 2-year-period. The results indicated that mecA-targeted oligonucleotide probes can be effectively used for in situ monitoring of methicillin resistant isolates inhabiting surface waters. PMID:27156085

  10. Ultrasensitive Detection of Ebola Virus Oligonucleotide Based on Upconversion Nanoprobe/Nanoporous Membrane System.

    Tsang, Ming-Kiu; Ye, WeiWei; Wang, Guojing; Li, Jingming; Yang, Mo; Hao, Jianhua

    2016-01-26

    Ebola outbreaks are currently of great concern, and therefore, development of effective diagnosis methods is urgently needed. The key for lethal virus detection is high sensitivity, since early-stage detection of virus may increase the probability of survival. Here, we propose a luminescence scheme of assay consisting of BaGdF5:Yb/Er upconversion nanoparticles (UCNPs) conjugated with oligonucleotide probe and gold nanoparticles (AuNPs) linked with target Ebola virus oligonucleotide. As a proof of concept, a homogeneous assay was fabricated and tested, yielding a detection limit at picomolar level. The luminescence resonance energy transfer is ascribed to the spectral overlapping of upconversion luminescence and the absorption characteristics of AuNPs. Moreover, we anchored the UCNPs and AuNPs on a nanoporous alumina (NAAO) membrane to form a heterogeneous assay. Importantly, the detection limit was greatly improved, exhibiting a remarkable value at the femtomolar level. The enhancement is attributed to the increased light-matter interaction throughout the nanopore walls of the NAAO membrane. The specificity test suggested that the nanoprobes were specific to Ebola virus oligonucleotides. The strategy combining UCNPs, AuNPs, and NAAO membrane provides new insight into low-cost, rapid, and ultrasensitive detection of different diseases. Furthermore, we explored the feasibility of clinical application by using inactivated Ebola virus samples. The detection results showed great potential of our heterogeneous design for practical application. PMID:26720408

  11. Richness-abundance relationships for zooplankton in ballast water: temperate versus Arctic comparisons

    Chan, F T; Briski, Elizabeta; S. A. Bailey; MacIsaac, H. J.

    2014-01-01

    Species richness and abundance are two commonly measured parameters used to characterize invasion risk associated with transport vectors, especially those capable of transferring large species assemblages. Understanding the relationship between these two variables can further improve our ability to predict future invasions by identifying conditions where high-risk (i.e. species-rich or high abundance or both) and low-risk (i.e. species-poor and low abundance) introduction events are expected....

  12. Designing a shipboard line transect survey to estimate cetacean abundance off the Azores archipelago

    Faustino, Cláudia E. S.; Silva, Mónica A.; Marques, Tiago A.; Thomas, Len

    2010-01-01

    Management schemes dedicated to the conservation of wildlife populations rely on the effective monitoring of population size, and this may require the accurate and precise estimation of this parameter. Line transect distance sampling can be an effective approach for estimating abundance. Little information is available regarding cetacean abundance in the Azores. This paper had two aims: 1) to design a line transect shipboard survey to estimate the absolute abundance of the most common ceta...

  13. Prickly business: abundance of sea urchins on breakwaters and coral reefs in Dubai.

    Bauman, Andrew G; Dunshea, Glenn; Feary, David A; Hoey, Andrew S

    2016-04-30

    Echinometra mathaei is a common echinoid on tropical reefs and where abundant plays an important role in the control of algal communities. Despite high prevalence of E. mathaei on southern Persian/Arabian Gulf reefs, their abundance and distribution is poorly known. Spatial and temporal patterns in population abundance were examined at 12 sites between breakwater and natural reef habitats in Dubai (UAE) every 3months from 2008 to 2010. Within the breakwater habitat, densities were greatest at shallow wave-exposed sites, and reduced with both decreasing wave-exposure and increasing depth. Interestingly, E. mathaei were significantly more abundant on exposed breakwaters than natural reef sites, presumably due to differences in habitat structure and benthic cover. Population abundances differed seasonally, with peak abundances during summer (July-September) and lower abundances in winter (December-February). Seasonal fluctuations are likely the result of peak annual recruitment pulses coupled with increased fish predation from summer to winter. PMID:26563547

  14. Synthesis of Biotin Linkers with the Activated Triple Bond Donor [p-(N-propynoylaminotoluic Acid] (PATA for Efficient Biotinylation of Peptides and Oligonucleotides

    Martina Jezowska

    2012-11-01

    Full Text Available Biotin is an important molecule for modern biological studies including, e.g., cellular transport. Its exclusive affinity to fluorescent streptavidin/avidin proteins allows ready and specific detection. As a consequence methods for the attachment of biotin to various biological targets are of high importance, especially when they are very selective and can also proceed in water. One useful method is Hüisgen dipolar [3+2]-cycloaddition, commonly referred to as “click chemistry”. As we reported recently, the activated triple bond donor p-(N-propynoylaminotoluic acid (PATA gives excellent results when used for conjugations at submicromolar concentrations. Thus, we have designed and synthesized two biotin linkers, with different lengths equipped with this activated triple bond donor and we proceeded with biotinylation of oligonucleotides and C-myc peptide both in solution and on solid support with excellent yields of conversion.

  15. Anti-Urokinase Receptor Antisense Oligonucleotide (uPAR-aODN) to Prevent and Cure Long-Term Space Exploration-Related Retinal Pathological Angiogenesis

    Lazzarano, Stefano; Lulli, Matteo; Fibbi, Gabriella; Margheri, Francesca; Papucci, Laura; Serrati, Simona; Witort, Ewa; Chilla, Anastasia; Lapucci, Andrea; Donnini, Martino; Quaglierini, Paolo; Romiti, Alice; Specogna, Rebecca; Del Rosso, Mario; Capaccioli, Sergio

    2008-06-01

    Angiogenesis underlies a variety of physiological processes and its possible deregulation during long term space exploration needs to be investigated. Angiogenesis is a multistep process of new blood capillary formation, where degradation of the extracellular matrix (ECM) by proteolytic enzymes, including uPA (urokinase plasminogen activator) and opening the way to migration of endothelial cells (EC), is critical. Plasminogen activation system regulates angiogenesis by both uPA-driven ECM degradation and uPA receptor (uPAR). Microgravity and low dose irradiations promote tissue neoangiogeenesis and neovascularization is often common occurence in ophthalmologic pathologies. We have designed and patented the uPAR antisense oligonucleotide (aODN) and evaluated its antiangiogenetic activity by EC cellular migration and capillary morphogenesis assays. The uPAR aODN treatment caused a 75% inhibition of human microvascular EC migration and a complete inhibition of capillary morphogenesis, suggesting its therapeutic application to prevent neoangiogenesis-related ophthalmologic pathologies during space exploration.

  16. Use of Polymerase Chain Reaction Enzyme Linked Oligonucleotide Sorbent Assay (Pcr-Elosa for Detection of Disease Agents

    Simson Tarigan

    2016-03-01

    Full Text Available Diagnostic tool comprises one of the vital components in the control of infectious diseases. One of the most common techniques in the diagnosis of infectious disease currently available is the polymerase chain reaction (PCR because this technique is very sensitive, specific, and rapid. This technique requires an adjunct technique to indicate the formation of the right reaction product. Agarose gel electrophoresis has been the most common technique to visualise the PCR product or amplicon. Enzyme linked oligonucleotide sorbent assay (ELOSA is an alternative technique which is more sensitive and gives more important identity of the amplicon. This technique can be more than 100 times as sensitive as a gel agarose electrophoresis, and very specific since confirmation of the amplicon is carried out by DNA hibridisation. The capacity of the ELOSA can also be extended to the detection of disease-causal agent at subtype level, or detection of mutation at particular location in a gene. Since the equipment used for ELOSA is similar to that for ELISA (enzyme linked immunosorbent assay, a large number of samples can be accomplished rapidly. As in ELISA, a number of variation can be made in ELOSA depend on the requirement. Nucleotide can be immobilised on the microwell plate either by passive adsorbtion, by first conjugation of nucleotide with biotin then immobilisation on streptavidin-coated microwell plate, or immobilisaion by covalent bonding. The PCR and ELOSA can be performed at separate or in a single tube by first immobilising the PCR primers on the surface of microwell plates.

  17. A note on the abundance conjecture

    Dorsch, Tobias; Lazić, Vladimir

    2014-01-01

    We prove that the abundance conjecture for non-uniruled klt pairs in dimension $n$ implies the abundance conjecture for uniruled klt pairs in dimension $n$, assuming the Minimal Model Program in lower dimensions.

  18. I. A comparative study of ribo-, deoxyribo-, and hybrid oligonucleotide helices by nuclear magnetic resonance. II. Optical studies of ethidium binding to oligonucleotides

    Pardi, A.

    1980-11-01

    The conformations and the helix-to-coil transitions in the following oligonucleotides: (I) a DNA duplex dCT/sub 5/G + dCA/sub 5/G; (II) an RNA duplex rCU/sub 5/G + rCA/sub 5/G; (III) a DNA-RNA hybrid duplex dCT/sub 5/G + rCA/sub 5/G; and (IV) a DNA-RNA hybrid triplex rCU/sub 5/G + dCA/sub 5/G were studied. The first three mixtures all form stable double helical structures at 5/sup 0/C, whereas IV forms a triple strand with a ratio of 2:1 rCU/sub 5/G:dCA/sub 5/G. The chemical shifts of the imino protons in the double strands indicate that I, II, and III have different conformations in solution. This implies a significant change in helical parameters. The chemical shift and sugar pucker data are consistent with helix I having B form geometry, whereas II and III have A (or A') geometry. The chemical shifts of the base protons in system I had transition midpoints of 28 to 30/sup 0/C indicating an all-or-none transition. The exchange lifetimes of the imino protons on helix I were a factor of two longer, for the interior A.T base pairs, than those on helix III. This reflects the greater stability of the DNA helix compared to the hybrid helix. The two terminal C.G base pairs in helix I were also found to have much different exchange rates, indicative of a sequence dependence for these exchange rates. The thermodynamic properties of ethidium binding to several oligonucleotides were investigated. The order of the stability for the 2:1 oligonucleotide:ethidium complexes was found to be rCpG > dCpG > rCpUpG approx. = rUpA > rGpUpG + rCpC. These complexes present models for a possible mechanism for frameshift mutagenesis by ethidium. A large positive induced circular dichroism (CD) has been observed for ethidium upon intercalation into nucleic acids. (ERB)

  19. Solar System chemical abundances corrected for systematics

    Gonzalez, Guillermo

    2014-01-01

    The relative chemical abundances between CI meteorites and the solar photosphere exhibit a significant trend with condensation temperature. A trend with condensation temperature is also seen when the solar photospheric abundances are compared to those of nearby solar twins. We use both these trends to determine the alteration of the elemental abundances of the meteorties and the photosphere by fractionation and calculate a new set of primordial Solar System abundances.

  20. Zooplankton of the southwest coast of India: abundance, composition, temporal and spatial variability in 1987

    Madhupratap, M.; Haridas, P.; Ramaiah, Neelam; Achuthankutty, C.T.

    During early southwest and northeast monsoon periods of 1967, zooplankton standing stocks and abundances were high all along the west coast of India. Swarms of zooplankton were common in the shelf areas resulting in a low diversity-high biomass...

  1. Common Reactions After Trauma

    ... here Enter ZIP code here Common Reactions After Trauma Public This section is for Veterans, General Public, Family, & Friends Common Reactions After Trauma Available in Spanish: Reacciones Comunes Después de un ...

  2. Urban warming drives insect pest abundance on street trees.

    Meineke, Emily K; Dunn, Robert R; Sexton, Joseph O; Frank, Steven D

    2013-01-01

    Cities profoundly alter biological communities, favoring some species over others, though the mechanisms that govern these changes are largely unknown. Herbivorous arthropod pests are often more abundant in urban than in rural areas, and urban outbreaks have been attributed to reduced control by predators and parasitoids and to increased susceptibility of stressed urban plants. These hypotheses, however, leave many outbreaks unexplained and fail to predict variation in pest abundance within cities. Here we show that the abundance of a common insect pest is positively related to temperature even when controlling for other habitat characteristics. The scale insect Parthenolecanium quercifex was 13 times more abundant on willow oak trees in the hottest parts of Raleigh, NC, in the southeastern United States, than in cooler areas, though parasitism rates were similar. We further separated the effects of heat from those of natural enemies and plant quality in a greenhouse reciprocal transplant experiment. P. quercifex collected from hot urban trees became more abundant in hot greenhouses than in cool greenhouses, whereas the abundance of P. quercifex collected from cooler urban trees remained low in hot and cool greenhouses. Parthenolecanium quercifex living in urban hot spots succeed with warming, and they do so because some demes have either acclimatized or adapted to high temperatures. Our results provide the first evidence that heat can be a key driver of insect pest outbreaks on urban trees. Since urban warming is similar in magnitude to global warming predicted in the next 50 years, pest abundance on city trees may foreshadow widespread outbreaks as natural forests also grow warmer. PMID:23544087

  3. Urban warming drives insect pest abundance on street trees.

    Emily K Meineke

    Full Text Available Cities profoundly alter biological communities, favoring some species over others, though the mechanisms that govern these changes are largely unknown. Herbivorous arthropod pests are often more abundant in urban than in rural areas, and urban outbreaks have been attributed to reduced control by predators and parasitoids and to increased susceptibility of stressed urban plants. These hypotheses, however, leave many outbreaks unexplained and fail to predict variation in pest abundance within cities. Here we show that the abundance of a common insect pest is positively related to temperature even when controlling for other habitat characteristics. The scale insect Parthenolecanium quercifex was 13 times more abundant on willow oak trees in the hottest parts of Raleigh, NC, in the southeastern United States, than in cooler areas, though parasitism rates were similar. We further separated the effects of heat from those of natural enemies and plant quality in a greenhouse reciprocal transplant experiment. P. quercifex collected from hot urban trees became more abundant in hot greenhouses than in cool greenhouses, whereas the abundance of P. quercifex collected from cooler urban trees remained low in hot and cool greenhouses. Parthenolecanium quercifex living in urban hot spots succeed with warming, and they do so because some demes have either acclimatized or adapted to high temperatures. Our results provide the first evidence that heat can be a key driver of insect pest outbreaks on urban trees. Since urban warming is similar in magnitude to global warming predicted in the next 50 years, pest abundance on city trees may foreshadow widespread outbreaks as natural forests also grow warmer.

  4. Effect of disjunct size distributions on foraminiferal species abundance determinations

    Martin, R.E.; Liddell, W.D.

    1988-02-01

    Studies of foraminiferal distribution and abundance have typically employed a procedure (standard method) that entails counting approximately 300 specimens from a size range greater than some specified minimum (commonly 63 or 125 ..mu..m). This method fails to take into account that foraminifera may be found only within certain size fractions, either because of species specific size ranges or taphonomic processes (sorting, transport, abrasion). Use of a modified counting procedure (sieve method) takes into account foraminiferal size distributions. The sieve method uses counts of up to 300 specimens in each sand-size fraction (0.125-0.25, 0.25-0.5, 0.5-1, 1-2 mm) of each sample. Counts are then totaled for each sample (up to 1200 specimens per site) and used in determination of species abundances for each site. The sieve method has been of considerable utility in recognition of a foraminiferal bathymetric zonation preserved in sediment assemblages from fringing reef environments at Discovery Bay, north Jamaica. Well-documented reef zones (based on corals and physiography) are clearly defined in Q-mode cluster analysis (UPGMA) of species abundances determined using the sieve method. In contrast, individual fore reef zones are not recognized in cluster analysis of foraminiferal species abundances based on the standard method, nor by cluster analysis of species abundances within individual size fractions.

  5. [Commemorative lecture of receiving Imamura Memorial Prize. II. Mode of action of oligonucleotide fraction extracted from Mycobacterium bovis BCG].

    Yamamoto, S

    1994-09-01

    A fraction extracted from Mycobacterium bovis BCG was found to exhibit strong antitumor activity. This fraction, which was designated MY-1, caused some animal tumors to regress and/or prevent metastasis very effectively. MY-1 after digestion with DNase had almost completely reduced activity, while MY-1 digested with RNase did not. MY-1 also augmented natural killer (NK) cell activity of mouse spleen cells in vitro, and produced factors which showed anti-viral activity and rendered macrophages cytotoxic towards tumor cells. The function of the factor to activate macrophages was destroyed by treatment with anti-interferon (IFN)-gamma antibody, while the anti-viral activity was destroyed by treatment with anti-INF alpha/beta antibody. The oligonucleotides contained in MY-1 distributed in a broad range of molecular size, and peaked at 45 nucleotides. We synthesized 13 kinds of 45-mer nucleotides with sequence present in the known cDNA encoding various BCG proteins. Six out of these oligonucleotides, which contained one or more hexameric palindromic structures, showed strong antitumor activity, while the other without palindrome did not. These active oligonucleotides possessed the capability to induce IFN and to augment NK cell activity of mouse spleen cells by coincubation in vitro. When a portion of the sequence of the inactive oligonucleotides was substituted with either palindromic sequence of GACGTC, AGCGCT or AACGTT, the oligonucleotide acquired the ability to augment NK activity. In contrast, the oligonucleotides substituted with another palindromic sequence such as ACCGGT was without effect. Furthermore, exchange of two neighboring mononucleotides within, but not outside, the active palindromic sequence destroyed the ability of the oligonucleotide to augment NK activity.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7526022

  6. A high density COX1 barcode oligonucleotide array for identification and detection of species of Penicillium subgenus Penicillium.

    Chen, W; Seifert, K A; Lévesque, C A

    2009-05-01

    We developed a COX1 barcode oligonucleotide array based on 358 sequences, including 58 known and two new species of Penicillium subgenus Penicillium, and 12 allied species. The array was robotically spotted at near microarray density on membranes. Species and clade-specific oligonucleotides were selected using the computer programs SigOli and Array Designer. Robotic spotting allowed 768 spots with duplicate sets of perfect match and the corresponding mismatch and positive control oligonucleotides, to be printed on 2 × 6 cm(2) nylon membranes. The array was validated with hybridizations between the array and digoxigenin (DIG)-labelled COX1 polymerase chain reaction amplicons from 70 pure DNA samples, and directly from environmental samples (cheese and plants) without culturing. DNA hybridization conditions were optimized, but undesired cross-reactions were detected frequently, reflecting the relatively high sequence similarity of the COX1 gene among Penicillium species. Approximately 60% of the perfect match oligonucleotides were rejected because of low specificity and 76 delivered useful group-specific or species-specific reactions and could be used for detecting certain species of Penicillium in environmental samples. In practice, the presence of weak signals on arrays exposed to amplicons from environmental samples, which could have represented weak detections or weak cross reactions, made interpretation difficult for over half of the oligonucleotides. DNA regions with very few single nucleotide polymorphisms or lacking insertions/deletions among closely related species are not ideal for oligonucleotide-based diagnostics, and supplementing the COX1-based array with oligonucleotides derived from additional genes would result in a more robust hierarchical identification system. PMID:21564971

  7. RNA Interference-Guided Targeting of Hepatitis C Virus Replication with Antisense Locked Nucleic Acid-Based Oligonucleotides Containing 8-oxo-dG Modifications

    Mutso, Margit; Nikonov, Andrei; Pihlak, Arno; Žusinaite, Eva; Viru, Liane; Selyutina, Anastasia; Reintamm, Tõnu; Kelve, Merike; Saarma, Mart; Karelson, Mati; Merits, Andres

    2015-01-01

    The inhibitory potency of an antisense oligonucleotide depends critically on its design and the accessibility of its target site. Here, we used an RNA interference-guided approach to select antisense oligonucleotide target sites in the coding region of the highly structured hepatitis C virus (HCV) RNA genome. We modified the conventional design of an antisense oligonucleotide containing locked nucleic acid (LNA) residues at its termini (LNA/DNA gapmer) by inserting 8-oxo-2’-deoxyguanosine (8-...

  8. 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.

    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

  9. Surface abundances of ON stars

    Martins, F.; Simón-Díaz, S.; Palacios, A.; Howarth, I.; Georgy, C.; Walborn, N. R.; Bouret, J.-C.; Barbá, R.

    2015-06-01

    Context. Massive stars burn hydrogen through the CNO cycle during most of their evolution. When mixing is efficient or when mass transfer in binary systems occurs, chemically processed material is observed at the surface of O and B stars. Aims: ON stars show stronger lines of nitrogen than morphologically normal counterparts. Whether this corresponds to the presence of material processed through the CNO cycle is not known. Our goal is to answer this question. Methods: We performed a spectroscopic analysis of a sample of ON stars with atmosphere models. We determined the fundamental parameters as well as the He, C, N, and O surface abundances. We also measured the projected rotational velocities. We compared the properties of the ON stars to those of normal O stars. Results: We show that ON stars are usually rich in helium. Their CNO surface abundances are fully consistent with predictions of nucleosynthesis. ON stars are more chemically evolved and rotate - on average - faster than normal O stars. Evolutionary models including rotation cannot account for the extreme enrichment observed among ON main sequence stars. Some ON stars are members of binary systems, but others are single stars as indicated by stable radial velocities. Mass transfer is therefore not a simple explanation for the observed chemical properties. Conclusions: We conclude that ON stars show extreme chemical enrichment at their surface, consistent with nucleosynthesis through the CNO cycle. Its origin is not clear at present. Based on observations obtained 1) at the Anglo-Australian Telescope; 2) at the Canada-France-Hawaii Telescope (CFHT), which is operated by the National Research Council (NRC) of Canada, the Institut National des Science de l'Univers of the Centre National de la Recherche Scientifique (CNRS) of France, and the University of Hawaii; 3) at the ESO/La Silla Observatory under programs 081.D-2008, 083.D-0589, 086.D-0997; 4) the Nordic Optical Telescope, operated on the island of La

  10. Detection of hepatitis B virus genotypes using oligonucleotide chip among hepatitis B virus carriers in Eastern China

    Xiang-Rong Tang; Ji-Shen Zhang; Hui Zhao; Yu-Hua Gong; Yong-Zhong Wang; Jian-Long Zhao

    2007-01-01

    AIM: To determine the genotype distribution of hepatitis B virus (HBV) with a newly oligonucleotide chip assay among the HBV carriers in Eastern China.METHODS: An assay using oligonucleotide chip was developed for detection of HBV genotypes in serum samples from HBV DNA-positive patients in Eastern China. This method is based on the principle of reverse hybridization with Cy5-labeled amplicons hybridizing to type-specific oligonucleotide probes that are immobilized on slides. The results of 80 randomly chosen sera were confirmed by direct sequencing.RESULTS: HBV genotype B, C and mixed genotype were detected in 400 serum samples, accounting for8.3% (n = 33), 83.2% (n = 333), and 8.5% (n = 34),respectively. The evaluation of the oligonucleotide assay showed 100% concordance with the amplicon phylogenetic analysis except 9 mixed genotype infections undetected by sequencing.CONCLUSION: The study indicates that HBV genotype C and B prevail in the Eastern China. It is suggested that the oligonucleotide chip is a reliable and convenient tool for the detection of HBV genotyping.

  11. Characterization of the overall and internal dynamics of short oligonucleotides by depolarized dynamic light scattering and NMR relaxation measurements

    The dynamics of three synthetic oligonucleotides d(CG)4, d(CG)6, and d(CGCGTTGTTCGCG) of different length and shape were studied in solution by depolarized dynamic light scattering (DDLS) and time-resolved nuclear Overhauser effect cross-relaxation measurements. For cylindrically symmetric molecules the DDLS spectrum is dominated by the rotation of the main symmetry axis of the cylinder. The experimental correlation times describe the rotation of the oligonucleotides under hydrodynamic stick boundary conditions. It is shown that the hydrodynamic theory of Tirado and Garcia de la Torre gives good predictions of the rotational diffusion coefficients of cylindrically symmetric molecules of the small axial ratios studied here. These relations are used to calculate the solution dimensions of the DNA fragments from measured correlation times. The DDLS relaxations measurements provide a power method for distinguishing between different conformations of the oligonucleotides. Furthermore, the rotational correlation times are a very sensitive probe of the length of different fragments. The NMR results reflect the anisotropic motion of the molecules as well as the amount of local internal motion present. The experimental correlation time from NMR is determined by the rotation of both the short and long axes of the oligonucleotide. The authors results are compared with those from NMR relaxation measurements on other short oligonucleotides with lengths of up to 20 base pairs. Various dynamic models for the reorientation of the internuclear vector are applied to their interpretation

  12. NAA-modified DNA oligonucleotides with zwitterionic backbones: stereoselective synthesis of A-T phosphoramidite building blocks.

    Schmidtgall, Boris; Höbartner, Claudia; Ducho, Christian

    2015-01-01

    Modifications of the nucleic acid backbone are essential for the development of oligonucleotide-derived bioactive agents. The NAA-modification represents a novel artificial internucleotide linkage which enables the site-specific introduction of positive charges into the otherwise polyanionic backbone of DNA oligonucleotides. Following initial studies with the introduction of the NAA-linkage at T-T sites, it is now envisioned to prepare NAA-modified oligonucleotides bearing the modification at X-T motifs (X = A, C, G). We have therefore developed the efficient and stereoselective synthesis of NAA-linked 'dimeric' A-T phosphoramidite building blocks for automated DNA synthesis. Both the (S)- and the (R)-configured NAA-motifs were constructed with high diastereoselectivities to furnish two different phosphoramidite reagents, which were employed for the solid phase-supported automated synthesis of two NAA-modified DNA oligonucleotides. This represents a significant step to further establish the NAA-linkage as a useful addition to the existing 'toolbox' of backbone modifications for the design of bioactive oligonucleotide analogues. PMID:25670992

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

    Jee Justin

    2008-12-01

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

  14. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D.; Otero, Carolina

    2016-02-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.

  15. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake.

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D; Otero, Carolina

    2016-12-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes. PMID:26847692

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

    Lanfranchi Gerolamo

    2002-10-01

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

  17. Origin of Cosmic Chemical Abundances

    Maio, Umberto

    2015-01-01

    Cosmological N-body hydrodynamic computations following atomic and molecular chemistry (e$^-$, H, H$^+$, H$^-$, He, He$^+$, He$^{++}$, D, D$^+$, H$_2$, H$_2^+$, HD, HeH$^+$), gas cooling, star formation and production of heavy elements (C, N, O, Ne, Mg, Si, S, Ca, Fe, etc.) from stars covering a range of mass and metallicity are used to explore the origin of several chemical abundance patterns and to study both the metal and molecular content during simulated galaxy assembly. The resulting trends show a remarkable similarity to up-to-date observations of the most metal-poor damped Lyman-$\\alpha$ absorbers at redshift $z\\gtrsim 2$. These exhibit a transient nature and represent collapsing gaseous structures captured while cooling is becoming effective in lowering the temperature below $\\sim 10^4\\,\\rm K$, before they are disrupted by episodes of star formation or tidal effects. Our theoretical results agree with the available data for typical elemental ratios, such as [C/O], [Si/Fe], [O/Fe], [Si/O], [Fe/H], [O/...

  18. Significant biases affecting abundance determinations

    Wesson, Roger

    2015-08-01

    I have developed two highly efficient codes to automate analyses of emission line nebulae. The tools place particular emphasis on the propagation of uncertainties. The first tool, ALFA, uses a genetic algorithm to rapidly optimise the parameters of gaussian fits to line profiles. It can fit emission line spectra of arbitrary resolution, wavelength range and depth, with no user input at all. It is well suited to highly multiplexed spectroscopy such as that now being carried out with instruments such as MUSE at the VLT. The second tool, NEAT, carries out a full analysis of emission line fluxes, robustly propagating uncertainties using a Monte Carlo technique.Using these tools, I have found that considerable biases can be introduced into abundance determinations if the uncertainty distribution of emission lines is not well characterised. For weak lines, normally distributed uncertainties are generally assumed, though it is incorrect to do so, and significant biases can result. I discuss observational evidence of these biases. The two new codes contain routines to correctly characterise the probability distributions, giving more reliable results in analyses of emission line nebulae.

  19. Simulated population responses of common carp to commercial exploitation

    Weber, Michael J.; Hennen, Matthew J.; Brown, Michael L.

    2011-12-01

    Common carp Cyprinus carpio is a widespread invasive species that can become highly abundant and impose deleterious ecosystem effects. Thus, aquatic resource managers are interested in controlling common carp populations. Control of invasive common carp populations is difficult, due in part to the inherent uncertainty of how populations respond to exploitation. To understand how common carp populations respond to exploitation, we evaluated common carp population dynamics (recruitment, growth, and mortality) in three natural lakes in eastern South Dakota. Common carp exhibited similar population dynamics across these three systems that were characterized by consistent recruitment (ages 3 to 15 years present), fast growth (K = 0.37 to 0.59), and low mortality (A = 1 to 7%). We then modeled the effects of commercial exploitation on size structure, abundance, and egg production to determine its utility as a management tool to control populations. All three populations responded similarly to exploitation simulations with a 575-mm length restriction, representing commercial gear selectivity. Simulated common carp size structure modestly declined (9 to 37%) in all simulations. Abundance of common carp declined dramatically (28 to 56%) at low levels of exploitation (0 to 20%) but exploitation >40% had little additive effect and populations were only reduced by 49 to 79% despite high exploitation (>90%). Maximum lifetime egg production was reduced from 77 to 89% at a moderate level of exploitation (40%), indicating the potential for recruitment overfishing. Exploitation further reduced common carp size structure, abundance, and egg production when simulations were not size selective. Our results provide insights to how common carp populations may respond to exploitation. Although commercial exploitation may be able to partially control populations, an integrated removal approach that removes all sizes of common carp has a greater chance of controlling population abundance

  20. Charge Transport in DNA Oligonucleotides with Various Base-Pairing Patterns

    Kratochvílová, Irena; Todorciuc, Tatiana; Král, Karel; Němec, Hynek; Bunček, M.; Šebera, Jakub; Záliš, Stanislav; Vokáčová, Zuzana; Sychrovský, Vladimír; Bednárová, Lucie; Mojzeš, P.; Schneider, Bohdan

    2010-01-01

    Roč. 17, 1a (2010), L7-L7. ISSN 1211-5894. [Discussions in Structural Molecular Biology /8./. 18.03.2010-20.03.2010, Nové Hrady] R&D Projects: GA AV ČR KAN401770651; GA ČR GA203/08/1594 Institutional research plan: CEZ:AV0Z40500505; CEZ:AV0Z40550506; CEZ:AV0Z10100520; CEZ:AV0Z40400503; CEZ:AV0Z50520701 Keywords : base-pairing patterns * oligonucleotides * DNA Subject RIV: CF - Physical ; Theoretical Chemistry

  1. Efficient activation of nucleoside phosphoramidites with 4,5-dicyanoimidazole during oligonucleotide synthesis.

    Vargeese, C; Carter, J; Yegge, J; Krivjansky, S; Settle, A.; Kropp, E; Peterson, K.; Pieken, W

    1998-01-01

    A new activator for the coupling of phosphoramidites to the 5'-hydroxyl group during oligonucleotide synthesis is introduced. The observed time to complete coupling is twice as fast with 4, 5-dicyanoimidazole (DCI) as the activator, compared with 1 H -tetrazole. The effectiveness of DCI is thought to be based on its nucleophilicity. DCI is soluble in acetonitrile up to 1.1 M at room temperature and can be used as the sole coupling activator during routine automated solid phase synthesis of ol...

  2. Information Limited Oligonucleotide Amplification Assay for Affinity-Based, Parallel Detection Studies.

    Harish Bokkasam

    Full Text Available Molecular communication systems encounter similar constraints as telecommunications. In either case, channel crosstalk at the receiver end will result in information loss that statistical analysis cannot compensate. This is because in any communication channel there is a physical limit to the amount of information that can be transmitted. We present a novel and simple modified end amplification (MEA technique to generate reduced and defined amounts of specific information in form of short fragments from an oligonucleotide source that also contains unrelated and redundant information. Our method can be a valuable tool to investigate information overflow and channel capacity in biomolecular recognition systems.

  3. MODEST: a web-based design tool for oligonucleotide-mediated genome engineering and recombineering

    Bonde, Mads; Klausen, Michael S.; Anderson, Mads Valdemar;

    2014-01-01

    combinatorial cell libraries. Manual design of oligonucleotides for these approaches can be tedious, time-consuming, and may not be practical for larger projects targeting many genomic sites. At present, the change from a desired phenotype (e.g. altered expression of a specific protein) to a designed MAGE oligo......Recombineering and multiplex automated genome engineering (MAGE) offer the possibility to rapidly modify multiple genomic or plasmid sites at high efficiencies. This enables efficient creation of genetic variants including both single mutants with specifically targeted modifications as well as...

  4. Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer.

    Scheiermann, Julia; Klinman, Dennis M

    2014-11-12

    Synthetic oligonucleotides (ODN) that express unmethylated "CpG motifs" trigger cells that express Toll-like receptor 9. In humans this includes plasmacytoid dendritic cells and B cells. CpG ODN induce an innate immune response characterized by the production of Th1 and pro-inflammatory cytokines. Their utility as vaccine adjuvants was evaluated in a number of clinical trials. Results indicate that CpG ODN improve antigen presentation and the generation of vaccine-specific cellular and humoral responses. This work provides an up-to-date overview of the utility of CpG ODN as adjuvants for vaccines targeting infectious agents and cancer. PMID:24975812

  5. Rescue of dystrophin expression in mdx mouse muscle by RNA/DNA oligonucleotides

    Rando, Thomas A.; Disatnik, Marie-Helene; Zhou, Lucy Z.-H.

    2000-01-01

    Chimeric RNA/DNA oligonucleotides (“chimeraplasts”) have been shown to induce single base alterations in genomic DNA both in vitro and in vivo. The mdx mouse strain has a point mutation in the dystrophin gene, the consequence of which is a muscular dystrophy resulting from deficiency of the dystrophin protein in skeletal muscle. To test the feasibility of chimeraplast-mediated gene therapy for muscular dystrophies, we used a chimeraplast (designated “MDX1”) designed to correct the point mutat...

  6. Optical Properties and In Vitro Biological Studies of Oligonucleotide-Modified Quantum Dots

    Valérie A. Gérard

    2013-01-01

    Full Text Available Water-soluble semiconducting nanocrystals or quantum dots (QDs have attracted much interest in recent years due to their tuneable emission and potential applications in photonics and biological imaging. Fluorescence resonance energy transfer (FRET processes are very important for elucidating biochemical mechanisms in vitro, and QDs constitute an excellent substrate for this purpose. In this work, new oligonucleotide-functionalised CdTe-based QDs were prepared, characterised and biologically tested. These QDs demonstrated interesting optical properties as well as remarkable in vitro behaviour and potential for a range of biological applications.

  7. Chromosomal mutations induced by triplex-forming oligonucleotides in mammalian cells.

    Vasquez, K M; Wang, G; Havre, P A; Glazer, P M

    1999-01-01

    Specific recognition of a region of duplex DNA by triplex-forming oligonucleotides (TFOs) provides an attractive strategy for genetic manipulation. Based on this, we have investigated the ability of the triplex-directed approach to induce mutations at a chromosomal locus in living cells. A mouse fibroblast cell line was constructed containing multiple chromosomal copies of the lambdasupFG1 vector carrying the supFG1 mutation-reporter gene. Cells were treated with specific (psoAG30) or control...

  8. Optimal design of parallel triplex forming oligonucleotides containing Twisted Intercalating Nucleic Acids--TINA

    Schneider, Uffe V; Mikkelsen, Nikolaj D; Jøhnk, Nina;

    2010-01-01

    Twisted intercalating nucleic acid (TINA) is a novel intercalator and stabilizer of Hoogsteen type parallel triplex formations (PT). Specific design rules for position of TINA in triplex forming oligonucleotides (TFOs) have not previously been presented. We describe a complete collection of easy...... on PT is remarkably high (between 7.4 and 15.2 degrees C) compared to antiparallel duplexes (between 3.8 and 9.4 degrees C). The specificity of PT by Delta T(m) increases when shorter TFOs and higher pH are chosen. To increase Delta Tms, base mismatches should be placed in the center of the TFO and...

  9. TRANSPLATIN-CONJUGATED TRIPLEX-FORMING OLIGONUCLEOTIDES FORM ADDUCTS WITH BOTH STRANDS OF DNA

    Campbell, Meghan A.; Miller, Paul S.

    2009-01-01

    Triplex-forming oligonucleotides (TFOs) can bind to polypurine•polypyrimidine tracts in DNA and as a consequence, perturb normal functioning of a targeted gene. The effectiveness of such anti-gene TFOs can potentially be enhanced by covalent attachment of the TFO to its DNA target. Here we report that attachment of N-7-platinated guanine nucleosides to the 3′- and/or 5′-ends of oligopyrimidine TFOs enables these TFOs to form highly stable adducts with target DNA deoxyguanosines or deoxyadenos...

  10. Synthesis and triplex formation of oligonucleotides containing 8-thioxodeoxyadenosine and 5-methyl-2-thiodeoxycytosine.

    Ohkubo, Akihiro; Miyata, Kenichi; Tsunoda, Hirosuke; Seio, Kohji; Sekine, Mitsuo

    2009-01-01

    For more effective DNA triplex formation under neutral conditions, we synthesized triplex-forming oligonucleotides (TFO) containing 8-thioxodeoxyadenine (s(8)A) residues in place of the protonated cytosines (Cs) required for the third base pairing with DNA duplexes. Consequently, it was found that s(8)A exhibited much stronger hybridization ability than C under neutral conditions when four s(8)A bases were arranged in a consecutive sequence. Moreover, we also synthesized TFOs containing 5-methyl-2-thiocytosines and examined their ability of triplex formation. PMID:19749240

  11. Sequence-specific DNA double-strand breaks induced by triplex forming 125I labeled oligonucleotides.

    Panyutin, I G; Neumann, R D

    1994-01-01

    A triplex-forming oligonucleotide (TFO) complementary to the polypurine-polypyrimidine region of the nef gene of the Human Immunodeficiency Virus (HIV) was labeled with 125I at the C5 position of a single deoxycytosine residue. Labeled TFO was incubated with a plasmid containing a fragment of the nef gene. Decay of 125I was found to cause double-strand breaks (DSB) within the nef gene upon triplex formation in a sequence specific manner. No DSB were detected after incubation at ionic conditio...

  12. Cytotoxic effects and specific gene expression alterations induced by I-125-labeled triplex-forming oligonucleotides

    Dahmen, Volker; Kriehuber, Ralf

    2012-01-01

    Purpose: Triplex-forming oligonucleotides (TFO) bind to the DNA double helix in a sequence-specific manner. Therefore, TFO seem to be a suitable carrier for Auger electron emitters to damage exclusively targeted DNA sequences, e.g., in tumor cells. We studied the influence of I-125 labeled TFO with regard to cell survival and induction of DNA double-strand breaks (DSB) using TFO with different genomic targets and target numbers. Furthermore, the ability of TFO to alter the gene expression of ...

  13. Biophysical and RNA Interference Inhibitory Properties of Oligonucleotides Carrying Tetrathiafulvalene Groups at Terminal Positions

    Pérez-Rentero, S.; Somoza, A.; Grijalvo, S.; Janoušek, Jiří; Bělohradský, Martin; Stará, Irena G.; Starý, Ivo; Eritja, R.

    2013-01-01

    Roč. 2013, June (2013), 650610/1-650610/11. ISSN 2090-9063 R&D Projects: GA ČR GAP207/10/2214; GA MŠk 7E09054 EU Projects: European Commission(XE) 213382 - FUNMOL Grant ostatní: European Commission(XE) NMP4-LA-2011-262943 MULTIFUN Institutional support: RVO:61388963 Keywords : DNA * derivates * tetrathiafulvalene * TTF-oligonucleotide conjugates Subject RIV: CC - Organic Chemistry http://dx.doi.org/10.1155/2013/650610

  14. Study of DNA oligonucleotides interactions with ethidium bromide by partial-filling affinity capillary electrophoresis

    Růžička, Martin; Koval, Dušan; Kašička, Václav

    Olomouc: Palacký University, 2014 - (Maier, V.; Ševčík, J.), s. 194-195 ISBN 978-80-244-3950-1. ISSN 0232-0061. [Advances in Chromatography and Electrophoresis & Chiranal 2014. Olomouc (CZ), 10.02.2014-14.02.2014] R&D Projects: GA ČR(CZ) GAP206/12/0453; GA ČR(CZ) GA13-17224S; GA ČR GA13-32974S Institutional support: RVO:61388963 Keywords : affinity capillary electrophoresis * binding constant * oligonucleotide Subject RIV: CB - Analytical Chemistry, Separation

  15. Development and validation of an oligonucleotide ligation assay to detect lamivudine resistance in hepatitis B virus.

    Beck, Ingrid A; Payant, Rachel; Ngo-Giang-Huong, Nicole; Khamduang, Woottichai; Laomanit, Laddawan; Jourdain, Gonzague; Frenkel, Lisa M

    2016-07-01

    Treatment of chronic hepatitis B virus (HBV) infection with lamivudine-monotherapy rapidly selects mutant variants in a high proportion of individuals. Monitoring lamivudine resistance by consensus sequencing is costly and insensitive for detection of minority variants. An oligonucleotide ligation assay (OLA) for HBV lamivudine-resistance was developed and compared to consensus sequencing. Both assays detected drug resistance mutations in 35/64 (54.7%) specimens evaluated, and OLA detected minority mutants in an additional six (9.4%). OLA may offer a sensitive and inexpensive alternative to consensus sequencing for detection of HBV drug resistance in resource-limited settings. PMID:27025356

  16. Identification of Cystic Fibrosis Variants by Polymerase Chain Reaction/Oligonucleotide Ligation Assay

    Schwartz, Karen M.; Pike-Buchanan, Lisa L.; Muralidharan, Kasinathan; Redman, Joy B.; Wilson, Jean Amos; Jarvis, Michael; Cura, M. Grace; Pratt, Victoria M.

    2009-01-01

    The purpose of this work is to define rare variants of cystic fibrosis (CF) that are potential sources of error and can confound molecular genetic testing methods. We performed routine, clinical CF mutation screening using a laboratory-developed test and the oligonucleotide ligation assay reagents from Abbott/Celera. In this report, we describe 11 unique allele drop outs [3849 + 10kb C>T (NM_000492.2:c.3718-2477C>T), V520F (c.1558G>T), 1078delT (c.948delT), A455E (c.1364C>A), R347P (c.1040G>C...

  17. In situ Synthesis of Oligonucleotides on Plasma Treated Polypropylene Microporous Membrane

    Nong Yue HE; Jian Xin TANG; Song LI; Hong CHEN; An Cun ZHOU

    2005-01-01

    Polypropylene microporous membranes were treated with plasma in a mixture of N2and H2 (1:2 in volume). Attenuated total reflectance Fourier transform infrared spectroscopy(ATR-FTIR), X-ray photoelectron spectroscopy (XPS) and ultra-violet (UV) spectra demonstratedthe success of grafting amino groups. The density of the polar amino groups on the membrane surface is about 0.59 μmol/cm2. The as-treated membranes were successively applied to the in situ synthesis of oligonucleotides and an average coupling yield was more than 98%. The surface feature of the treated membrane is suggested to be responsible for its advantage over a glass slide.

  18. Large-scale mRNA expression profiling in the common ice plant, Mesembryanthemum crystallinum, performing C3 photosynthesis and Crassulacean acid metabolism (CAM).

    Cushman, John C; Tillett, Richard L; Wood, Joshua A; Branco, Joshua M; Schlauch, Karen A

    2008-01-01

    The common ice plant (Mesembryanthemum crystallinum L.) has emerged as a useful model for molecular genetic studies of Crassulacean acid metabolism (CAM) because CAM can be induced in this species by water deficit or salinity stress. Non-redundant sequence information from expressed sequence tag data was used to fabricate a custom oligonucleotide microarray to compare large-scale mRNA expression patterns in M. crystallinum plants conducting C(3) photosynthesis versus CAM. Samples were collected every 4 h over a 24 h time period at the start of the subjective second day from plants grown under constant light and temperature conditions in order to capture variation in mRNA expression due to salinity stress and circadian clock control. Of 8455 genes, a total of 2343 genes (approximately 28%) showed a significant change as judged by analysis of variance (ANOVA) in steady-state mRNA abundance at one or more time points over the 24 h period. Of these, 858 (10%) and 599 (7%) exhibited a greater than two-fold ratio (TFR) increase or decrease in mRNA abundance, respectively. Functional categorization of these TFR genes revealed that many genes encoding products that function in CAM-related C(4) acid carboxylation/decarboxylation, glycolysis/gluconeogenesis, polysaccharide, polyol, and starch biosynthesis/degradation, protein degradation, transcriptional activation, signalling, stress response, and transport facilitation, and novel, unclassified proteins exhibited stress-induced increases in mRNA abundance. In contrast, salt stress resulted in a significant decrease in transcript abundance for genes encoding photosynthetic functions, protein synthesis, and cellular biogenesis functions. Many genes with CAM-related functions exhibited phase shifts in their putative circadian expression patterns following CAM induction. This report establishes an extensive catalogue of gene expression patterns for future investigations aimed at understanding the complex, transcriptional

  19. The Science Commons Project

    Martin, Juan Carlos

    2007-01-01

    Science Commons serves the advancement of science by contributing to the removal of unnecessary legal and technical barriers to scientific collaboration and innovation. Built on the promise of Open Access to scholarly literature and data, Science Commons identifies and eases key barriers to the movement of information, tools and data through the scientific research cycle. The long term vision of Science Commons is to provide more than just useful contracts. We will combine our publishin...

  20. Creative Commons International

    Catharina Maracke

    2010-01-01

    When Creative Commons (CC) was founded in 2001, the core Creative Commons licenses were drafted according to United States Copyright Law. Since their first introduction in December 2002, Creative Commons licenses have been enthusiastically adopted by many creators, authors, and other content producers – not only in the United States, but in many other jurisdictions as well. Global interest in the CC licenses prompted a discussion about the need for national versions of the CC licenses. To bes...

  1. Phosphorothioate oligonucleotide inhibits tissue factor expression in endothelial cells induced by blood flow shear stress in rats

    Li Qianning; Yang Yimin; Ying Dajun; Cheng Rongchuan; Gong Zili; Liu Yong; Zhou Zhujuan; Zheng Jian

    2008-01-01

    Objective: To determine the effect of antiparallel phosphorothioate triplex-forming oligonucleotide (apsTFO),which was designed according to shear stress response element (SSRE) in tissue factor (TF) gene promoter region, on the expression of endothelial TF in carotid artery stenosis rats. Methods: Rat model of severe carotid artery stenosis were inflicted by silica gel tube ligation. Half an hour before the model infliction, GT20-apsTFO, GT20-psTFO and GT21-apsTFO labeled with green fluorescence (FITC) were injected into the vena caudalis of rat at a dose of 0.5 mg/kg.Half an hour, 4 or 9 h after the ligation, the distribution of TFO in the common carotid artery, the liver and the kidney was detected with aid of fluorescence microscopy. And the mRNA and protein expressions of TF, Egr-1 and Spl in the above-mentioned organs were determined with in situ hybridization and immunohistoehemical assay respectively in 6 h after the model establishment, and the results were analyzed with an image analysis system. Results: Only in 1 h after TFO injection, fluorescent granules appeared in the liver, the kidney and the vascular wall and lumen of carotid artery,and then in 4.5 h, they still deposited in above sites except the vascular lumen. GT20-apsTFO and GT21-apsTFO significant down-regulated the mRNA and protein expressions of TF compared to the rats without treatment (P0.05).The 3 TFOs had no inhibition on the mRNA and protein expressions of Egr-I and Spl. Conclusion: Pretreated apsTFO can partly come into the vascular endothelial cells, and inhibit TF expression induced by shear stress, but had no effect on Egr-1 and Spl gene expressions.

  2. The oligonucleotide binding (OB-fold domain of KREPA4 is essential for stable incorporation into editosomes.

    Smriti Kala

    Full Text Available Most mitochondrial mRNAs in trypanosomatid parasites require uridine insertion/deletion RNA editing, a process mediated by guide RNA (gRNA and catalyzed by multi-protein complexes called editosomes. The six oligonucleotide/oligosaccharide binding (OB-fold proteins (KREPA1-A6, are a part of the common core of editosomes. They form a network of interactions among themselves as well as with the insertion and deletion sub-complexes and are essential for the stability of the editosomes. KREPA4 and KREPA6 proteins bind gRNA in vitro and are known to interact directly in yeast two-hybrid analysis. In this study, using several approaches we show a minimal interaction surface of the KREPA4 protein that is required for this interaction. By screening a series of N- and C-terminally truncated KREPA4 fragments, we show that a predicted α-helix of KREPA4 OB-fold is required for its interaction with KREPA6. An antibody against the KREPA4 α-helix or mutations of this region can eliminate association with KREPA6; while a peptide fragment corresponding to the α-helix can independently interact with KREPA6, thereby supporting the identification of KREPA4-KREPA6 interface. We also show that the predicted OB-fold of KREPA4; independent of its interaction with gRNA, is responsible for the stable integration of KREPA4 in the editosomes, and editing complexes co-purified with the tagged OB-fold can catalyze RNA editing. Therefore, we conclude that while KREPA4 interacts with KREPA6 through the α-helix region of its OB-fold, the entire OB-fold is required for its integration in the functional editosome, through additional protein-protein interactions.

  3. Classifying degrees of species commonness: North Sea fish as a case study

    Coro, Gianpaolo; Webb, Thomas J.; Appeltans, Ward; Bailly, Nicolas; Cattrijsse, Andr?; Pagano, Pasquale (ISTI-CNR)

    2015-01-01

    Species commonness is often related to abundance and species conservation status. Intuitively, a “common species” is a species that is abundant in a certain area, widespread and at low risk of extinction. Analysing and classifying species commonness can help discovering indicators of ecosystem status and can prevent sudden changes in biodiversity. However, it is challenging to quantitatively define this concept. This paper presents a procedure to automatically characterize species commonness ...

  4. Hydrocarbon Reserves: Abundance or Scarcity

    IFP and the OAPEC jointly organize a regular international seminar dealing with world oil-related problems appearing in the news. For the first time, this seminar has been opened to oil and gas company specialists, service companies, research centers and independents. This year's theme concerns oil and gas reserves: are they abundant or are we headed towards the shortages announced by some experts? This theme is especially topical in that: oil and gas currently meet two thirds of world energy needs and almost completely dominate the transport sector; the reserves declared by the OAPEC countries account for nearly half of world reserves; the price of a barrel of oil went through the roof in 2004; world energy demand is growing fast and alternative sources of energy are far from ready to take over from oil and gas in the next few decades. Since the reserves correspond to the volume it is technically and economically viable to produce, the seminar has, of course, dealt with the technical and economic questions that arise in connection with exploration and production, but it has also considered changes in the geopolitical context. Presentations by the leading companies of the OAPEC countries and by the IFP group were completed by presentation from the International Energy Agency (IEA), the United States Geological Survey (USGS), the IHS Energy Group, Total and Gaz de France. This document gathers the transparencies of the following presentations: Hydrocarbon reserves in OAPEC members countries: current and future (M. Al-Lababidi); Non OAPEC liquid reserves and production forecasts (Y. Mathieu); World oil and gas resources and production outlook (K. Chew); Global investments in the upstream (F. Birol); Total's policy in the oil and gas sector (C. de Margerie); Gaz de France's policy in the oil and gas sector (J. Abiteboul); NOC/IOC's opportunities in OPEC countries (I. Sandrea); Relationships between companies, countries and investors: How they may impact on the growth

  5. Hydrocarbon Reserves: Abundance or Scarcity

    NONE

    2005-07-01

    IFP and the OAPEC jointly organize a regular international seminar dealing with world oil-related problems appearing in the news. For the first time, this seminar has been opened to oil and gas company specialists, service companies, research centers and independents. This year's theme concerns oil and gas reserves: are they abundant or are we headed towards the shortages announced by some experts? This theme is especially topical in that: oil and gas currently meet two thirds of world energy needs and almost completely dominate the transport sector; the reserves declared by the OAPEC countries account for nearly half of world reserves; the price of a barrel of oil went through the roof in 2004; world energy demand is growing fast and alternative sources of energy are far from ready to take over from oil and gas in the next few decades. Since the reserves correspond to the volume it is technically and economically viable to produce, the seminar has, of course, dealt with the technical and economic questions that arise in connection with exploration and production, but it has also considered changes in the geopolitical context. Presentations by the leading companies of the OAPEC countries and by the IFP group were completed by presentation from the International Energy Agency (IEA), the United States Geological Survey (USGS), the IHS Energy Group, Total and Gaz de France. This document gathers the transparencies of the following presentations: Hydrocarbon reserves in OAPEC members countries: current and future (M. Al-Lababidi); Non OAPEC liquid reserves and production forecasts (Y. Mathieu); World oil and gas resources and production outlook (K. Chew); Global investments in the upstream (F. Birol); Total's policy in the oil and gas sector (C. de Margerie); Gaz de France's policy in the oil and gas sector (J. Abiteboul); NOC/IOC's opportunities in OPEC countries (I. Sandrea); Relationships between companies, countries and investors: How they may

  6. The Sulfur Abundance Anomaly in Planetary Nebulae

    Henry, R B C; Kwitter, K B; Milingo, M B

    2006-01-01

    The failure of S and O abundances in most planetary nebulae to display the same strong direct correlation that is observed in extragalactic H II regions represents one of the most perplexing problems in the area of PN abundances today. Galactic chemical evolution models as well as large amounts of observational evidence from H II region studies support the contention that cosmic abundances of alpha elements such as O, Ne, S, Cl, and Ar increase together in lockstep. Yet abundance results from the Henry, Kwitter, & Balick (2004) database show a strong tendency for most PNe to have S abundances that are significantly less than expected from the observed level of O. One reasonable hypothesis for the sulfur anomaly is the past failure to properly measure the abundances of unseen ionization stages above S^+2. Future observations with Spitzer will allow us to test this hypothesis.

  7. Oxygen abundances in nearby dwarf irregular galaxies

    Oxygen abundances are obtained by optical spectrophotometry of H II regions in seven nearby dwarf irregular galaxies. All of these yield oxygen abundances of less than 1/10 of the solar value, and most are in the range of 3-5 percent of the solar value. This suggests that observations of nearby dwarf galaxies may provide an effective means for studying the chemical evolution of low-mass galaxies and, possibly, the primordial helium abundance. A strong correlation is found between the oxygen abundances and absolute magnitudes for nearby irregular galaxies. This correlation will be useful for estimating abundances of irregular galaxies without observable H II regions, and possibly as a distance indicator for irregular galaxies with known abundances. It is inferred from this relationship that infall is no more important in irregular galaxies with extremely large H I halos than in typical irregular galaxies. 72 refs

  8. Stellar abundances of beryllium and CUBES

    Smiljanic, R

    2014-01-01

    Stellar abundances of beryllium are useful in different areas of astrophysics, including studies of the Galactic chemical evolution, of stellar evolution, and of the formation of globular clusters. Determining Be abundances in stars is, however, a challenging endeavor. The two Be II resonance lines useful for abundance analyses are in the near UV, a region strongly affected by atmospheric extinction. CUBES is a new spectrograph planned for the VLT that will be more sensitive than current instruments in the near UV spectral region. It will allow the observation of fainter stars, expanding the number of targets where Be abundances can be determined. Here, a brief review of stellar abundances of Be is presented together with a discussion of science cases for CUBES. In particular, preliminary simulations of CUBES spectra are presented, highlighting its possible impact in investigations of Be abundances of extremely metal-poor stars and of stars in globular clusters.

  9. FLUORESCENT OLIGONUCLEOTIDES CONTAINING A NOVEL PERYLENE 2 '-AMINO-alpha-L-LNA MONOMER: SYNTHESIS AND ANALYTICAL POTENTIAL

    Astakhova, I. V.; Kumar, T. S.; Wengel, J.

    2011-01-01

    Herein, a novel fluorescent nucleotide analogue, perylene-2'-amino-alpha-L-LNA, has been prepared and studied within synthetic oligonucleotides of different sequences. The phosphoramidite reagent was synthesized in 85% overall yield starting from 2'-amino-alpha-L-LNA nucleoside. Incorporation...... efficiency of the resulting perylene-2'-amino-alpha-L-LNA monomer (T*) into synthetic oligonucleotides was significantly improved by replacement of the typically used 1H-tetrazole activator with pyridine hydrochloride. Generally, oligonucleotides containing monomer T* showed high binding affinity towards...... a covalent link between two T* monomers in the double-labeled probe provides a remarkable degree of rigidity in the double helix which enforces positioning of the bulky perylene moieties in the nonpolar groove resulting in reduced fluorescence quenching....

  10. Automated synthesis of an 18F-labelled pyridine-based alkylating agent for high yield oligonucleotide conjugation

    Alkylating agents have been shown to be very promising for the radiolabelling of oligonucleotides with fluorine-18. In this report we describe the fully automated synthesis of 2-bromo-N-[3-(2-[18F]fluoropyridin-3-yloxy)propyl]acetamide ([18F]FPyBrA) utilizing a modular synthesis unit. Reaction conditions for the coupling of this pyridine-based alkylating agent at the 5' end of a fully phosphorothioated random 20-mer DNA sequence were optimized to achieve very high radiochemical yields (>90%) and a maximum specific activity of 5-6 GBq/μmoL. The potential for rapid purification by solid phase extraction without need of chromatographic isolation of the radiolabelled oligonucleotide presents an overall benefit for the application of oligonucleotides in preclinical studies and potential clinical applications.

  11. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field.

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds' vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field. PMID:26999445

  12. Structural Aspects of the Antiparallel and Parallel Duplexes Formed by DNA, 2’-O-Methyl RNA and RNA Oligonucleotides

    Szabat, Marta; Pedzinski, Tomasz; Czapik, Tomasz; Kierzek, Elzbieta; Kierzek, Ryszard

    2015-01-01

    This study investigated the influence of the nature of oligonucleotides on the abilities to form antiparallel and parallel duplexes. Base pairing of homopurine DNA, 2’-O-MeRNA and RNA oligonucleotides with respective homopyrimidine DNA, 2’-O-MeRNA and RNA as well as chimeric oligonucleotides containing LNA resulted in the formation of 18 various duplexes. UV melting, circular dichroism and fluorescence studies revealed the influence of nucleotide composition on duplex structure and thermal stability depending on the buffer pH value. Most duplexes simultaneously adopted both orientations. However, at pH 5.0, parallel duplexes were more favorable. Moreover, the presence of LNA nucleotides within a homopyrimidine strand favored the formation of parallel duplexes. PMID:26579720

  13. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds’ vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field. PMID:26999445

  14. Tragedy of the Commons

    Nørgaard, Jørgen

    individualistic and selfish attitude this would collaps, since each single citizen could benefit from putting more sheep on the common, which would eventually collapse by overgrazing. The metaphore is applied to our common planet, and our ability to built up institutions, economics and ethics, geared for sharing...

  15. Monitoring Butterfly Abundance: Beyond Pollard Walks

    Pellet, Jérôme; Bried, Jason T.; Parietti, David; Gander, Antoine; Heer, Patrick O.; Cherix, Daniel; Arlettaz, Raphaël

    2012-01-01

    Most butterfly monitoring protocols rely on counts along transects (Pollard walks) to generate species abundance indices and track population trends. It is still too often ignored that a population count results from two processes: the biological process (true abundance) and the statistical process (our ability to properly quantify abundance). Because individual detectability tends to vary in space (e.g., among sites) and time (e.g., among years), it remains unclear whether index counts truly...

  16. In silico screening based on predictive algorithms as a design tool for exon skipping oligonucleotides in Duchenne muscular dystrophy.

    Yusuke Echigoya

    Full Text Available The use of antisense 'splice-switching' oligonucleotides to induce exon skipping represents a potential therapeutic approach to various human genetic diseases. It has achieved greatest maturity in exon skipping of the dystrophin transcript in Duchenne muscular dystrophy (DMD, for which several clinical trials are completed or ongoing, and a large body of data exists describing tested oligonucleotides and their efficacy. The rational design of an exon skipping oligonucleotide involves the choice of an antisense sequence, usually between 15 and 32 nucleotides, targeting the exon that is to be skipped. Although parameters describing the target site can be computationally estimated and several have been identified to correlate with efficacy, methods to predict efficacy are limited. Here, an in silico pre-screening approach is proposed, based on predictive statistical modelling. Previous DMD data were compiled together and, for each oligonucleotide, some 60 descriptors were considered. Statistical modelling approaches were applied to derive algorithms that predict exon skipping for a given target site. We confirmed (1 the binding energetics of the oligonucleotide to the RNA, and (2 the distance in bases of the target site from the splice acceptor site, as the two most predictive parameters, and we included these and several other parameters (while discounting many into an in silico screening process, based on their capacity to predict high or low efficacy in either phosphorodiamidate morpholino oligomers (89% correctly predicted and/or 2'O Methyl RNA oligonucleotides (76% correctly predicted. Predictions correlated strongly with in vitro testing for sixteen de novo PMO sequences targeting various positions on DMD exons 44 (R² 0.89 and 53 (R² 0.89, one of which represents a potential novel candidate for clinical trials. We provide these algorithms together with a computational tool that facilitates screening to predict exon skipping efficacy at each

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

    Tomcin S

    2014-11-01

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

  18. Energy Transfer Assays Using Quantum Dot-Gold Nanoparticle Complexes: Optimizing Oligonucleotide Assay Configuration Using Monovalently Conjugated Quantum Dots.

    Uddayasankar, Uvaraj; Krull, Ulrich J

    2015-07-28

    The energy transfer between quantum dots (QDs) and gold nanoparticles (AuNPs) represents a popular transduction scheme in analytical assays that use nanomaterials. The impact of the spatial arrangement of the two types of nanoparticles on analytical performance has now been evaluated using a nucleic acid strand displacement assay. The first spatial arrangement (configuration 1) involved the assembly of a number of monovalently functionalized QD-oligonucleotide conjugates around a single central AuNP that was functionalized with complementary oligonucleotide sequences. The assembly of these complexes, and subsequent disassembly via target oligonucleotide-mediated displacement, were used to evaluate energy transfer efficiencies. Furthermore, the inner filter effect of AuNPs on the fluorescence intensity of the QD was studied. AuNPs of three different diameters (6, 13, and 30 nm) were used in these studies. Configuration 2 was based on the placement of monovalently functionalized AuNP-oligonucleotide conjugates around a single QD that was functionalized with a complementary oligonucleotide. The optimal assay configuration, established by evaluating energy transfer efficiencies and inner filter effects, was obtained by arranging at most 15 QDs around the 13 nm AuNP (configuration 1). These assays provided a 2.5-fold change in fluorescence intensity in the presence of target oligonucleotides. To obtain the same response with configuration 2 required the placement of three 6 nm AuNPs around the QD. This resulted in configuration 2 having a 5-fold lower fluorescence intensity when compared to configuration 1. The use of low-cost detection systems (digital camera) further emphasized the higher analytical performance of configuration 1. Response curves obtained using these detection systems demonstrated that configuration 1 had a 10-fold higher sensitivity when compared to configuration 2. This study provides an important framework for the development of sensitive assays

  19. Small molecule aptamer assays based on fluorescence anisotropy signal-enhancer oligonucleotides.

    Perrier, Sandrine; Bouilloud, Prisca; De Oliveira Coelho, Gisella; Henry, Mickael; Peyrin, Eric

    2016-08-15

    Herein, we design novel fluorescence anisotropy (FA) aptamer sensing platforms dedicated to small molecule detection. The assay strategy relied on enhanced fluctuations of segmental motion dynamics of the aptamer tracer mediated by an unlabelled, partially complementary oligonucleotide. The signal-enhancer oligonucleotide (SEO) essentially served as a free probe fraction revealer. By targeting specific regions of the signalling functional nucleic acid, the SEO binding to the unbound aptamer triggered perturbations of both the internal DNA flexibility and the localized dye environment upon the free probe to duplex structure transition. This potentiating effect determined increased FA variations between the duplex and target bound states of the aptameric probe. FA assay responses were obtained with both pre-structured (adenosine) and unstructured (tyrosinamide) aptamers and with dyes of different photochemical properties (fluorescein and texas red). The multiplexed analysis ability was further demonstrated through the simultaneous multicolour detection of the two small targets. The FA method appears to be especially simple, sensitive and widely applicable. PMID:27085946

  20. Anti-microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways.

    Gomez, Ivan G; MacKenna, Deidre A; Johnson, Bryce G; Kaimal, Vivek; Roach, Allie M; Ren, Shuyu; Nakagawa, Naoki; Xin, Cuiyan; Newitt, Rick; Pandya, Shweta; Xia, Tai-He; Liu, Xueqing; Borza, Dorin-Bogdan; Grafals, Monica; Shankland, Stuart J; Himmelfarb, Jonathan; Portilla, Didier; Liu, Shiguang; Chau, B Nelson; Duffield, Jeremy S

    2015-01-01

    MicroRNA-21 (miR-21) contributes to the pathogenesis of fibrogenic diseases in multiple organs, including the kidneys, potentially by silencing metabolic pathways that are critical for cellular ATP generation, ROS production, and inflammatory signaling. Here, we developed highly specific oligonucleotides that distribute to the kidney and inhibit miR-21 function when administered subcutaneously and evaluated the therapeutic potential of these anti-miR-21 oligonucleotides in chronic kidney disease. In a murine model of Alport nephropathy, miR-21 silencing did not produce any adverse effects and resulted in substantially milder kidney disease, with minimal albuminuria and dysfunction, compared with vehicle-treated mice. miR-21 silencing dramatically improved survival of Alport mice and reduced histological end points, including glomerulosclerosis, interstitial fibrosis, tubular injury, and inflammation. Anti-miR-21 enhanced PPARα/retinoid X receptor (PPARα/RXR) activity and downstream signaling pathways in glomerular, tubular, and interstitial cells. Moreover, miR-21 silencing enhanced mitochondrial function, which reduced mitochondrial ROS production and thus preserved tubular functions. Inhibition of miR-21 was protective against TGF-β-induced fibrogenesis and inflammation in glomerular and interstitial cells, likely as the result of enhanced PPARα/RXR activity and improved mitochondrial function. Together, these results demonstrate that inhibition of miR-21 represents a potential therapeutic strategy for chronic kidney diseases including Alport nephropathy. PMID:25415439

  1. Anti–microRNA-21 oligonucleotides prevent Alport nephropathy progression by stimulating metabolic pathways

    Gomez, Ivan G.; MacKenna, Deidre A.; Johnson, Bryce G.; Kaimal, Vivek; Roach, Allie M.; Ren, Shuyu; Nakagawa, Naoki; Xin, Cuiyan; Newitt, Rick; Pandya, Shweta; Xia, Tai-He; Liu, Xueqing; Borza, Dorin-Bogdan; Grafals, Monica; Shankland, Stuart J.; Himmelfarb, Jonathan; Portilla, Didier; Liu, Shiguang; Chau, B. Nelson; Duffield, Jeremy S.

    2014-01-01

    MicroRNA-21 (miR-21) contributes to the pathogenesis of fibrogenic diseases in multiple organs, including the kidneys, potentially by silencing metabolic pathways that are critical for cellular ATP generation, ROS production, and inflammatory signaling. Here, we developed highly specific oligonucleotides that distribute to the kidney and inhibit miR-21 function when administered subcutaneously and evaluated the therapeutic potential of these anti–miR-21 oligonucleotides in chronic kidney disease. In a murine model of Alport nephropathy, miR-21 silencing did not produce any adverse effects and resulted in substantially milder kidney disease, with minimal albuminuria and dysfunction, compared with vehicle-treated mice. miR-21 silencing dramatically improved survival of Alport mice and reduced histological end points, including glomerulosclerosis, interstitial fibrosis, tubular injury, and inflammation. Anti–miR-21 enhanced PPARα/retinoid X receptor (PPARα/RXR) activity and downstream signaling pathways in glomerular, tubular, and interstitial cells. Moreover, miR-21 silencing enhanced mitochondrial function, which reduced mitochondrial ROS production and thus preserved tubular functions. Inhibition of miR-21 was protective against TGF-β–induced fibrogenesis and inflammation in glomerular and interstitial cells, likely as the result of enhanced PPARα/RXR activity and improved mitochondrial function. Together, these results demonstrate that inhibition of miR-21 represents a potential therapeutic strategy for chronic kidney diseases including Alport nephropathy. PMID:25415439

  2. Peeling single-stranded DNA from graphite surface to determine oligonucleotide binding energy by force spectroscopy.

    Manohar, Suresh; Mantz, Amber R; Bancroft, Kevin E; Hui, Chung-Yuen; Jagota, Anand; Vezenov, Dmitri V

    2008-12-01

    We measured the force required to peel single-stranded DNA molecules from single-crystal graphite using chemical force microscopy. Force traces during retraction of a tip chemically modified with oligonucleotides displayed characteristic plateaus with abrupt force jumps, which we interpreted as a steady state peeling process punctuated by complete detachment of one or more molecules. We were able to differentiate between bases in pyrimidine homopolymers; peeling forces were 85.3 - 4.7 pN for polythymine and 60.8 +/- 5.5 pN for polycytosine, substantially independent of salt concentration and the rate of detachment. We developed a model for peeling a freely jointed chain from the graphite surface and estimated the average binding energy per monomer to be 11.5 +/- 0.6 k(B)T and 8.3 +/- 0.7 k(B)T in the cases of thymine and cytosine nucleotides, respectively. The equilibrium free-energy profile simulated using molecular dynamics had a potential well of 18.9 k(B)T for thymidine, showing that nonelectrostatic interactions dominate the binding. The discrepancy between the experiment and theory indicates that not all bases are adsorbed on the surface or that there is a population of conformations in which they adsorb. Force spectroscopy using oligonucleotides covalently linked to AFM tips provides a flexible and unambiguous means to quantify the strength of interactions between DNA and a number of substrates, potentially including nanomaterials such as carbon nanotubes. PMID:19368004

  3. Reliable Assessment and Quantification of the Fluorescence-Labeled Antisense Oligonucleotides In Vivo

    Maria Chiara Munisso

    2014-01-01

    Full Text Available The availability of fluorescent dyes and the advances in the optical systems for in vivo imaging have stimulated an increasing interest in developing new methodologies to study and quantify the biodistribution of labeled agents. However, despite these great achievements, we are facing significant challenges in determining if the observed fluorescence does correspond to the quantity of the dye in the tissues. In fact, although the far-red and near-infrared lights can propagate through several centimetres of tissue, they diffuse within a few millimetres as consequence of the elastic scattering of photons. In addition, when dye-labeled oligonucleotides form stable complex with cationic carriers, a large change in the fluorescence intensity of the dye is observed. Therefore, the measured fluorescence intensity is altered by the tissue heterogeneity and by the fluctuation of dye intensity. Hence, in this study a quantification strategy for fluorescence-labeled oligonucleotides was developed to solve these disadvantageous effects. Our results proved that upon efficient homogenization and dilution with chaotropic agents, such as guanidinium thiocyanate, it is possible to achieve a complete fluorescence intensity recovery. Furthermore, we demonstrated that this method has the advantage of good sensitivity and reproducibility, as well as easy handling of the tissue samples.

  4. Multivalent linkers for improved covalent binding of oligonucleotides to dye-doped silica nanoparticles

    Kelleher, S. M.; Nooney, R. I.; Flynn, S. P.; Clancy, E.; Burke, M.; Daly, S.; Smith, T. J.; Daniels, S.; McDonagh, C.

    2015-09-01

    This paper describes the fabrication of oligonucleotide-coated Cy5-doped silica nanoparticles using a combination of multivalent linkers and their use in surface-based DNA sandwich hybridization assays. Dipodal silane is introduced as a means to fabricate amine-coated silica nanoparticles and its advantages compared to monopodal silanes are discussed. The use of dipodal silane in conjunction with three different polymer linkers (oxidized dextran, linear and 8-arm polyethylene glycol (PEG)) to immobilize single-stranded DNA to Cy5-doped nanoparticles is investigated and dynamic light scattering measurements and Fourier transform infrared spectroscopy are used to follow the progression of the functionalization of the nanoparticles. We observe a significant improvement in the binding stability of the single-stranded DNA when the dipodal silane and 8-arm PEG are used in combination, when compared to alternative conjugation strategies. Both 8mer and 22mer oligonucleotides are securely conjugated to the high-brightness nanoparticles and their availability to hybridize with a complementary strand is confirmed using solution-based DNA hybridization experiments. In addition, a full surface-based sandwich assay demonstrates the potential these nanoparticles have in the detection of less than 500 femtomolar of a DNA analogue of micro RNA, miR-451.

  5. Clinical expert panel on monitoring potential lung toxicity of inhaled oligonucleotides: consensus points and recommendations.

    Alton, Eric W; Boushey, Homer A; Garn, Holger; Green, Francis H; Hodges, Michael; Martin, Richard J; Murdoch, Robert D; Renz, Harald; Shrewsbury, Stephen B; Seguin, Rosanne; Johnson, Graham; Parry, Joel D; Tepper, Jeff; Renzi, Paolo; Cavagnaro, Joy; Ferrari, Nicolay

    2012-08-01

    Oligonucleotides (ONs) are an emerging class of drugs being developed for the treatment of a wide variety of diseases including the treatment of respiratory diseases by the inhalation route. As a class, their toxicity on human lungs has not been fully characterized, and predictive toxicity biomarkers have not been identified. To that end, identification of sensitive methods and biomarkers that can detect toxicity in humans before any long term and/or irreversible side effects occur would be helpful. In light of the public's greater interests, the Inhalation Subcommittee of the Oligonucleotide Safety Working Group (OSWG) held expert panel discussions focusing on the potential toxicity of inhaled ONs and assessing the strengths and weaknesses of different monitoring techniques for use during the clinical evaluation of inhaled ON candidates. This white paper summarizes the key discussions and captures the panelists' perspectives and recommendations which, we propose, could be used as a framework to guide both industry and regulatory scientists in future clinical research to characterize and monitor the short and long term lung response to inhaled ONs. PMID:22809313

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

    Hanczyc, Piotr

    2012-04-24

    We report that short, synthetic, double- as well as single-stranded DNA can be aligned in stretched humid poly(vinyl alcohol) (PVA) matrix, and the secondary structure (nucleobase orientation) can be characterized with linear dichroism (LD) spectroscopy. Oligonucleotides of lengths varying between 10 (3.4 nm) and 60 bases (20.4 nm) were investigated with respect to structural properties in the gel-like polymer environment. The DNA conformation as a function of relative humidity reveals a strong dependence of helical structure of DNA on PVA hydration level, results of relevance for nanotechnical studies of DNA-based supramolecular systems. Also, the PVA gel could provide possibilities to test models for nucleic acid interactions and distribution in cell contexts, including structural stability of genetic material in the cell and PVA-packaging for gene delivery. A method by which duplex oligonucleotides, with sequences designed to provide specific binding sites, become amenable to polarized-light spectroscopy opens up new possibilities for studying structure in DNA complexes with small adduct molecules as well as proteins. © 2012 American Chemical Society.

  7. Antisense oligonucleotide therapy for the treatment of C9ORF72 ALS/FTD diseases.

    Riboldi, Giulietta; Zanetta, Chiara; Ranieri, Michela; Nizzardo, Monica; Simone, Chiara; Magri, Francesca; Bresolin, Nereo; Comi, Giacomo P; Corti, Stefania

    2014-12-01

    Motor neuron disorders, and particularly amyotrophic lateral sclerosis (ALS), are fatal diseases that are due to the loss of motor neurons in the brain and spinal cord, with progressive paralysis and premature death. It has been recently shown that the most frequent genetic cause of ALS, frontotemporal dementia (FTD), and other neurological diseases is the expansion of a hexanucleotide repeat (GGGGCC) in the non-coding region of the C9ORF72 gene. The pathogenic mechanisms that produce cell death in the presence of this expansion are still unclear. One of the most likely hypotheses seems to be the gain-of-function that is achieved through the production of toxic RNA (able to sequester RNA-binding protein) and/or toxic proteins. In recent works, different authors have reported that antisense oligonucleotides complementary to the C9ORF72 RNA transcript sequence were able to significantly reduce RNA foci generated by the expanded RNA, in affected cells. Here, we summarize the recent findings that support the idea that the buildup of "toxic" RNA containing the GGGGCC repeat contributes to the death of motor neurons in ALS and also suggest that the use of antisense oligonucleotides targeting this transcript is a promising strategy for treating ALS/frontotemporal lobe dementia (FTLD) patients with the C9ORF72 repeat expansion. These data are particularly important, given the state of the art antisense technology, and they allow researchers to believe that a clinical application of these discoveries will be possible soon. PMID:24809691

  8. Improved bioactivity of G-rich triplex-forming oligonucleotides containing modified guanine bases.

    Rogers, Faye A; Lloyd, Janice A; Tiwari, Meetu Kaushik

    2014-01-01

    Triplex structures generated by sequence-specific triplex-forming oligonucleotides (TFOs) have proven to be promising tools for gene targeting strategies. In addition, triplex technology has been highly utilized to study the molecular mechanisms of DNA repair, recombination and mutagenesis. However, triplex formation utilizing guanine-rich oligonucleotides as third strands can be inhibited by potassium-induced self-association resulting in G-quadruplex formation. We report here that guanine-rich TFOs partially substituted with 8-aza-7-deaza-guanine (PPG) have improved target site binding in potassium compared with TFOs containing the natural guanine base. We designed PPG-substituted TFOs to bind to a polypurine sequence in the supFG1 reporter gene. The binding efficiency of PPG-substituted TFOs to the target sequence was analyzed using electrophoresis mobility gel shift assays. We have determined that in the presence of potassium, the non-substituted TFO, AG30 did not bind to its target sequence, however binding was observed with the PPG-substituted AG30 under conditions with up to 140 mM KCl. The PPG-TFOs were able to maintain their ability to induce genomic modifications as measured by an assay for gene-targeted mutagenesis. In addition, these compounds were capable of triplex-induced DNA double strand breaks, which resulted in activation of apoptosis. PMID:25483840

  9. Antiproliferation effects of an androgen receptor triple-helix forming oligonucleotide on prostate cancer cells

    Objective: To provide experimental basis for antigene radiation therapy through exploring the effects of antigene strategy on androgen receptor (AR) expression and proliferation of prostate cancer cells. Methods: The triple-helix forming oligonucleotide (TFO) targeting 2447-2461nt of AR cDNA was designed and transfected LNCaP prostate cancer cells with liposome. 24-72 h after transfection, the cellular proliferation was detected by 3H-thymidine (TdR) incorporation test, the expression of AR gene was examined by reverse transcription-polymerase chain reaction (RT-PCR) and expression of AR protein was performed by radioligand binding assay. The results of TFO were compared with antisense oligonucleotide (ASON). Results: At all time points, the AR expression levels in TFO group were markedly lower than that of ASON group (P<0.05). The inhibitory rate of TFO for cellular proliferation was significantly higher than that of ASON (P<0.05). Conclusion: The TFO was a potent inhibitor for AR expression and cell proliferation of LNCaP cells , and could be used in antigene radiotherapy. (authors)

  10. Detection of adenosine 5'-triphosphate by fluorescence variation of oligonucleotide-templated silver nanoclusters.

    Lee, Jennifer Daneen; Cang, Jinshun; Chen, Ying-Chieh; Chen, Wei-Yu; Ou, Chung-Mao; Chang, Huan-Tsung

    2014-08-15

    Oligonucleotide-templated Ag nanoclusters (DNA-Ag NCs) prepared from AgNO3 using an oligonucleotide (5'-TAACCCCTAACCCCT-3') as a template and NaBH4 as a reducing agent have been used for sensing of adenosine 5'-triphosphate (ATP). The fluorescence intensity and emission wavelength of DNA-Ag NCs are dependent on the pH value and ATP concentration. At pH 3.0 and 11.0, ATP shows greater effects on fluorescence of the DNA-Ag NCs. Upon increasing ATP concentration from 10 to 50μM, their emission wavelength at pH 3.0 shifts from 525 to 585nm. At pH 11.0, their fluorescence intensity (510nm) increases upon increasing ATP concentration. The circular dichroism (CD), electrospray ionization-mass spectrometry (ESI-MS), absorption, and fluorescence results indicate that ATP and pH affect the interactions between DNAs and Ag atoms, resulting in changes in their fluorescence. The DNA-Ag NCs allow detection of ATP over a concentration range of 0.1-10μM, with a limit of detection 33nM. Practicality of the DNA-Ag NCs probe has been validated with the determination of ATP concentrations in the lysate of MDA-MB-231 breast carcinoma cells. PMID:24657647

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

    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

  12. Small antisense oligonucleotides against G-quadruplexes: specific mRNA translational switches

    Rouleau, Samuel G.; Beaudoin, Jean-Denis; Bisaillon, Martin; Perreault, Jean-Pierre

    2015-01-01

    G-quadruplexes (G4) are intricate RNA structures found throughout the transcriptome. Because they are associated with a variety of biological cellular mechanisms, these fascinating structural motifs are seen as potential therapeutic targets against many diseases. While screening of chemical compounds specific to G4 motifs has yielded interesting results, no single compound successfully discriminates between G4 motifs based on nucleotide sequences alone. This level of specificity is best attained using antisense oligonucleotides (ASO). Indeed, oligonucleotide-based strategies are already used to modulate DNA G4 folding in vitro. Here, we report that, in human cells, the use of short ASO to promote and inhibit RNA G4 folding affects the translation of specific mRNAs, including one from the 5′UTR of the H2AFY gene, a histone variant associated with cellular differentiation and cancer. These results suggest that the relatively high specificity of ASO-based strategies holds significant potential for applications aimed at modulating G4-motif folding. PMID:25510493

  13. Oligonucleotide-Mediated Genome Editing Provides Precision and Function to Engineered Nucleases and Antibiotics in Plants.

    Sauer, Noel J; Narváez-Vásquez, Javier; Mozoruk, Jerry; Miller, Ryan B; Warburg, Zachary J; Woodward, Melody J; Mihiret, Yohannes A; Lincoln, Tracey A; Segami, Rosa E; Sanders, Steven L; Walker, Keith A; Beetham, Peter R; Schöpke, Christian R; Gocal, Greg F W

    2016-04-01

    Here, we report a form of oligonucleotide-directed mutagenesis for precision genome editing in plants that uses single-stranded oligonucleotides (ssODNs) to precisely and efficiently generate genome edits at DNA strand lesions made by DNA double strand break reagents. Employing a transgene model in Arabidopsis (Arabidopsis thaliana), we obtained a high frequency of precise targeted genome edits when ssODNs were introduced into protoplasts that were pretreated with the glycopeptide antibiotic phleomycin, a nonspecific DNA double strand breaker. Simultaneous delivery of ssODN and a site-specific DNA double strand breaker, either transcription activator-like effector nucleases (TALENs) or clustered, regularly interspaced, short palindromic repeats (CRISPR/Cas9), resulted in a much greater targeted genome-editing frequency compared with treatment with DNA double strand-breaking reagents alone. Using this site-specific approach, we applied the combination of ssODN and CRISPR/Cas9 to develop an herbicide tolerance trait in flax (Linum usitatissimum) by precisely editing the 5'-ENOLPYRUVYLSHIKIMATE-3-PHOSPHATE SYNTHASE (EPSPS) genes. EPSPS edits occurred at sufficient frequency that we could regenerate whole plants from edited protoplasts without employing selection. These plants were subsequently determined to be tolerant to the herbicide glyphosate in greenhouse spray tests. Progeny (C1) of these plants showed the expected Mendelian segregation of EPSPS edits. Our findings show the enormous potential of using a genome-editing platform for precise, reliable trait development in crop plants. PMID:26864017

  14. Mesoporous Silica Nanoparticles Decorated with Carbosilane Dendrons as New Non-viral Oligonucleotide Delivery Carriers.

    Martínez, Ángel; Fuentes-Paniagua, Elena; Baeza, Alejandro; Sánchez-Nieves, Javier; Cicuéndez, Mónica; Gómez, Rafael; de la Mata, F Javier; González, Blanca; Vallet-Regí, María

    2015-10-26

    A novel nanosystem based on mesoporous silica nanoparticles covered with carbosilane dendrons grafted on the external surface of the nanoparticles is reported. This system is able to transport single-stranded oligonucleotide into cells, avoiding an electrostatic repulsion between the cell membrane and the negatively charged nucleic acids thanks to the cationic charge provided by the dendron coating under physiological conditions. Moreover, the presence of the highly ordered pore network inside the silica matrix would make possible to allocate other therapeutic agents within the mesopores with the aim of achieving a double delivery. First, carbosilane dendrons of second and third generation possessing ammonium or tertiary amine groups as peripheral functional groups were prepared. Hence, different strategies were tested in order to obtain their suitable grafting on the outer surface of the nanoparticles. As nucleic acid model, a single-stranded DNA oligonucleotide tagged with a fluorescent Cy3 moiety was used to evaluate the DNA adsorption capacity. The hybrid material functionalised with the third generation of a neutral dendron showed excellent DNA binding properties. Finally, the cytotoxicity as well as the capability to deliver DNA into cells, was tested in vitro by using a human osteoblast-like cell line, achieving good levels of internalisation of the vector DNA/carbosilane dendron-functionalised material without affecting the cellular viability. PMID:26361378

  15. Oligonucleotide-mediated gene modification and its promise for animal agriculture.

    Laible, Götz; Wagner, Stefan; Alderson, Jon

    2006-01-17

    One of the great aspirations in modern biology is the ability to utilise the expanding knowledge of the genetic basis of phenotypic diversity through the purposeful tailoring of the mammalian genome. A number of technologies are emerging which have the capacity to modify genes in their chromosomal context. Not surprisingly, the major thrust in this area has come from the evaluation of gene therapy applications to correct mutations implicated in human genetic diseases. The recent development of somatic cell nuclear transfer (SCNT) provides access to these technologies for the purposeful modification of livestock animals. The enormous phenotypic variety existent in contemporary livestock animals has in many cases been linked to quantitative trait loci (QTL) and their underlying point mutations, often referred to as single-nucleotide polymorphisms (SNPs). Thus, the ability for the targeted genetic modification of livestock animals constitutes an attractive opportunity for future agricultural applications. In this review, we will summarize attempts and approaches for oligonucleotide-mediated gene modification (OGM) strategies for the site-specific modification of the genome, with an emphasis on chimeric RNA-DNA oligonucleotides (RDOs) and single-stranded oligonucletides (ssODNs). The potential of this approach for the directed genetic improvement of livestock animals is illustrated through examples, outlining the effects of point mutations on important traits, including meat and milk production, reproductive performance, disease resistance and superior models of human diseases. Current technological hurdles and potential strategies that might remove these barriers in the future are discussed. PMID:16330159

  16. Detection of enterotoxigenic Escherichia coli in stool samples by using nonradioactively labeled oligonucleotide DNA probes and PCR.

    Schultsz, C.; Pool, G J; Van Ketel, R; DE WEVER, B; Speelman, P; Dankert, J.

    1994-01-01

    The detection of heat-labile enterotoxin LT-A and heat-stable enterotoxin ST Ia and ST Ib genes from enterotoxigenic Escherichia coli (ETEC) by using oligonucleotide DNA probes and the PCR was evaluated in reconstruction experiments and by testing stool specimens from 29 healthy subjects and from 50 patients with diarrhea who had returned from the (sub)tropics. ETEC strains were detected in concentrations ranging from 10(6) to 10(8) CFU/g of feces when oligonucleotide probes were applied to c...

  17. 2'-Aminoethoxy-2-amino-3-methylpyridine in triplex-forming oligonucleotides: high affinity, selectivity and resistance to enzymatic degradation.

    Lou, Chenguang; Shelbourne, Montserrat; Fox, Keith R; Brown, Tom

    2011-12-23

    The phosphoramidite monomer of the C-nucleoside 2'-aminoethoxy-2-amino-3-methylpyridine (AE-MAP) has been synthesized for the first time and incorporated into triplex-forming oligonucleotides (TFOs). Ultraviolet melting and DNase I footprinting studies show that AE-MAP is a potent triplex-stabilizing monomer that is selective for GC base pairs. TFOs containing AE-MAP bind with high affinity to duplexes but only weakly to single stranded DNA. In addition, AE-MAP confers high nuclease resistance on oligonucleotides. TFOs containing AE-MAP have potential for gene knock-out and gene expression studies. PMID:22127905

  18. The abundance and isotopic composition of water in eucrites

    Barrett, T. J.; Barnes, J. J.; TartèSe, R.; Anand, M.; Franchi, I. A.; Greenwood, R. C.; Charlier, B. L. A.; Grady, M. M.

    2016-06-01

    Volatile elements play a key role in the dynamics of planetary evolution. Extensive work has been carried out to determine the abundance, distribution, and source(s) of volatiles in planetary bodies such as the Earth, Moon, and Mars. A recent study showed that the water in apatite from eucrites has similar hydrogen isotopic compositions compared to water in terrestrial rocks and carbonaceous chondrites, suggesting that water accreted very early in the inner solar system given the ancient crystallization ages (~4.5 Ga) of eucrites. Here, the measurements of water (reported as equivalent H2O abundances) and the hydrogen isotopic composition (δD) of apatite from five basaltic eucrites and one cumulate eucrite are reported. Apatite H2O abundances range from ~30 to ~3500 ppm and are associated with a weighted average δD value of -34 ± 67‰. No systematic variations or correlations are observed in H2O abundance or δD value with eucrite geochemical trend or metamorphic grade. These results extend the range of previously published hydrogen isotope data for eucrites and confirm the striking homogeneity in the H-isotopic composition of water in eucrites, which is consistent with a common source for water in the inner solar system.

  19. Invasive lionfish reduce native fish abundance on a regional scale.

    Ballew, Nicholas G; Bacheler, Nathan M; Kellison, G Todd; Schueller, Amy M

    2016-01-01

    Invasive lionfish pose an unprecedented threat to biodiversity and fisheries throughout Atlantic waters off of the southeastern United States, the Caribbean, and the Gulf of Mexico. Here, we employ a spatially replicated Before-After-Control-Impact analysis with temporal pairing to quantify for the first time the impact of the lionfish invasion on native fish abundance across a broad regional scale and over the entire duration of the lionfish invasion (1990-2014). Our results suggest that 1) lionfish-impacted areas off of the southeastern United States are most prevalent off-shore near the continental shelf-break but are also common near-shore and 2) in impacted areas, lionfish have reduced tomtate (a native forage fish) abundance by 45% since the invasion began. Tomtate served as a model native fish species in our analysis, and as such, it is likely that the lionfish invasion has had similar impacts on other species, some of which may be of economic importance. Barring the development of a control strategy that reverses the lionfish invasion, the abundance of lionfish in the Atlantic, Caribbean, and Gulf of Mexico will likely remain at or above current levels. Consequently, the effect of lionfish on native fish abundance will likely continue for the foreseeable future. PMID:27578096

  20. The abundance and isotopic composition of water in eucrites

    Barrett, T. J.; Barnes, J. J.; TartèSe, R.; Anand, M.; Franchi, I. A.; Greenwood, R. C.; Charlier, B. L. A.; Grady, M. M.

    2016-05-01

    Volatile elements play a key role in the dynamics of planetary evolution. Extensive work has been carried out to determine the abundance, distribution, and source(s) of volatiles in planetary bodies such as the Earth, Moon, and Mars. A recent study showed that the water in apatite from eucrites has similar hydrogen isotopic compositions compared to water in terrestrial rocks and carbonaceous chondrites, suggesting that water accreted very early in the inner solar system given the ancient crystallization ages (~4.5 Ga) of eucrites. Here, the measurements of water (reported as equivalent H2O abundances) and the hydrogen isotopic composition (δD) of apatite from five basaltic eucrites and one cumulate eucrite are reported. Apatite H2O abundances range from ~30 to ~3500 ppm and are associated with a weighted average δD value of -34 ± 67‰. No systematic variations or correlations are observed in H2O abundance or δD value with eucrite geochemical trend or metamorphic grade. These results extend the range of previously published hydrogen isotope data for eucrites and confirm the striking homogeneity in the H-isotopic composition of water in eucrites, which is consistent with a common source for water in the inner solar system.

  1. Cross-scale interactions and the distribution-abundance relationship.

    Werner, Earl E; Davis, Christopher J; Skelly, David K; Relyea, Rick A; Benard, Michael F; McCauley, Shannon J

    2014-01-01

    Positive interspecific relationships between local abundance and extent of regional distribution are among the most ubiquitous patterns in ecology. Although multiple hypotheses have been proposed, the mechanisms underlying distribution-abundance (d-a) relationships remain poorly understood. We examined the intra- and interspecific distribution-abundance relationships for a metacommunity of 13 amphibian species sampled for 15 consecutive years. Mean density of larvae in occupied ponds was positively related to number of ponds occupied by species; employing the fraction of ponds uniquely available to each species this same relationship sharply decelerates. The latter relationship suggested that more abundant species inhabited most available habitats annually, whereas rarer species were dispersal limited. We inferred the mechanisms responsible for this pattern based on the dynamics of one species, Pseudacris triseriata, which transitioned between a rare, narrowly distributed species to a common, widely distributed species and then back again. Both transitions were presaged by marked changes in mean local densities driven by climatic effects on habitat quality. We identified threshold densities separating these population regime shifts that differed with landscape configuration. Our data suggest that these transitions were caused by strong cross-scale interactions between local resource/niche processes and larger scale metapopulation processes. The patterns we observed have relevance for understanding the mechanisms of interspecific d-a relationships and critical thresholds associated with habitat fragmentation. PMID:24875899

  2. Microbial distribution and abundance in the digestive system of five shipworm species (Bivalvia: Teredinidae.

    Meghan A Betcher

    Full Text Available Marine bivalves of the family Teredinidae (shipworms are voracious consumers of wood in marine environments. In several shipworm species, dense communities of intracellular bacterial endosymbionts have been observed within specialized cells (bacteriocytes of the gills (ctenidia. These bacteria are proposed to contribute to digestion of wood by the host. While the microbes of shipworm gills have been studied extensively in several species, the abundance and distribution of microbes in the digestive system have not been adequately addressed. Here we use Fluorescence In-Situ Hybridization (FISH and laser scanning confocal microscopy with 16S rRNA directed oligonucleotide probes targeting all domains, domains Bacteria and Archaea, and other taxonomic groups to examine the digestive microbiota of 17 specimens from 5 shipworm species (Bankia setacea, Lyrodus pedicellatus, Lyrodus massa, Lyrodus sp. and Teredo aff. triangularis. These data reveal that the caecum, a large sac-like appendage of the stomach that typically contains large quantities of wood particles and is considered the primary site of wood digestion, harbors only very sparse microbial populations. However, a significant number of bacterial cells were observed in fecal pellets within the intestines. These results suggest that due to low abundance, bacteria in the caecum may contribute little to lignocellulose degradation. In contrast, the comparatively high population density of bacteria in the intestine suggests a possible role for intestinal bacteria in the degradation of lignocellulose.

  3. Microbial Distribution and Abundance in the Digestive System of Five Shipworm Species (Bivalvia: Teredinidae)

    Betcher, Meghan A.; Fung, Jennifer M.; Han, Andrew W.; O’Connor, Roberta; Seronay, Romell; Concepcion, Gisela P.; Distel, Daniel L.; Haygood, Margo G.

    2012-01-01

    Marine bivalves of the family Teredinidae (shipworms) are voracious consumers of wood in marine environments. In several shipworm species, dense communities of intracellular bacterial endosymbionts have been observed within specialized cells (bacteriocytes) of the gills (ctenidia). These bacteria are proposed to contribute to digestion of wood by the host. While the microbes of shipworm gills have been studied extensively in several species, the abundance and distribution of microbes in the digestive system have not been adequately addressed. Here we use Fluorescence In-Situ Hybridization (FISH) and laser scanning confocal microscopy with 16S rRNA directed oligonucleotide probes targeting all domains, domains Bacteria and Archaea, and other taxonomic groups to examine the digestive microbiota of 17 specimens from 5 shipworm species (Bankia setacea, Lyrodus pedicellatus, Lyrodus massa, Lyrodus sp. and Teredo aff. triangularis). These data reveal that the caecum, a large sac-like appendage of the stomach that typically contains large quantities of wood particles and is considered the primary site of wood digestion, harbors only very sparse microbial populations. However, a significant number of bacterial cells were observed in fecal pellets within the intestines. These results suggest that due to low abundance, bacteria in the caecum may contribute little to lignocellulose degradation. In contrast, the comparatively high population density of bacteria in the intestine suggests a possible role for intestinal bacteria in the degradation of lignocellulose. PMID:23028923

  4. Common Ground and Delegation

    Dobrajska, Magdalena; Foss, Nicolai Juul; Lyngsie, Jacob

    preconditions of increasing delegation. We argue that key HR practices?namely, hiring, training and job-rotation?are associated with delegation of decision-making authority. These practices assist in the creation of shared knowledge conditions between managers and employees. In turn, such a ?common ground......-scale questionnaire survey with unique population-wide employer-employee data. We find evidence of a direct and positive influence of hiring decisions (proxied by common educational background), and the training and job rotation of employees on delegation. Moreover, we find a positive interaction between common...

  5. Common Hair Problems

    ... the top of the head. Women may develop female pattern baldness in which the hair becomes thin over the ... is most commonly seen in children. Hair Loss, Female Pattern Baldness (Female Pattern Alopecia) Female pattern baldness (alopecia) is ...

  6. Common Knowledge on Networks

    Liddell, Torrin M

    2015-01-01

    Common knowledge of intentions is crucial to basic social tasks ranging from cooperative hunting to oligopoly collusion, riots, revolutions, and the evolution of social norms and human culture. Yet little is known about how common knowledge leaves a trace on the dynamics of a social network. Here we show how an individual's network properties---primarily local clustering and betweenness centrality---provide strong signals of the ability to successfully participate in common knowledge tasks. These signals are distinct from those expected when practices are contagious, or when people use less-sophisticated heuristics that do not yield true coordination. This makes it possible to infer decision rules from observation. We also find that tasks that require common knowledge can yield significant inequalities in success, in contrast to the relative equality that results when practices spread by contagion alone.

  7. Common Conditions in Newborns

    ... Prenatal Baby Bathing & Skin Care Breastfeeding Crying & Colic Diapers & Clothing Feeding & Nutrition Preemie Sleep Teething & Tooth Care Toddler Preschool Gradeschool Teen Young Adult Healthy Children > Ages & Stages > Baby > Common Conditions in ...

  8. Common Foot Problems

    ... and rashes clinical tools newsletter | contact Share | Common Foot Problems A A A Trauma, infection, skin disease, ... the sole of the front part of the foot and on the toes. Foot infections include warts; ...

  9. Five Common Glaucoma Tests

    ... About Us Donate In This Section Five Common Glaucoma Tests en Español email Send this article to ... year or two after age 35. A Comprehensive Glaucoma Exam To be safe and accurate, five factors ...

  10. UNDERSTANDING THE GLOBAL COMMONS

    Bromley, Daniel W.; Cochrane, Jeffrey A.

    1994-01-01

    We want to clarify the way in which we think about the global commons, particularly the problem of global warming caused by greenhouse gas emissions and tropical deforestation. We develop a policy framework in which the policy goal is the sustainability of the earth's ability to absorb greenhouse gases. The framework considers the unequal incidence of benefits and costs of particular policies. We identify several resource management regimes and suggest that management under a common property ...

  11. Common Warehouse Metamodel

    Jersák, Pavel

    2008-01-01

    The main issue of this work is Common Warehouse Metamodel (CWM) which is used both as the standard for metadata in data warehousing and business intelligence and as means of exchange of metadata between tools in DWH. As this issue of CWM and metadata management has not been published in Czech-language so far, the main aim of this work is to introduce Common Warehouse Metamodel, its utilization in BI and also to present advantages of metadata management implementation based on CWM.

  12. Timely Common Knowledge

    Yannai A. Gonczarowski; Moses, Yoram

    2013-01-01

    Coordinating activities at different sites of a multi-agent system typically imposes epistemic constraints on the participants. Specifying explicit bounds on the relative times at which actions are performed induces combined temporal and epistemic constraints on when agents can perform their actions. This paper characterises the interactive epistemic state that arises when actions must meet particular temporal constraints. The new state, called timely common knowledge, generalizes common know...

  13. Taking species abundance distributions beyond individuals

    Morlon, Helene; White, Ethan P.; Etienne, Rampal S.; Green, Jessica L.; Ostling, Annette; Alonso, David; Enquist, Brian J.; He, Fangliang; Hurlbert, Allen; Magurran, Anne E.; Maurer, Brian A.; McGill, Brian J.; Olff, Han; Storch, David; Zillio, Tommaso; Chave, Jérôme

    2009-01-01

    The species abundance distribution (SAD) is one of the few universal patterns in ecology. Research on this fundamental distribution has primarily focused on the study of numerical counts, irrespective of the traits of individuals. Here we show that considering a set of Generalized Species Abundance

  14. Methanol abundance in low mass protostars

    Maret, S

    2004-01-01

    Methanol lines observations of a sample of low mass Class 0 protostars are presented. Using a 1D radiative transfer model, I show that several protostars have large abundance jumps in the inner hot and dense region of envelopes, probably because of thermal grain mantle evaporation. These abundances are compared with a grain surface chemistry model.

  15. Solar Energetic Particles: Sampling Coronal Abundances

    Reames, Donald V.

    1998-05-01

    In the large solar energetic particle (SEP) events, coronal mass ejections (CMEs) drive shock waves out through the corona that accelerate elements of the ambient material to MeV energies in a fairly democratic, temperature-independent manner. These events provide the most complete source of information on element abundances in the corona. Relative abundances of 22 elements from H through Zn display the well-known dependence on the first ionization potential (FIP) that distinguishes coronal and photospheric material. For most elements, the main abundance variations depend upon the gyrofrequency, and hence on the charge-to-mass ratio, Q/A, of the ion. Abundance variations in the dominant species, H and He, are not Q/A dependent, presumably because of non-linear wave-particle interactions of H and He during acceleration. Impulsive flares provide a different sample of material that confirms the Ne:Mg:Si and He/C abundances in the corona.

  16. The Abundance of Circumbinary Exoplanets

    Armstrong, David J.

    2015-12-01

    Circumbinary planets, planets orbiting around binary stars, represent a new angle of information on planet formation theories, showing us how the different processes that form a planet react to the torque of a central host binary. I will present recently published observational constraints on the occurrence rates and orbital element distributions of circumbinary planets. This work utilises the Kepler dataset of ~2000 eclipsing binaries, along with an independently developed tailored search algorithm, to debias the dataset and find the underlying frequency of these planets. We discover that circumbinary planets have a similar occurrence rate to planets around single stars, but only if they are preferentially coplanar with their host binary. If they are more misaligned, they must be significantly more common. This effect is strong enough that following a reductio ad absurdum argument, we confirm the coplanar preference for these planets. Along with these results, we confirm the previously noted tendency for circumbinary planets to not be found around the closest (P(binary) < ~7 days) host binaries. This last result may be a marker of the binary star formation process.

  17. Fish and phytoplankton exhibit contrasting temporal species abundance patterns in a dynamic north temperate lake.

    Gretchen J A Hansen

    Full Text Available Temporal patterns of species abundance, although less well-studied than spatial patterns, provide valuable insight to the processes governing community assembly. We compared temporal abundance distributions of two communities, phytoplankton and fish, in a north temperate lake. We used both 17 years of observed relative abundance data as well as resampled data from Monte Carlo simulations to account for the possible effects of non-detection of rare species. Similar to what has been found in other communities, phytoplankton and fish species that appeared more frequently were generally more abundant than rare species. However, neither community exhibited two distinct groups of "core" (common occurrence and high abundance and "occasional" (rare occurrence and low abundance species. Both observed and resampled data show that the phytoplankton community was dominated by occasional species appearing in only one year that exhibited large variation in their abundances, while the fish community was dominated by core species occurring in all 17 years at high abundances. We hypothesize that the life-history traits that enable phytoplankton to persist in highly dynamic environments may result in communities dominated by occasional species capable of reaching high abundances when conditions allow. Conversely, longer turnover times and broad environmental tolerances of fish may result in communities dominated by core species structured primarily by competitive interactions.

  18. Estimating abundance in the presence of species uncertainty

    Chambert, Thierry A; Hossack, Blake R.; Fishback, LeeAnn; Davenport, Jon M.

    2016-01-01

    1.N-mixture models have become a popular method for estimating abundance of free-ranging animals that are not marked or identified individually. These models have been used on count data for single species that can be identified with certainty. However, co-occurring species often look similar during one or more life stages, making it difficult to assign species for all recorded captures. This uncertainty creates problems for estimating species-specific abundance and it can often limit life stages to which we can make inference. 2.We present a new extension of N-mixture models that accounts for species uncertainty. In addition to estimating site-specific abundances and detection probabilities, this model allows estimating probability of correct assignment of species identity. We implement this hierarchical model in a Bayesian framework and provide all code for running the model in BUGS-language programs. 3.We present an application of the model on count data from two sympatric freshwater fishes, the brook stickleback (Culaea inconstans) and the ninespine stickleback (Pungitius pungitius), ad illustrate implementation of covariate effects (habitat characteristics). In addition, we used a simulation study to validate the model and illustrate potential sample size issues. We also compared, for both real and simulated data, estimates provided by our model to those obtained by a simple N-mixture model when captures of unknown species identification were discarded. In the latter case, abundance estimates appeared highly biased and very imprecise, while our new model provided unbiased estimates with higher precision. 4.This extension of the N-mixture model should be useful for a wide variety of studies and taxa, as species uncertainty is a common issue. It should notably help improve investigation of abundance and vital rate characteristics of organisms’ early life stages, which are sometimes more difficult to identify than adults.

  19. Few constraints limit the design of quinone methide-oligonucleotide self-adducts for directing DNA alkylation†

    Rossiter, Clifford S.; Modica, Emilia; Kumar, Dalip; Rokita, Steven E

    2010-01-01

    Nucleotide sequences minimally containing adenosine, cytosine or guanosine are sufficient to form intrastrand oligonucleotide quinone methide self-adducts reversibly for subsequent alkylation of complementary DNA. The general lack of sequence restrictions should now allow for alkylation of most any target of interest although reaction is most efficient when the self-adducts contain guanine residues and do not form hairpin structures.

  20. Group-specific 16S rRNA-targeted oligonucleotide probes to identify thermophilic bacteria in marine hydrothermal vents

    Harmsen, HJM; Prieur, D; Jeanthon, C

    1997-01-01

    Four 16S rRNA-targeted oligonucleotide probes were designed for the detection of thermophilic members of the domain Bacteria known to thrive in marine hydrothermal systems, We developed and characterized probes encompassing most of the thermophilic members of the genus Bacillus, most species of the