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Sample records for oligonucleotide array discovery

  1. Oligonucleotide array discovery of polymorphisms in cultivated tomato (Solanum lycopersicum L.) reveals patterns of SNP variation associated with breeding.

    Science.gov (United States)

    Sim, Sung-Chur; Robbins, Matthew D; Chilcott, Charles; Zhu, Tong; Francis, David M

    2009-10-09

    Cultivated tomato (Solanum lycopersicum L.) has narrow genetic diversity that makes it difficult to identify polymorphisms between elite germplasm. We explored array-based single feature polymorphism (SFP) discovery as a high-throughput approach for marker development in cultivated tomato. Three varieties, FL7600 (fresh-market), OH9242 (processing), and PI114490 (cherry) were used as a source of genomic DNA for hybridization to oligonucleotide arrays. Identification of SFPs was based on outlier detection using regression analysis of normalized hybridization data within a probe set for each gene. A subset of 189 putative SFPs was sequenced for validation. The rate of validation depended on the desired level of significance (alpha) used to define the confidence interval (CI), and ranged from 76% for polymorphisms identified at alpha or= 2 SNPs per locus. We used a subset of validated SNPs for genetic diversity analysis of 92 tomato varieties and accessions. Pairwise estimation of theta (Fst) suggested significant differentiation between collections of fresh-market, processing, vintage, Latin American (landrace), and S. pimpinellifolium accessions. The fresh-market and processing groups displayed high genetic diversity relative to vintage and landrace groups. Furthermore, the patterns of SNP variation indicated that domestication and early breeding practices have led to progressive genetic bottlenecks while modern breeding practices have reintroduced genetic variation into the crop from wild species. Finally, we examined the ratio of non-synonymous (Ka) to synonymous substitutions (Ks) for 20 loci with multiple SNPs (>or= 4 per locus). Six of 20 loci showed ratios of Ka/Ks >or= 0.9. Array-based SFP discovery was an efficient method to identify a large number of molecular markers for genetics and breeding in elite tomato germplasm. Patterns of sequence variation across five major tomato groups provided insight into to the effect of human selection on genetic variation.

  2. Oligonucleotide array discovery of polymorphisms in cultivated tomato (Solanum lycopersicum L. reveals patterns of SNP variation associated with breeding

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    Zhu Tong

    2009-10-01

    Full Text Available Abstract Background Cultivated tomato (Solanum lycopersicum L. has narrow genetic diversity that makes it difficult to identify polymorphisms between elite germplasm. We explored array-based single feature polymorphism (SFP discovery as a high-throughput approach for marker development in cultivated tomato. Results Three varieties, FL7600 (fresh-market, OH9242 (processing, and PI114490 (cherry were used as a source of genomic DNA for hybridization to oligonucleotide arrays. Identification of SFPs was based on outlier detection using regression analysis of normalized hybridization data within a probe set for each gene. A subset of 189 putative SFPs was sequenced for validation. The rate of validation depended on the desired level of significance (α used to define the confidence interval (CI, and ranged from 76% for polymorphisms identified at α ≤ 10-6 to 60% for those identified at α ≤ 10-2. Validation percentage reached a plateau between α ≤ 10-4 and α ≤ 10-7, but failure to identify known SFPs (Type II error increased dramatically at α ≤ 10-6. Trough sequence validation, we identified 279 SNPs and 27 InDels in 111 loci. Sixty loci contained ≥ 2 SNPs per locus. We used a subset of validated SNPs for genetic diversity analysis of 92 tomato varieties and accessions. Pairwise estimation of θ (Fst suggested significant differentiation between collections of fresh-market, processing, vintage, Latin American (landrace, and S. pimpinellifolium accessions. The fresh-market and processing groups displayed high genetic diversity relative to vintage and landrace groups. Furthermore, the patterns of SNP variation indicated that domestication and early breeding practices have led to progressive genetic bottlenecks while modern breeding practices have reintroduced genetic variation into the crop from wild species. Finally, we examined the ratio of non-synonymous (Ka to synonymous substitutions (Ks for 20 loci with multiple SNPs (≥ 4 per

  3. Design Considerations for Array CGH to OligonucleotideArrays

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    Baldocchi, R.A.; Glynne, R.J.; Chin, K.; Kowbel, D.; Collins, C.; Mack, D.H.; Gray, J.W.

    2005-03-04

    Background: Representational oligonucleotide microarray analysis has been developed for detection of single nucleotide polymorphisms and/or for genome copy number changes. In this process, the intensity of hybridization to oligonucleotides arrays is increased by hybridizing a polymerase chain reaction (PCR)-amplified representation of reduced genomic complexity. However, hybridization to some oligonucleotides is not sufficiently high to allow precise analysis of that portion of the genome. Methods: In an effort to identify aspects of oligonucleotide hybridization affecting signal intensity, we explored the importance of the PCR product strand to which each oligonucleotide is homologous and the sequence of the array oligonucleotides. We accomplished this by hybridizing multiple PCR-amplified products to oligonucleotide arrays carrying two sense and two antisense 50-mer oligonucleotides for each PCR amplicon. Results: In some cases, hybridization intensity depended more strongly on the PCR amplicon strand (i.e., sense vs. antisense) than on the detection oligonucleotide sequence. In other cases, the oligonucleotide sequence seemed to dominate. Conclusion: Oligonucleotide arrays for analysis of DNA copy number or for single nucleotide polymorphism content should be designed to carry probes to sense and antisense strands of each PCR amplicon to ensure sufficient hybridization and signal intensity.

  4. Mapping genomic library clones using oligonucleotide arrays

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    Sapolsky, R.J.; Lipshutz, R.J. [Affymetrix, Santa Clara, CA (United States)

    1996-05-01

    We have developed a high-density DNA probe array and accompanying biochemical and informatic methods to order clones from genomic libraries. This approach involves a series of enzymatic steps for capturing a set of short dispersed sequence markers scattered throughout a high-molecular-weight DNA. By this process, all the ambiguous sequences lying adjacent to a given Type IIS restriction site are ligated between two DNA adaptors. These markers, once amplified and labeled by PCR, can be hybridized and detected on a high-density olligonucleotide array bearing probes complementary to all possible markers. The array is synthesized using light-directed combinatorial chemistry. For each clone in a genomic library, a characteristic set of sequence markers can be determined. On the basis of the similarity between the marker sets for each pair of clones, their relative overlap can be measured. The library can be sequentially ordered into a contig map using this overlap information. This new methodology does not require gel-based methods or prior sequence information and involves manipulations that should allow for easy adaptation to automated processing and data collection. 28 refs., 9 figs., 2 tabs.

  5. Typing of enteroviruses by use of microwell oligonucleotide arrays.

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    Susi, P; Hattara, L; Waris, M; Luoma-Aho, T; Siitari, H; Hyypiä, T; Saviranta, P

    2009-06-01

    We have developed a straightforward assay for the rapid typing of enteroviruses using oligonucleotide arrays in microtiter wells. The viral nucleic acids are concomitantly amplified and labeled during reverse transcription-PCR, and unpurified PCR products are used for hybridization. DNA strands are separated by alkaline denaturation, and hybridization is started by neutralization. The microarray hybridization reactions and the subsequent washes are performed in standard 96-well microtiter plates, which makes the method easily adaptable to high-throughput analysis. We describe here the assay principle and its potential in clinical laboratory use by correctly identifying 10 different enterovirus reference strains. Furthermore, we explore the detection of unknown sequence variants using serotype consensus oligonucleotide probes. With just two consensus probes for the coxsackievirus A9 (CVA9) serotype, we detected 23 out of 25 highly diverse CVA9 isolates. Overall, the assay involves several features aiming at ease of performance, robustness, and applicability to large-scale studies.

  6. Oligonucleotide array outperforms SNP array on formalin-fixed paraffin-embedded clinical samples.

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    Nasri, Soroush; Anjomshoaa, Ahmad; Song, Sarah; Guilford, Parry; McNoe, Les; Black, Michael; Phillips, Vicky; Reeve, Anthony; Humar, Bostjan

    2010-04-01

    Compromised quality of formalin-fixed paraffin-embedded (FFPE)-derived DNA has compounded the use of archival specimens for array-based genomic studies. Recent technological advances have led to first successes in this field; however, there is currently no general agreement on the most suitable platform for the array-based analysis of FFPE DNA. In this study, FFPE and matched fresh-frozen (FF) specimens were separately analyzed with Affymetrix single nucleotide polymorphism (SNP) 6.0 and Agilent 4x44K oligonucleotide arrays to compare the genomic profiles from the two tissue sources and to assess the relative performance of the two platforms on FFPE material. Genomic DNA was extracted from matched FFPE-FF pairs of normal intestinal epithelium from four patients and were applied to the SNP and oligonucleotide platforms according to the manufacturer-recommended protocols. On the Affymetrix platform, a substantial increase in apparent copy number alterations was observed in all FFPE tissues relative to their matched FF counterparts. In contrast, FFPE and matched FF genomic profiles obtained via the Agilent platform were very similar. Both the SNP and the oligonucleotide platform performed comparably on FF material. This study demonstrates that Agilent oligonucleotide array comparative genomic hybridization generates reliable results from FFPE extracted DNA, whereas the Affymetrix SNP-based array seems less suitable for the analysis of FFPE material.

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

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    Kong Sek

    2004-10-01

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

  8. Utilization of a labeled tracking oligonucleotide for visualization and quality control of spotted 70-mer arrays

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    Khan Shehnaz

    2004-02-01

    Full Text Available Abstract Background Spotted 70-mer oligonucleotide arrays offer potentially greater specificity and an alternative to expensive cDNA library maintenance and amplification. Since microarray fabrication is a considerable source of data variance, we previously directly tagged cDNA probes with a third fluorophore for prehybridization quality control. Fluorescently modifying oligonucleotide sets is cost prohibitive, therefore, a co-spotted Staphylococcus aureus-specific fluorescein-labeled "tracking" oligonucleotide is described to monitor fabrication variables of a Mycobacterium tuberculosis oligonucleotide microarray. Results Significantly (p M. tuberculosis H37Rv and M. tuberculosis mprA. Linearity between the mean log Cy3/Cy5 ratios of genes differentially expressed from arrays either possessing or lacking the tracking oligonucleotide was observed (R2 = 0.90, p Conclusions This novel approach enables prehybridization array visualization for spotted oligonucleotide arrays and sets the stage for more sophisticated slide qualification and data filtering applications.

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

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    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

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

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    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.

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

    Science.gov (United States)

    Li, Hao; Wood, Constance L; Getchell, Thomas V; Getchell, Marilyn L; Stromberg, Arnold J

    2004-12-30

    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. 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 alpha-level (alphanew = 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 alphanew to control the family-wise error rate (FWER) for each gene examined. 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.

  12. Genomic analysis by oligonucleotide array Comparative Genomic Hybridization utilizing formalin-fixed, paraffin-embedded tissues.

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    Savage, Stephanie J; Hostetter, Galen

    2011-01-01

    Formalin fixation has been used to preserve tissues for more than a hundred years, and there are currently more than 300 million archival samples in the United States alone. The application of genomic protocols such as high-density oligonucleotide array Comparative Genomic Hybridization (aCGH) to formalin-fixed, paraffin-embedded (FFPE) tissues, therefore, opens an untapped resource of available tissues for research and facilitates utilization of existing clinical data in a research sample set. However, formalin fixation results in cross-linking of proteins and DNA, typically leading to such a significant degradation of DNA template that little is available for use in molecular applications. Here, we describe a protocol to circumvent formalin fixation artifact by utilizing enzymatic reactions to obtain quality DNA from a wide range of FFPE tissues for successful genome-wide discovery of gene dosage alterations in archival clinical samples.

  13. Managing the sequence-specificity of antisense oligonucleotides in drug discovery.

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    Hagedorn, Peter H; Hansen, Bo R; Koch, Troels; Lindow, Morten

    2017-03-17

    All drugs perturb the expression of many genes in the cells that are exposed to them. These gene expression changes can be divided into effects resulting from engaging the intended target and effects resulting from engaging unintended targets. For antisense oligonucleotides, developments in bioinformatics algorithms, and the quality of sequence databases, allow oligonucleotide sequences to be analyzed computationally, in terms of the predictability of their interactions with intended and unintended RNA targets. Applying these tools enables selection of sequence-specific oligonucleotides where no- or only few unintended RNA targets are expected. To evaluate oligonucleotide sequence-specificity experimentally, we recommend a transcriptomics protocol where two or more oligonucleotides targeting the same RNA molecule, but with entirely different sequences, are evaluated together. This helps to clarify which changes in cellular RNA levels result from downstream processes of engaging the intended target, and which are likely to be related to engaging unintended targets. As required for all classes of drugs, the toxic potential of oligonucleotides must be evaluated in cell- and animal models before clinical testing. Since potential adverse effects related to unintended targeting are sequence-dependent and therefore species-specific, in vitro toxicology assays in human cells are especially relevant in oligonucleotide drug discovery. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  14. Refinement of light-responsive transcript lists using rice oligonucleotide arrays: evaluation of gene-redundancy.

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    Ki-Hong Jung

    Full Text Available 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 biological significance of the data by analyzing sources of variation and confirming expression trends with reverse transcription polymerase chain reaction. We examined trends in the data by evaluating enrichment of gene ontology terms at multiple false discovery rate thresholds. To compare data generated with the NSF45K array with published results, we developed publicly available, web-based tools (www.ricearray.org. The Oligo and EST Anatomy Viewer enables visualization of EST-based expression profiling data for all genes on the array. The Rice Multi-platform Microarray Search Tool facilitates comparison of gene expression profiles across multiple rice microarray platforms. Finally, we incorporated gene expression and biochemical pathway data to reduce the number of candidate gene products putatively participating in the eight steps of the photorespiration pathway from 52 to 10, based on expression levels of putatively functionally redundant genes. We confirmed the efficacy of this method to cope with redundancy by correctly predicting participation in photorespiration of a gene with five paralogs. Applying these methods will accelerate rice functional genomics.

  15. Strategies in the preparation of DNA oligonucleotide arrays for diagnostic applications.

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    Beaucage, S L

    2001-08-01

    This report emphasizes the interfacial chemistry that is required to ensure proper attachment of oligonucleotides onto the surface of microarrays. For example, strategies for the covalent attachment of pre-synthesized oligonucleotides to glass slides, gold films, polyacrylamide gel pads, polypyrrole films, and optical fibers are surveyed in an attempt to better define the parameters for optimal formation and detection of DNA hybrids. These parameters include among others, the nature and length of the linkers attaching oligonucleotides to the arrays, and the surface density of oligonucleotides required for unhindered hybridization with DNA targets. Sensitive detection methods such as the use of light-scattering techniques, molecular beacons, surface plasmon resonance, attenuated total internal reflection-FTIR, and the evanescent field excitation of fluorescence from surface-bound fluorophores have been developed to study the kinetics and specificity of hybridization events. Finally, the synthesis of oligonucleotides directly on glass surfaces and polypropylene sheets has been investigated to enable DNA sequencing by hybridization and achieve oligonucleotide densities of ca. 10(6) sequences per cm(2) on DNA chips.

  16. Membrane-based oligonucleotide array developed from multiple markers for the detection of many Phytophthora species.

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    Chen, Wen; Djama, Zeinab Robleh; Coffey, Michael D; Martin, Frank N; Bilodeau, Guillaume J; Radmer, Lorien; Denton, Geoff; Lévesque, C André

    2013-01-01

    Most Phytophthora spp. are destructive plant pathogens; therefore, effective monitoring and accurate early detection are important means of preventing potential epidemics and outbreaks of diseases. In the current study, a membrane-based oligonucleotide array was developed that can detect Phytophthora spp. reliably using three DNA regions; namely, the internal transcribed spacer (ITS), the 5' end of cytochrome c oxidase 1 gene (cox1), and the intergenic region between cytochrome c oxidase 2 gene (cox2) and cox1 (cox2-1 spacer). Each sequence data set contained ≈250 sequences representing 98 described and 15 undescribed species of Phytophthora. The array was validated with 143 pure cultures and 35 field samples. Together, nonrejected oligonucleotides from all three markers have the ability to reliably detect 82 described and 8 undescribed Phytophthora spp., including several quarantine or regulated pathogens such as Phytophthora ramorum. Our results showed that a DNA array containing signature oligonucleotides designed from multiple genomic regions provided robustness and redundancy for the detection and differentiation of closely related taxon groups. This array has the potential to be used as a routine diagnostic tool for Phytophthora spp. from complex environmental samples without the need for extensive growth of cultures.

  17. Comparative analysis of copy number detection by whole-genome BAC and oligonucleotide array CGH

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    Bejjani Bassem A

    2010-06-01

    Full Text Available Abstract Background Microarray-based comparative genomic hybridization (aCGH is a powerful diagnostic tool for the detection of DNA copy number gains and losses associated with chromosome abnormalities, many of which are below the resolution of conventional chromosome analysis. It has been presumed that whole-genome oligonucleotide (oligo arrays identify more clinically significant copy-number abnormalities than whole-genome bacterial artificial chromosome (BAC arrays, yet this has not been systematically studied in a clinical diagnostic setting. Results To determine the difference in detection rate between similarly designed BAC and oligo arrays, we developed whole-genome BAC and oligonucleotide microarrays and validated them in a side-by-side comparison of 466 consecutive clinical specimens submitted to our laboratory for aCGH. Of the 466 cases studied, 67 (14.3% had a copy-number imbalance of potential clinical significance detectable by the whole-genome BAC array, and 73 (15.6% had a copy-number imbalance of potential clinical significance detectable by the whole-genome oligo array. However, because both platforms identified copy number variants of unclear clinical significance, we designed a systematic method for the interpretation of copy number alterations and tested an additional 3,443 cases by BAC array and 3,096 cases by oligo array. Of those cases tested on the BAC array, 17.6% were found to have a copy-number abnormality of potential clinical significance, whereas the detection rate increased to 22.5% for the cases tested by oligo array. In addition, we validated the oligo array for detection of mosaicism and found that it could routinely detect mosaicism at levels of 30% and greater. Conclusions Although BAC arrays have faster turnaround times, the increased detection rate of oligo arrays makes them attractive for clinical cytogenetic testing.

  18. Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array

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    Shiu Lin-Yi

    2011-04-01

    Full Text Available Abstract Background Airborne fungi play an important role in causing allergy and infections in susceptible people. Identification of these fungi, based on morphological characteristics, is time-consuming, expertise-demanding, and could be inaccurate. Methods We developed an oligonucleotide array that could accurately identify 21 important airborne fungi (13 genera that may cause adverse health problems. The method consisted of PCR amplification of the internal transcribed spacer (ITS regions, hybridization of the PCR products to a panel of oligonucleotide probes immobilized on a nylon membrane, and detection of the hybridization signals with alkaline phosphatase-conjugated antibodies. Results A collection of 72 target and 66 nontarget reference strains were analyzed by the array. Both the sensitivity and specificity of the array were 100%, and the detection limit was 10 pg of genomic DNA per assay. Furthermore, 70 fungal isolates recovered from air samples were identified by the array and the identification results were confirmed by sequencing of the ITS and D1/D2 domain of the large-subunit RNA gene. The sensitivity and specificity of the array for identification of the air isolates was 100% (26/26 and 97.7% (43/44, respectively. Conclusions Identification of airborne fungi by the array was cheap and accurate. The current array may contribute to decipher the relationship between airborne fungi and adverse health effect.

  19. Rapid identification of allergenic and pathogenic molds in environmental air by an oligonucleotide array

    OpenAIRE

    2011-01-01

    Abstract Background Airborne fungi play an important role in causing allergy and infections in susceptible people. Identification of these fungi, based on morphological characteristics, is time-consuming, expertise-demanding, and could be inaccurate. Methods We developed an oligonucleotide array that could accurately identify 21 important airborne fungi (13 genera) that may cause adverse health problems. The method consisted of PCR amplification of the internal transcribed spacer (ITS) region...

  20. Discovery and development of the G-rich oligonucleotide AS1411 as a novel treatment for cancer.

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    Bates, Paula J; Laber, Damian A; Miller, Donald M; Thomas, Shelia D; Trent, John O

    2009-06-01

    Certain guanine-rich (G-rich) DNA and RNA molecules can associate intermolecularly or intramolecularly to form four stranded or "quadruplex" structures, which have unusual biophysical and biological properties. Several synthetic G-rich quadruplex-forming oligodeoxynucleotides have recently been investigated as therapeutic agents for various human diseases. We refer to these biologically active G-rich oligonucleotides as aptamers because their activities arise from binding to protein targets via shape-specific recognition (analogous to antibody-antigen binding). As therapeutic agents, the G-rich aptamers may have some advantages over monoclonal antibodies and other oligonucleotide-based approaches. For example, quadruplex oligonucleotides are non-immunogenic, heat stable and they have increased resistance to serum nucleases and enhanced cellular uptake compared to unstructured sequences. In this review, we describe the characteristics and activities of G-rich oligonucleotides. We also give a personal perspective on the discovery and development of AS1411, an antiproliferative G-rich phosphodiester oligonucleotide that is currently being tested as an anticancer agent in Phase II clinical trials. This molecule functions as an aptamer to nucleolin, a multifunctional protein that is highly expressed by cancer cells, both intracellularly and on the cell surface. Thus, the serendipitous discovery of the G-rich oligonucleotides also led to the identification of nucleolin as a new molecular target for cancer therapy.

  1. chipD: a web tool to design oligonucleotide probes for high-density tiling arrays

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    Dufour, Yann S.; Wesenberg, Gary E.; Tritt, Andrew J.; Glasner, Jeremy D.; Perna, Nicole T.; Mitchell, Julie C.; Donohue, Timothy J.

    2010-01-01

    chipD is a web server that facilitates design of DNA oligonucleotide probes for high-density tiling arrays, which can be used in a number of genomic applications such as ChIP-chip or gene-expression profiling. The server implements a probe selection algorithm that takes as an input, in addition to the target sequences, a set of parameters that allow probe design to be tailored to specific applications, protocols or the array manufacturer’s requirements. The algorithm optimizes probes to meet three objectives: (i) probes should be specific; (ii) probes should have similar thermodynamic properties; and (iii) the target sequence coverage should be homogeneous and avoid significant gaps. The output provides in a text format, the list of probe sequences with their genomic locations, targeted strands and hybridization characteristics. chipD has been used successfully to design tiling arrays for bacteria and yeast. chipD is available at http://chipd.uwbacter.org/. PMID:20529880

  2. Rapid and specific detection of Lassa virus by reverse transcription-PCR coupled with oligonucleotide array hybridization.

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    Olschläger, Stephan; Günther, Stephan

    2012-07-01

    To facilitate sequence-specific detection of DNA amplified in a diagnostic reverse transcription (RT)-PCR for Lassa virus, we developed an array featuring 47 oligonucleotide probes for post-PCR hybridization of the amplicons. The array procedure may be performed with low-tech equipment and does not take longer than agarose gel detection.

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

    DEFF Research Database (Denmark)

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

    2003-01-01

    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 ......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...

  4. A statistical multiprobe model for analyzing cis and trans genes in genetical genomics experiments with short-oligonucleotide arrays

    NARCIS (Netherlands)

    Alberts, Rudi; Terpstra, Peter; Bystrykh, Leonid V.; Haan, Gerald de; Jansen, Ritsert C.

    2005-01-01

    Short-oligonucleotide arrays typically contain multiple probes per gene. In genetical genomics applications a statistical model for the individual probe signals can help in separating ‘‘true’’ differential mRNA expression from ‘‘ghost’’ effects caused by polymorphisms, misdesigned probes, and batch

  5. A sensitive method to detect the hepatitis B virus mutations by using solid phase PCR on oligonucleotide array

    Institute of Scientific and Technical Information of China (English)

    YAN QIN LU; JIN XIANG HAN; ZHONG LIN SHEN; CHUAN XI WANG

    2006-01-01

    A sensitive method based on solid phase PCR on oligonucleotide array was established to detect two point mutations 1896G-A and 1901G-A in hepatitis B virus (HBV) DNA, in which 6 probes including these two point mutations were immobilized on modified glass slides through 5' terminal linker,while the 3' terminal was made to be free. The mutated loci were designed to locate on the last nucleotides of 3' terminal respectively, and the positive control probes lacked the last nucleotide of 3' terminal in comparison with the probes used for detection. Probes fixed on oligonucleotide array were also the solid phase amplification primers. One pair of liquid primers was used to amplify the short template product from whole HBV DNA. Using target DNA as template, the solid primers were extended under the action of Taq DNA polymerase and incorporated with Cy-3dCTP as marker. During the thermal cycling reaction,probes served as solid phase amplification primers and amplification products bound to the oligonucleotide array, which could be visualized by incorporation with fluorescent dyes. After amplification, the oligonucleotide array was washed, performed with laser scanning, and then used for quantitative analysis to obtain the information for mutations. The hybridization results were compared with DNA sequencing. It was demonstrated that in case of sample A, the ratios of fluorescence intensities in wide type and in the muin these two loci. These results correlated to those obtained from DNA sequencing analysis in which the fluorescence intensity ratios in wide type and in the mutated types of 1996G-A and 1901D-A mutations in using solid phase PCR based on oligonucleotide array appears to be a sensitive and promising way to detect mutations with low-density.

  6. Array-Based Discovery of Aptamer Pairs

    Science.gov (United States)

    2014-12-11

    18460−18465. (25) Liu, Y.; Adams, J. D.; Turner, K.; Cochran, F. V.; Gambhir, S. S.; Soh, H. T. Lab Chip 2009, 9, 1033−1036. (26) Katilius, E.; Flores, C...discovery of aptamer pairs. We use microfluidic selection and high-throughput sequencing to obtain an enriched pool of aptamer sequences. Next, we...overcomes this problem to achieve efficient discovery of aptamer pairs. We use microfluidic selection and high- throughput sequencing to obtain an

  7. Complete genome sequence of Treponema pallidum ssp. pallidum strain SS14 determined with oligonucleotide arrays

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    Sodergren Erica

    2008-05-01

    Full Text Available 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 resulting sequence were filled with targeted dideoxy-terminators (DDT sequencing and the sequence was confirmed by whole genome fingerprinting (WGF. When compared to the Nichols strain, 327 single nucleotide substitutions (224 transitions, 103 transversions, 14 deletions, and 18 insertions were found. On the proteome level, the highest frequency of amino acid-altering substitution polymorphisms was in novel genes, while the lowest was in housekeeping genes, as expected by their evolutionary conservation. Evidence was also found for hypervariable regions and multiple regions showing intrastrain heterogeneity in the T. pallidum chromosome. Conclusion The observed genetic changes do not have influence on the ability of Treponema pallidum to cause syphilitic infection, since both SS14 and Nichols are virulent in rabbit. However, this is the first assessment of the degree of variation between the two syphilis pathogens and paves the way for phylogenetic studies of this fascinating organism.

  8. Evaluation of methods for oligonucleotide array data via quantitative real-time PCR

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    Morris Daryl E

    2006-01-01

    Full Text Available Abstract Background There are currently many different methods for processing and summarizing probe-level data from Affymetrix oligonucleotide arrays. It is of great interest to validate these methods and identify those that are most effective. There is no single best way to do this validation, and a variety of approaches is needed. Moreover, gene expression data are collected to answer a variety of scientific questions, and the same method may not be best for all questions. Only a handful of validation studies have been done so far, most of which rely on spike-in datasets and focus on the question of detecting differential expression. Here we seek methods that excel at estimating relative expression. We evaluate methods by identifying those that give the strongest linear association between expression measurements by array and the "gold-standard" assay. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR is generally considered the "gold-standard" assay for measuring gene expression by biologists and is often used to confirm findings from microarray data. Here we use qRT-PCR measurements to validate methods for the components of processing oligo array data: background adjustment, normalization, mismatch adjustment, and probeset summary. An advantage of our approach over spike-in studies is that methods are validated on a real dataset that was collected to address a scientific question. Results We initially identify three of six popular methods that consistently produced the best agreement between oligo array and RT-PCR data for medium- and high-intensity genes. The three methods are generally known as MAS5, gcRMA, and the dChip mismatch mode. For medium- and high-intensity genes, we identified use of data from mismatch probes (as in MAS5 and dChip mismatch and a sequence-based method of background adjustment (as in gcRMA as the most important factors in methods' performances. However, we found poor reliability for methods

  9. Comparative linkage analysis and visualization of high-density oligonucleotide SNP array data

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    Smith Richard JH

    2005-02-01

    Full Text Available Abstract Background The identification of disease-associated genes using single nucleotide polymorphisms (SNPs has been increasingly reported. In particular, the Affymetrix Mapping 10 K SNP microarray platform uses one PCR primer to amplify the DNA samples and determine the genotype of more than 10,000 SNPs in the human genome. This provides the opportunity for large scale, rapid and cost-effective genotyping assays for linkage analysis. However, the analysis of such datasets is nontrivial because of the large number of markers, and visualizing the linkage scores in the context of genome maps remains less automated using the current linkage analysis software packages. For example, the haplotyping results are commonly represented in the text format. Results Here we report the development of a novel software tool called CompareLinkage for automated formatting of the Affymetrix Mapping 10 K genotype data into the "Linkage" format and the subsequent analysis with multi-point linkage software programs such as Merlin and Allegro. The new software has the ability to visualize the results for all these programs in dChip in the context of genome annotations and cytoband information. In addition we implemented a variant of the Lander-Green algorithm in the dChipLinkage module of dChip software (V1.3 to perform parametric linkage analysis and haplotyping of SNP array data. These functions are integrated with the existing modules of dChip to visualize SNP genotype data together with LOD score curves. We have analyzed three families with recessive and dominant diseases using the new software programs and the comparison results are presented and discussed. Conclusions The CompareLinkage and dChipLinkage software packages are freely available. They provide the visualization tools for high-density oligonucleotide SNP array data, as well as the automated functions for formatting SNP array data for the linkage analysis programs Merlin and Allegro and calling

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

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    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

  11. Screening the Single Nucleotide Polymorphisms in Patients with Internal Carotid Artery Stenosis by Oligonucleotide-Based Custom DNA Array

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    Kenji Nakai

    2007-01-01

    Full Text Available Early screening of individuals considered to be at risk for severe internal carotid artery (ICA stenosis is an important strategy for preventing ischemic cerebral stroke. The purpose of this study is to screening candidate single nucleotide polymorphisms (SNPs associated with severe ICA stenosis using a newly developed oligonucleotide-based custom DNA array. The subjects consisted of 47 controls and 46 patients with severe ICA stenosis (70% who underwent carotid endarterectomy (CEA. Subjects gave informed consent and we obtained samples of blood and genomic DNA. We studied 8 candidate genes: renin-angiotensin system [angiotensinogen (AGT, angiotensin II receptor type 1 (AGTR1, nitric oxide synthase 3 (NOS3]; growth factor [hepatocyte growth factor (HGF]; transgelin (SM22; cytokine [chemokine receptor 2 (CCR2]; coagulation-fibrinolysis system [5,10-methylenetetrahydrofolate reductase (MTHFR]; and plasminogen activator inhibitor 1 (PAI-1. Genotyping of candidate SNPs was done with a line probe assay (LiPA based on an oligonucleotide-based DNA array. Results: The allele frequency of PAI-1 –1965 delG (odds ratio (OR, 0.3; 95% confidence interval (CI, 0.2–0.6 and MTHFR (OR 1.3, 95% CI, 1.0–1.5 were significantly different between controls and cases with ICA stenosis by Fisher’s exact test. Multiple logistic analysis revealed that diabetes mellitus (DM, SNPs in PAI-1 –1965 delG and MTHFR were an independent risk for ICA stenosis. In conclusion, genetic factors of coagulation-fibrinolysis as well as diabetes mellitus (DM were relevant in ICA stenosis.

  12. Pathogen receptor discovery with a microfluidic human membrane protein array

    Science.gov (United States)

    Glick, Yair; Ben-Ari, Ya’ara; Drayman, Nir; Pellach, Michal; Neveu, Gregory; Boonyaratanakornkit, Jim; Avrahami, Dorit; Einav, Shirit; Oppenheim, Ariella

    2016-01-01

    The discovery of how a pathogen invades a cell requires one to determine which host cell receptors are exploited. This determination is a challenging problem because the receptor is invariably a membrane protein, which represents an Achilles heel in proteomics. We have developed a universal platform for high-throughput expression and interaction studies of membrane proteins by creating a microfluidic-based comprehensive human membrane protein array (MPA). The MPA is, to our knowledge, the first of its kind and offers a powerful alternative to conventional proteomics by enabling the simultaneous study of 2,100 membrane proteins. We characterized direct interactions of a whole nonenveloped virus (simian virus 40), as well as those of the hepatitis delta enveloped virus large form antigen, with candidate host receptors expressed on the MPA. Selected newly discovered membrane protein–pathogen interactions were validated by conventional methods, demonstrating that the MPA is an important tool for cellular receptor discovery and for understanding pathogen tropism. PMID:27044079

  13. How to decide? Different methods of calculating gene expression from short oligonucleotide array data will give different results

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    Voesenek Laurentius ACJ

    2006-03-01

    Full Text Available Abstract Background Short oligonucleotide arrays for transcript profiling have been available for several years. Generally, raw data from these arrays are analysed with the aid of the Microarray Analysis Suite or GeneChip Operating Software (MAS or GCOS from Affymetrix. Recently, more methods to analyse the raw data have become available. Ideally all these methods should come up with more or less the same results. We set out to evaluate the different methods and include work on our own data set, in order to test which method gives the most reliable results. Results Calculating gene expression with 6 different algorithms (MAS5, dChip PMMM, dChip PM, RMA, GC-RMA and PDNN using the same (Arabidopsis data, results in different calculated gene expression levels. Consequently, depending on the method used, different genes will be identified as differentially regulated. Surprisingly, there was only 27 to 36% overlap between the different methods. Furthermore, 47.5% of the genes/probe sets showed good correlation between the mismatch and perfect match intensities. Conclusion After comparing six algorithms, RMA gave the most reproducible results and showed the highest correlation coefficients with Real Time RT-PCR data on genes identified as differentially expressed by all methods. However, we were not able to verify, by Real Time RT-PCR, the microarray results for most genes that were solely calculated by RMA. Furthermore, we conclude that subtraction of the mismatch intensity from the perfect match intensity results most likely in a significant underestimation for at least 47.5% of the expression values. Not one algorithm produced significant expression values for genes present in quantities below 1 pmol. If the only purpose of the microarray experiment is to find new candidate genes, and too many genes are found, then mutual exclusion of the genes predicted by contrasting methods can be used to narrow down the list of new candidate genes by 64 to 73%.

  14. Discovery and mapping of single feature polymorphisms in wheat using Affymetrix arrays

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    Hu Shengwa

    2009-05-01

    Full Text Available Abstract Background Wheat (Triticum aestivum L. is a staple food crop worldwide. The wheat genome has not yet been sequenced due to its huge genome size (~17,000 Mb and high levels of repetitive sequences; the whole genome sequence may not be expected in the near future. Available linkage maps have low marker density due to limitation in available markers; therefore new technologies that detect genome-wide polymorphisms are still needed to discover a large number of new markers for construction of high-resolution maps. A high-resolution map is a critical tool for gene isolation, molecular breeding and genomic research. Single feature polymorphism (SFP is a new microarray-based type of marker that is detected by hybridization of DNA or cRNA to oligonucleotide probes. This study was conducted to explore the feasibility of using the Affymetrix GeneChip to discover and map SFPs in the large hexaploid wheat genome. Results Six wheat varieties of diverse origins (Ning 7840, Clark, Jagger, Encruzilhada, Chinese Spring, and Opata 85 were analyzed for significant probe by variety interactions and 396 probe sets with SFPs were identified. A subset of 164 unigenes was sequenced and 54% showed polymorphism within probes. Microarray analysis of 71 recombinant inbred lines from the cross Ning 7840/Clark identified 955 SFPs and 877 of them were mapped together with 269 simple sequence repeat markers. The SFPs were randomly distributed within a chromosome but were unevenly distributed among different genomes. The B genome had the most SFPs, and the D genome had the least. Map positions of a selected set of SFPs were validated by mapping single nucleotide polymorphism using SNaPshot and comparing with expressed sequence tags mapping data. Conclusion The Affymetrix array is a cost-effective platform for SFP discovery and SFP mapping in wheat. The new high-density map constructed in this study will be a useful tool for genetic and genomic research in wheat.

  15. Identification of cryptic microaberrations in osteosarcoma by high-definition oligonucleotide array comparative genomic hybridization.

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    Selvarajah, Shamini; Yoshimoto, Maisa; Maire, Georges; Paderova, Jana; Bayani, Jane; Squire, Jeremy A; Zielenska, Maria

    2007-11-01

    Osteosarcoma (OS) is an aggressive bone tumor characterized by complex abnormal karyotypes and a high level of genomic instability. Using high-resolution array comparative genomic hybridization (aCGH), a novel class of localized copy number variations called microaberrations has been detected. These genomic anomalies typically involve DNA imbalances affecting 700 kb to 1 Mb DNA, and are often associated with some type of genetic syndromes. Because the origin of instability in OS is poorly understood, we used aCGH to determine whether microaberrations were a characteristic of four OS cell lines: U-2 OS, HOS, MG-63, and SAOS-2. TP53 is mutated in SAOS-2, a line in which 17 microaberrations were found. In contrast, U-2 OS, which has a wild-type TP53, had only six such anomalies, the lowest incidence. A 500-kb microaberration within a region of gain at 5p15.33 in SAOS-2 was confirmed by fluorescence in situ hybridization. Significantly, this genomic location is close to the TERT gene, a region of gain in all four cell lines. To our knowledge, this is the first systematic analysis of the incidence of microaberrations in OS. The high levels of these anomalies detected suggest that the instability processes in OS that lead to a highly abnormal karyotypes may also be associated with acquisition of genomic microaberrations.

  16. Using genomic DNA-based probe-selection to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species

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    Townsend Henrik J

    2005-11-01

    Full Text Available Abstract High-density oligonucleotide (oligo arrays are a powerful tool for transcript profiling. Arrays based on GeneChip® technology are amongst the most widely used, although GeneChip® arrays are currently available for only a small number of plant and animal species. Thus, we have developed a method to improve the sensitivity of high-density oligonucleotide arrays when applied to heterologous species and tested the method by analysing the transcriptome of Brassica oleracea L., a species for which no GeneChip® array is available, using a GeneChip® array designed for Arabidopsis thaliana (L. Heynh. Genomic DNA from B. oleracea was labelled and hybridised to the ATH1-121501 GeneChip® array. Arabidopsis thaliana probe-pairs that hybridised to the B. oleracea genomic DNA on the basis of the perfect-match (PM probe signal were then selected for subsequent B. oleracea transcriptome analysis using a .cel file parser script to generate probe mask files. The transcriptional response of B. oleracea to a mineral nutrient (phosphorus; P stress was quantified using probe mask files generated for a wide range of gDNA hybridisation intensity thresholds. An example probe mask file generated with a gDNA hybridisation intensity threshold of 400 removed > 68 % of the available PM probes from the analysis but retained >96 % of available A. thaliana probe-sets. Ninety-nine of these genes were then identified as significantly regulated under P stress in B. oleracea, including the homologues of P stress responsive genes in A. thaliana. Increasing the gDNA hybridisation intensity thresholds up to 500 for probe-selection increased the sensitivity of the GeneChip® array to detect regulation of gene expression in B. oleracea under P stress by up to 13-fold. Our open-source software to create probe mask files is freely available http://affymetrix.arabidopsis.info/xspecies/ and may be used to facilitate transcriptomic analyses of a wide range of plant and animal

  17. Combinatorial arrays and parallel screening for positive electrode discovery

    Science.gov (United States)

    Spong, A. D.; Vitins, G.; Guerin, S.; Hayden, B. E.; Russell, A. E.; Owen, John R.

    Combinatorial techniques have been applied to the preparation and screening of positive electrode candidates for lithium batteries. This work describes the automated parallel synthesis of 64-electrode arrays using a Packard Multiprobe II liquid handling system. A cell was constructed with a single lithium reference-counter electrode and 64, three-millimeter-diameter working electrodes containing Li xMn 2O 4 active material, PVdF-HFP binder and carbon black as a conducting additive. Eight duplicate electrodes, each of eight respective compositions, were deposited on the array and the mass fraction of carbon was varied in steps from 1 to 25%. The results showed a rapid increase in capacity at the percolation limit of 3% for most cells. Some groups of nominally identical cells showed random variations in capacity, especially at low carbon loadings. The overall result is a demonstration of advantages of the combinatorial concept, which were time-saving and an improved statistical significance of the results compared with on-off experiments.

  18. Evolutionary Adaptive Discovery of Phased Array Sensor Signal Identification

    Energy Technology Data Exchange (ETDEWEB)

    Timothy R. McJunkin; Milos Manic

    2011-05-01

    Tomography, used to create images of the internal properties and features of an object, from phased array ultasonics is improved through many sophisiticated methonds of post processing of data. One approach used to improve tomographic results is to prescribe the collection of more data, from different points of few so that data fusion might have a richer data set to work from. This approach can lead to rapid increase in the data needed to be stored and processed. It also does not necessarily lead to have the needed data. This article describes a novel approach to utilizing the data aquired as a basis for adapting the sensors focusing parameters to locate more precisely the features in the material: specifically, two evolutionary methods of autofocusing on a returned signal are coupled with the derivations of the forumulas for spatially locating the feature are given. Test results of the two novel methods of evolutionary based focusing (EBF) illustrate the improved signal strength and correction of the position of feature using the optimized focal timing parameters, called Focused Delay Identification (FoDI).

  19. Chromosomal Aberrations in ETV6/RUNX1-positive Childhood Acute Lymphoblastic Leukemia using 244K Oligonucleotide Array Comparative Genomic Hybridization

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    Zakaria Zubaidah

    2012-11-01

    Full Text Available Abstract Background Acute lymphoblastic leukemia (ALL is a heterogeneous form of hematological cancer consisting of various subtypes. We are interested to study the genetic aberration in precursor B-cell ALL with specific t(12;21 translocation in childhood ALL patients. A high resolution 244K array-based Comparative Genomic Hybridization (array-CGH was used to study eleven ETV6/RUNX1-positive childhood acute lymphoblastic leukemia (ALL patients. Result 155 chromosomal aberrations (119 losses, 36 gains were reported in the array findings, corresponding to 76.8% deletions and 23.2% amplifications. The ETV6 gene deletion occurred in 4 of the patients, corresponding to 45% of the sample. The most common alterations above 1 Mb were deletion 6q (13%, 12p (12% and 9p (8%, and duplication 4q (6% and Xq (4%. Other genes important in ALL were also identified in this study including RUNX1, CDKN2A, FHIT, and PAX5. The array-CGH technique was able to detect microdeletion as small as 400 bp. Conclusion The results demonstrate the usefulness of high resolution array-CGH as a complementary tool in the investigation of ALL.

  20. Discovery of novel variants in genotyping arrays improves genotype retention and reduces ascertainment bias

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    Didion John P

    2012-01-01

    Full Text Available Abstract Background High-density genotyping arrays that measure hybridization of genomic DNA fragments to allele-specific oligonucleotide probes are widely used to genotype single nucleotide polymorphisms (SNPs in genetic studies, including human genome-wide association studies. Hybridization intensities are converted to genotype calls by clustering algorithms that assign each sample to a genotype class at each SNP. Data for SNP probes that do not conform to the expected pattern of clustering are often discarded, contributing to ascertainment bias and resulting in lost information - as much as 50% in a recent genome-wide association study in dogs. Results We identified atypical patterns of hybridization intensities that were highly reproducible and demonstrated that these patterns represent genetic variants that were not accounted for in the design of the array platform. We characterized variable intensity oligonucleotide (VINO probes that display such patterns and are found in all hybridization-based genotyping platforms, including those developed for human, dog, cattle, and mouse. When recognized and properly interpreted, VINOs recovered a substantial fraction of discarded probes and counteracted SNP ascertainment bias. We developed software (MouseDivGeno that identifies VINOs and improves the accuracy of genotype calling. MouseDivGeno produced highly concordant genotype calls when compared with other methods but it uniquely identified more than 786000 VINOs in 351 mouse samples. We used whole-genome sequence from 14 mouse strains to confirm the presence of novel variants explaining 28000 VINOs in those strains. We also identified VINOs in human HapMap 3 samples, many of which were specific to an African population. Incorporating VINOs in phylogenetic analyses substantially improved the accuracy of a Mus species tree and local haplotype assignment in laboratory mouse strains. Conclusion The problems of ascertainment bias and missing

  1. Discovery of novel variants in genotyping arrays improves genotype retention and reduces ascertainment bias.

    Science.gov (United States)

    Didion, John P; Yang, Hyuna; Sheppard, Keith; Fu, Chen-Ping; McMillan, Leonard; de Villena, Fernando Pardo-Manuel; Churchill, Gary A

    2012-01-19

    High-density genotyping arrays that measure hybridization of genomic DNA fragments to allele-specific oligonucleotide probes are widely used to genotype single nucleotide polymorphisms (SNPs) in genetic studies, including human genome-wide association studies. Hybridization intensities are converted to genotype calls by clustering algorithms that assign each sample to a genotype class at each SNP. Data for SNP probes that do not conform to the expected pattern of clustering are often discarded, contributing to ascertainment bias and resulting in lost information - as much as 50% in a recent genome-wide association study in dogs. We identified atypical patterns of hybridization intensities that were highly reproducible and demonstrated that these patterns represent genetic variants that were not accounted for in the design of the array platform. We characterized variable intensity oligonucleotide (VINO) probes that display such patterns and are found in all hybridization-based genotyping platforms, including those developed for human, dog, cattle, and mouse. When recognized and properly interpreted, VINOs recovered a substantial fraction of discarded probes and counteracted SNP ascertainment bias. We developed software (MouseDivGeno) that identifies VINOs and improves the accuracy of genotype calling. MouseDivGeno produced highly concordant genotype calls when compared with other methods but it uniquely identified more than 786000 VINOs in 351 mouse samples. We used whole-genome sequence from 14 mouse strains to confirm the presence of novel variants explaining 28000 VINOs in those strains. We also identified VINOs in human HapMap 3 samples, many of which were specific to an African population. Incorporating VINOs in phylogenetic analyses substantially improved the accuracy of a Mus species tree and local haplotype assignment in laboratory mouse strains. The problems of ascertainment bias and missing information due to genotyping errors are widely recognized as

  2. A TMA de-arraying method for high throughput biomarker discovery in tissue research.

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    Yinhai Wang

    Full Text Available BACKGROUND: Tissue MicroArrays (TMAs represent a potential high-throughput platform for the analysis and discovery of tissue biomarkers. As TMA slides are produced manually and subject to processing and sectioning artefacts, the layout of TMA cores on the final slide and subsequent digital scan (TMA digital slide is often disturbed making it difficult to associate cores with their original position in the planned TMA map. Additionally, the individual cores can be greatly altered and contain numerous irregularities such as missing cores, grid rotation and stretching. These factors demand the development of a robust method for de-arraying TMAs which identifies each TMA core, and assigns them to their appropriate coordinates on the constructed TMA slide. METHODOLOGY: This study presents a robust TMA de-arraying method consisting of three functional phases: TMA core segmentation, gridding and mapping. The segmentation of TMA cores uses a set of morphological operations to identify each TMA core. Gridding then utilises a Delaunay Triangulation based method to find the row and column indices of each TMA core. Finally, mapping correlates each TMA core from a high resolution TMA whole slide image with its name within a TMAMap. CONCLUSION: This study describes a genuine robust TMA de-arraying algorithm for the rapid identification of TMA cores from digital slides. The result of this de-arraying algorithm allows the easy partition of each TMA core for further processing. Based on a test group of 19 TMA slides (3129 cores, 99.84% of cores were segmented successfully, 99.81% of cores were gridded correctly and 99.96% of cores were mapped with their correct names via TMAMaps. The gridding of TMA cores were also extensively tested using a set of 113 pseudo slide (13,536 cores with a variety of irregular grid layouts including missing cores, rotation and stretching. 100% of the cores were gridded correctly.

  3. A TMA de-arraying method for high throughput biomarker discovery in tissue research.

    Science.gov (United States)

    Wang, Yinhai; Savage, Kienan; Grills, Claire; McCavigan, Andrena; James, Jacqueline A; Fennell, Dean A; Hamilton, Peter W

    2011-01-01

    Tissue MicroArrays (TMAs) represent a potential high-throughput platform for the analysis and discovery of tissue biomarkers. As TMA slides are produced manually and subject to processing and sectioning artefacts, the layout of TMA cores on the final slide and subsequent digital scan (TMA digital slide) is often disturbed making it difficult to associate cores with their original position in the planned TMA map. Additionally, the individual cores can be greatly altered and contain numerous irregularities such as missing cores, grid rotation and stretching. These factors demand the development of a robust method for de-arraying TMAs which identifies each TMA core, and assigns them to their appropriate coordinates on the constructed TMA slide. This study presents a robust TMA de-arraying method consisting of three functional phases: TMA core segmentation, gridding and mapping. The segmentation of TMA cores uses a set of morphological operations to identify each TMA core. Gridding then utilises a Delaunay Triangulation based method to find the row and column indices of each TMA core. Finally, mapping correlates each TMA core from a high resolution TMA whole slide image with its name within a TMAMap. This study describes a genuine robust TMA de-arraying algorithm for the rapid identification of TMA cores from digital slides. The result of this de-arraying algorithm allows the easy partition of each TMA core for further processing. Based on a test group of 19 TMA slides (3129 cores), 99.84% of cores were segmented successfully, 99.81% of cores were gridded correctly and 99.96% of cores were mapped with their correct names via TMAMaps. The gridding of TMA cores were also extensively tested using a set of 113 pseudo slide (13,536 cores) with a variety of irregular grid layouts including missing cores, rotation and stretching. 100% of the cores were gridded correctly.

  4. Discovery of gigantic molecular nanostructures using a flow reaction array as a search engine.

    Science.gov (United States)

    Zang, Hong-Ying; de la Oliva, Andreu Ruiz; Miras, Haralampos N; Long, De-Liang; McBurney, Roy T; Cronin, Leroy

    2014-04-28

    The discovery of gigantic molecular nanostructures like coordination and polyoxometalate clusters is extremely time-consuming since a vast combinatorial space needs to be searched, and even a systematic and exhaustive exploration of the available synthetic parameters relies on a great deal of serendipity. Here we present a synthetic methodology that combines a flow reaction array and algorithmic control to give a chemical 'real-space' search engine leading to the discovery and isolation of a range of new molecular nanoclusters based on [Mo(2)O(2)S(2)](2+)-based building blocks with either fourfold (C4) or fivefold (C5) symmetry templates and linkers. This engine leads us to isolate six new nanoscale cluster compounds: 1, {Mo(10)(C5)}; 2, {Mo(14)(C4)4(C5)2}; 3, {Mo(60)(C4)10}; 4, {Mo(48)(C4)6}; 5, {Mo(34)(C4)4}; 6, {Mo(18)(C4)9}; in only 200 automated experiments from a parameter space spanning ~5 million possible combinations.

  5. Discovery of gigantic molecular nanostructures using a flow reaction array as a search engine

    Science.gov (United States)

    Zang, Hong-Ying; de La Oliva, Andreu Ruiz; Miras, Haralampos N.; Long, De-Liang; McBurney, Roy T.; Cronin, Leroy

    2014-04-01

    The discovery of gigantic molecular nanostructures like coordination and polyoxometalate clusters is extremely time-consuming since a vast combinatorial space needs to be searched, and even a systematic and exhaustive exploration of the available synthetic parameters relies on a great deal of serendipity. Here we present a synthetic methodology that combines a flow reaction array and algorithmic control to give a chemical ‘real-space’ search engine leading to the discovery and isolation of a range of new molecular nanoclusters based on [Mo2O2S2]2+-based building blocks with either fourfold (C4) or fivefold (C5) symmetry templates and linkers. This engine leads us to isolate six new nanoscale cluster compounds: 1, {Mo10(C5)}; 2, {Mo14(C4)4(C5)2}; 3, {Mo60(C4)10}; 4, {Mo48(C4)6}; 5, {Mo34(C4)4}; 6, {Mo18(C4)9}; in only 200 automated experiments from a parameter space spanning ~5 million possible combinations.

  6. SNP discovery and development of a high-density genotyping array for sunflower.

    Directory of Open Access Journals (Sweden)

    Eleni Bachlava

    Full Text Available Recent advances in next-generation DNA sequencing technologies have made possible the development of high-throughput SNP genotyping platforms that allow for the simultaneous interrogation of thousands of single-nucleotide polymorphisms (SNPs. Such resources have the potential to facilitate the rapid development of high-density genetic maps, and to enable genome-wide association studies as well as molecular breeding approaches in a variety of taxa. Herein, we describe the development of a SNP genotyping resource for use in sunflower (Helianthus annuus L.. This work involved the development of a reference transcriptome assembly for sunflower, the discovery of thousands of high quality SNPs based on the generation and analysis of ca. 6 Gb of transcriptome re-sequencing data derived from multiple genotypes, the selection of 10,640 SNPs for inclusion in the genotyping array, and the use of the resulting array to screen a diverse panel of sunflower accessions as well as related wild species. The results of this work revealed a high frequency of polymorphic SNPs and relatively high level of cross-species transferability. Indeed, greater than 95% of successful SNP assays revealed polymorphism, and more than 90% of these assays could be successfully transferred to related wild species. Analysis of the polymorphism data revealed patterns of genetic differentiation that were largely congruent with the evolutionary history of sunflower, though the large number of markers allowed for finer resolution than has previously been possible.

  7. SNP discovery and development of a high-density genotyping array for sunflower.

    Science.gov (United States)

    Bachlava, Eleni; Taylor, Christopher A; Tang, Shunxue; Bowers, John E; Mandel, Jennifer R; Burke, John M; Knapp, Steven J

    2012-01-01

    Recent advances in next-generation DNA sequencing technologies have made possible the development of high-throughput SNP genotyping platforms that allow for the simultaneous interrogation of thousands of single-nucleotide polymorphisms (SNPs). Such resources have the potential to facilitate the rapid development of high-density genetic maps, and to enable genome-wide association studies as well as molecular breeding approaches in a variety of taxa. Herein, we describe the development of a SNP genotyping resource for use in sunflower (Helianthus annuus L.). This work involved the development of a reference transcriptome assembly for sunflower, the discovery of thousands of high quality SNPs based on the generation and analysis of ca. 6 Gb of transcriptome re-sequencing data derived from multiple genotypes, the selection of 10,640 SNPs for inclusion in the genotyping array, and the use of the resulting array to screen a diverse panel of sunflower accessions as well as related wild species. The results of this work revealed a high frequency of polymorphic SNPs and relatively high level of cross-species transferability. Indeed, greater than 95% of successful SNP assays revealed polymorphism, and more than 90% of these assays could be successfully transferred to related wild species. Analysis of the polymorphism data revealed patterns of genetic differentiation that were largely congruent with the evolutionary history of sunflower, though the large number of markers allowed for finer resolution than has previously been possible.

  8. COLDz: Karl G. Jansky Very Large Array discovery of a gas-rich galaxy in COSMOS

    CERN Document Server

    Lentati, L; Carilli, C L; Riechers, D; Capak, P; Walter, F; Aravena, M; da Cunha, E; Hodge, J A; Ivison, R J; Smail, I; Sharon, C; Daddi, E; Decarli, R; Dickinson, M; Sargent, M; Scoville, N; Smolcic, V

    2014-01-01

    The broad spectral bandwidth at mm and cm-wavelengths provided by the recent upgrades to the Karl G. Jansky Very Large Array (VLA) has made it possible to conduct unbiased searches for molecular CO line emission at redshifts, z > 1.31. We present the discovery of a gas-rich, star-forming galaxy at z = 2.48, through the detection of CO(1-0) line emission in the COLDz survey, through a sensitive, Ka-band (31 to 39 GHz) VLA survey of a 6.5 square arcminute region of the COSMOS field. We argue that the broad line (FWHM ~570 +/- 80 km/s) is most likely to be CO(1-0) at z=2.48, as the integrated emission is spatially coincident with an infrared-detected galaxy with a photometric redshift estimate of z = 3.2 +/- 0.4. The CO(1-0) line luminosity is L'_CO = (2.2 +/- 0.3) x 10^{10} K km/s pc^2, suggesting a cold molecular gas mass of M_gas ~ (2 - 8)x10^{10}M_solar depending on the assumed value of the molecular gas mass to CO luminosity ratio alpha_CO. The estimated infrared luminosity from the (rest-frame) far-infrare...

  9. Analysis of Reverse Phase Protein Array Data: From Experimental Design towards Targeted Biomarker Discovery

    Science.gov (United States)

    Wachter, Astrid; Bernhardt, Stephan; Beissbarth, Tim; Korf, Ulrike

    2015-01-01

    Mastering the systematic analysis of tumor tissues on a large scale has long been a technical challenge for proteomics. In 2001, reverse phase protein arrays (RPPA) were added to the repertoire of existing immunoassays, which, for the first time, allowed a profiling of minute amounts of tumor lysates even after microdissection. A characteristic feature of RPPA is its outstanding sample capacity permitting the analysis of thousands of samples in parallel as a routine task. Until today, the RPPA approach has matured to a robust and highly sensitive high-throughput platform, which is ideally suited for biomarker discovery. Concomitant with technical advancements, new bioinformatic tools were developed for data normalization and data analysis as outlined in detail in this review. Furthermore, biomarker signatures obtained by different RPPA screens were compared with another or with that obtained by other proteomic formats, if possible. Options for overcoming the downside of RPPA, which is the need to steadily validate new antibody batches, will be discussed. Finally, a debate on using RPPA to advance personalized medicine will conclude this article. PMID:27600238

  10. Analysis of Reverse Phase Protein Array Data: From Experimental Design towards Targeted Biomarker Discovery

    Directory of Open Access Journals (Sweden)

    Astrid Wachter

    2015-11-01

    Full Text Available Mastering the systematic analysis of tumor tissues on a large scale has long been a technical challenge for proteomics. In 2001, reverse phase protein arrays (RPPA were added to the repertoire of existing immunoassays, which, for the first time, allowed a profiling of minute amounts of tumor lysates even after microdissection. A characteristic feature of RPPA is its outstanding sample capacity permitting the analysis of thousands of samples in parallel as a routine task. Until today, the RPPA approach has matured to a robust and highly sensitive high-throughput platform, which is ideally suited for biomarker discovery. Concomitant with technical advancements, new bioinformatic tools were developed for data normalization and data analysis as outlined in detail in this review. Furthermore, biomarker signatures obtained by different RPPA screens were compared with another or with that obtained by other proteomic formats, if possible. Options for overcoming the downside of RPPA, which is the need to steadily validate new antibody batches, will be discussed. Finally, a debate on using RPPA to advance personalized medicine will conclude this article.

  11. Molecular Mechanisms of Antisense Oligonucleotides.

    Science.gov (United States)

    Crooke, Stanley T

    2017-04-01

    In 1987, when I became interested in the notion of antisense technology, I returned to my roots in RNA biochemistry and began work to understand how oligonucleotides behave in biological systems. Since 1989, my research has focused primarily on this topic, although I have been involved in most areas of research in antisense technology. I believe that the art of excellent science is to frame large important questions that are perhaps not immediately answerable with existing knowledge and methods, and then conceive a long-term (multiyear) research strategy that begins by answering the most pressing answerable questions on the path to the long-term goals. Then, a step-by-step research pathway that will address the strategic questions posed must be implemented, adjusting the plan as new things are learned. This is the approach we have taken at Ionis. Obviously, to create antisense technology, we have had to address a wide array of strategic questions, for example, the medicinal chemistry of oligonucleotides, manufacturing and analytical methods, pharmacokinetics and toxicology, as well as questions about the molecular pharmacology of antisense oligonucleotides (ASOs). Each of these endeavors has consumed nearly three decades of scientific effort, is still very much a work-in-progress, and has resulted in hundreds of publications. As a recipient of the Lifetime Achievement Award 2016 granted by the Oligonucleotide Therapeutic Society, in this note, my goal is to summarize the contributions of my group to the efforts to understand the molecular mechanisms of ASOs.

  12. Molecular testing for familial hypercholesterolaemia-associated mutations in a UK-based cohort: development of an NGS-based method and comparison with multiplex polymerase chain reaction and oligonucleotide arrays.

    Science.gov (United States)

    Reiman, Anne; Pandey, Sarojini; Lloyd, Kate L; Dyer, Nigel; Khan, Mike; Crockard, Martin; Latten, Mark J; Watson, Tracey L; Cree, Ian A; Grammatopoulos, Dimitris K

    2016-11-01

    Background Detection of disease-associated mutations in patients with familial hypercholesterolaemia is crucial for early interventions to reduce risk of cardiovascular disease. Screening for these mutations represents a methodological challenge since more than 1200 different causal mutations in the low-density lipoprotein receptor has been identified. A number of methodological approaches have been developed for screening by clinical diagnostic laboratories. Methods Using primers targeting, the low-density lipoprotein receptor, apolipoprotein B, and proprotein convertase subtilisin/kexin type 9, we developed a novel Ion Torrent-based targeted re-sequencing method. We validated this in a West Midlands-UK small cohort of 58 patients screened in parallel with other mutation-targeting methods, such as multiplex polymerase chain reaction (Elucigene FH20), oligonucleotide arrays (Randox familial hypercholesterolaemia array) or the Illumina next-generation sequencing platform. Results In this small cohort, the next-generation sequencing method achieved excellent analytical performance characteristics and showed 100% and 89% concordance with the Randox array and the Elucigene FH20 assay. Investigation of the discrepant results identified two cases of mutation misclassification of the Elucigene FH20 multiplex polymerase chain reaction assay. A number of novel mutations not previously reported were also identified by the next-generation sequencing method. Conclusions Ion Torrent-based next-generation sequencing can deliver a suitable alternative for the molecular investigation of familial hypercholesterolaemia patients, especially when comprehensive mutation screening for rare or unknown mutations is required.

  13. Discovery of H I gas in a young radio galaxy at z = 0.44 using the Australian Square Kilometre Array Pathfinder

    NARCIS (Netherlands)

    Allison, J. R.; Sadler, E. M.; Moss, V. A.; Whiting, M. T.; Hunstead, R. W.; Pracy, M. B.; Curran, S. J.; Croom, S. M.; Glowacki, M.; Morganti, R.; Shabala, S. S.; Zwaan, M. A.; Allen, G.; Amy, S. W.; Axtens, P.; Ball, L.; Bannister, K. W.; Barker, S.; Bell, M. E.; Bock, D. C.-J.; Bolton, R.; Bowen, M.; Boyle, B.; Braun, R.; Broadhurst, S.; Brodrick, D.; Brothers, M.; Brown, A.; Bunton, J. D.; Cantrall, C.; Chapman, J.; Cheng, W.; Chippendale, A. P.; Chung, Y.; Cooray, F.; Cornwell, T.; DeBoer, D.; Diamond, P.; Edwards, P. G.; Ekers, R.; Feain, I.; Ferris, R. H.; Forsyth, R.; Gough, R.; Grancea, A.; Gupta, N.; Guzman, J. C.; Hampson, G.; Harvey-Smith, L.; Haskins, C.; Hay, S.; Hayman, D. B.; Heywood, I.; Hotan, A. W.; Hoyle, S.; Humphreys, B.; Indermuehle, B. T.; Jacka, C.; Jackson, C.; Jackson, S.; Jeganathan, K.; Johnston, S.; Joseph, J.; Kendall, R.; Kesteven, M.; Kiraly, D.; Koribalski, B. S.; Leach, M.; Lenc, E.; Lensson, E.; Mackay, S.; Macleod, A.; Marquarding, M.; Marvil, J.; McClure-Griffiths, N.; McConnell, D.; Mirtschin, P.; Norris, R. P.; Neuhold, S.; Ng, A.; O'Sullivan, J.; Pathikulangara, J.; Pearce, S.; Phillips, C.; Popping, A.; Qiao, R. Y.; Reynolds, J. E.; Roberts, P.; Sault, R. J.; Schinckel, A.; Serra, P.; Shaw, R.; Shields, M.; Shimwell, T.; Storey, M.; Sweetnam, T.; Troup, E.; Turner, B.; Tuthill, J.; Tzioumis, A.; Voronkov, M. A.; Westmeier, T.; Wilson, C. D.

    2015-01-01

    We report the discovery of a new 21-cm H I absorption system using commissioning data from the Boolardy Engineering Test Array of the Australian Square Kilometre Array Pathfinder (ASKAP). Using the 711.5-1015.5 MHz band of ASKAP we were able to conduct a blind search for the 21-cm line in a

  14. Template-Directed Ligation of Peptides to Oligonucleotides

    Science.gov (United States)

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

    1996-01-01

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

  15. Efficient high-resolution deletion discovery in Caenorhabditis elegans by array comparative genomic hybridization

    Science.gov (United States)

    Maydan, Jason S.; Flibotte, Stephane; Edgley, Mark L.; Lau, Joanne; Selzer, Rebecca R.; Richmond, Todd A.; Pofahl, Nathan J.; Thomas, James H.; Moerman, Donald G.

    2007-01-01

    We have developed array Comparative Genomic Hybridization for Caenorhabditis elegans as a means of screening for novel induced deletions in this organism. We designed three microarrays consisting of overlapping 50-mer probes to annotated exons and micro-RNAs, the first with probes to chromosomes X and II, the second with probes to chromosome II alone, and a third to the entire genome. These arrays were used to reliably detect both a large (50 kb) multigene deletion and a small (1 kb) single-gene deletion in homozygous and heterozygous samples. In one case, a deletion breakpoint was resolved to fewer than 50 bp. In an experiment designed to identify new mutations we used the X:II and II arrays to detect deletions associated with lethal mutants on chromosome II. One is an 8-kb deletion targeting the ast-1 gene on chromosome II and another is a 141-bp deletion in the gene C06A8.1. Others span large sections of the chromosome, up to >750 kb. As a further application of array Comparative Genomic Hybridization in C. elegans we used the whole-genome array to detect the extensive natural gene content variation (almost 2%) between the N2 Bristol strain and the strain CB4856, a strain isolated in Hawaii and JU258, a strain isolated in Madeira. PMID:17267812

  16. Random DNA fragmentation allows detection of single-copy, single-exon alterations of copy number by oligonucleotide array CGH in clinical FFPE samples.

    Science.gov (United States)

    Hostetter, Galen; Kim, Su Young; Savage, Stephanie; Gooden, Gerald C; Barrett, Michael; Zhang, Jian; Alla, Lalitamba; Watanabe, April; Einspahr, Janine; Prasad, Anil; Nickoloff, Brian J; Carpten, John; Trent, Jeffrey; Alberts, David; Bittner, Michael

    2010-01-01

    Genomic technologies, such as array comparative genomic hybridization (aCGH), increasingly offer definitive gene dosage profiles in clinical samples. Historically, copy number profiling was limited to large fresh-frozen tumors where intact DNA could be readily extracted. Genomic analyses of pre-neoplastic tumors and diagnostic biopsies are often limited to DNA processed by formalin-fixation and paraffin-embedding (FFPE). We present specialized protocols for DNA extraction and processing from FFPE tissues utilizing DNase processing to generate randomly fragmented DNA. The protocols are applied to FFPE clinical samples of varied tumor types, from multiple institutions and of varied block age. Direct comparative analyses with regression coefficient were calculated on split-sample (portion fresh/portion FFPE) of colorectal tumor samples. We show equal detection of a homozygous loss of SMAD4 at the exon-level in the SW480 cell line and gene-specific alterations in the split tumor samples. aCGH application to a set of archival FFPE samples of skin squamous cell carcinomas detected a novel hemizygous deletion in INPP5A on 10q26.3. Finally we present data on derivative of log ratio, a particular sensitive detector of measurement variance, for 216 sequential hybridizations to assess protocol reliability over a wide range of FFPE samples.

  17. Novel Altered Region for Biomarker Discovery in Hepatocellular Carcinoma (HCC Using Whole Genome SNP Array

    Directory of Open Access Journals (Sweden)

    Esraa M. Hashem

    2016-04-01

    Full Text Available cancer represents one of the greatest medical causes of mortality. The majority of Hepatocellular carcinoma arises from the accumulation of genetic abnormalities, and possibly induced by exterior etiological factors especially HCV and HBV infections. There is a need for new tools to analysis the large sum of data to present relevant genetic changes that may be critical for both understanding how cancers develop and determining how they could ultimately be treated. Gene expression profiling may lead to new biomarkers that may help develop diagnostic accuracy for detecting Hepatocellular carcinoma. In this work, statistical technique (discrete stationary wavelet transform for detection of copy number alternations to analysis high-density single-nucleotide polymorphism array of 30 cell lines on specific chromosomes, which are frequently detected in Hepatocellular carcinoma have been proposed. The results demonstrate the feasibility of whole-genome fine mapping of copy number alternations via high-density single-nucleotide polymorphism genotyping, Results revealed that a novel altered chromosomal region is discovered; region amplification (4q22.1 have been detected in 22 out of 30-Hepatocellular carcinoma cell lines (73%. This region strike, AFF1 and DSPP, tumor suppressor genes. This finding has not previously reported to be involved in liver carcinogenesis; it can be used to discover a new HCC biomarker, which helps in a better understanding of hepatocellular carcinoma.

  18. The delivery of therapeutic oligonucleotides.

    Science.gov (United States)

    Juliano, Rudolph L

    2016-08-19

    The oligonucleotide therapeutics field has seen remarkable progress over the last few years with the approval of the first antisense drug and with promising developments in late stage clinical trials using siRNA or splice switching oligonucleotides. However, effective delivery of oligonucleotides to their intracellular sites of action remains a major issue. This review will describe the biological basis of oligonucleotide delivery including the nature of various tissue barriers and the mechanisms of cellular uptake and intracellular trafficking of oligonucleotides. It will then examine a variety of current approaches for enhancing the delivery of oligonucleotides. This includes molecular scale targeted ligand-oligonucleotide conjugates, lipid- and polymer-based nanoparticles, antibody conjugates and small molecules that improve oligonucleotide delivery. The merits and liabilities of these approaches will be discussed in the context of the underlying basic biology. © The Author 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Single-nucleotide polymorphism discovery and validation in high-density SNP array for genetic analysis in European white oaks.

    Science.gov (United States)

    Lepoittevin, C; Bodénès, C; Chancerel, E; Villate, L; Lang, T; Lesur, I; Boury, C; Ehrenmann, F; Zelenica, D; Boland, A; Besse, C; Garnier-Géré, P; Plomion, C; Kremer, A

    2015-11-01

    An Illumina Infinium SNP genotyping array was constructed for European white oaks. Six individuals of Quercus petraea and Q. robur were considered for SNP discovery using both previously obtained Sanger sequences across 676 gene regions (1371 in vitro SNPs) and Roche 454 technology sequences from 5112 contigs (6542 putative in silico SNPs). The 7913 SNPs were genotyped across the six parental individuals, full-sib progenies (one within each species and two interspecific crosses between Q. petraea and Q. robur) and three natural populations from south-western France that included two additional interfertile white oak species (Q. pubescens and Q. pyrenaica). The genotyping success rate in mapping populations was 80.4% overall and 72.4% for polymorphic SNPs. In natural populations, these figures were lower (54.8% and 51.9%, respectively). Illumina genotype clusters with compression (shift of clusters on the normalized x-axis) were detected in ~25% of the successfully genotyped SNPs and may be due to the presence of paralogues. Compressed clusters were significantly more frequent for SNPs showing a priori incorrect Illumina genotypes, suggesting that they should be considered with caution or discarded. Altogether, these results show a high experimental error rate for the Infinium array (between 15% and 20% of SNPs potentially unreliable and 10% when excluding all compressed clusters), and recommendations are proposed when applying this type of high-throughput technique. Finally, results on diversity levels and shared polymorphisms across targeted white oaks and more distant species of the Quercus genus are discussed, and perspectives for future comparative studies are proposed.

  20. Development of a human mitochondrial oligonucleotide microarray (h-MitoArray and gene expression analysis of fibroblast cell lines from 13 patients with isolated F1Fo ATP synthase deficiency

    Directory of Open Access Journals (Sweden)

    Hansíková Hana

    2008-01-01

    Full Text Available Abstract Background To strengthen research and differential diagnostics of mitochondrial disorders, we constructed and validated an oligonucleotide microarray (h-MitoArray allowing expression analysis of 1632 human genes involved in mitochondrial biology, cell cycle regulation, signal transduction and apoptosis. Using h-MitoArray we analyzed gene expression profiles in 9 control and 13 fibroblast cell lines from patients with F1Fo ATP synthase deficiency consisting of 2 patients with mt9205ΔTA microdeletion and a genetically heterogeneous group of 11 patients with not yet characterized nuclear defects. Analysing gene expression profiles, we attempted to classify patients into expected defect specific subgroups, and subsequently reveal group specific compensatory changes, identify potential phenotype causing pathways and define candidate disease causing genes. Results Molecular studies, in combination with unsupervised clustering methods, defined three subgroups of patient cell lines – M group with mtDNA mutation and N1 and N2 groups with nuclear defect. Comparison of expression profiles and functional annotation, gene enrichment and pathway analyses of differentially expressed genes revealed in the M group a transcription profile suggestive of synchronized suppression of mitochondrial biogenesis and G1/S arrest. The N1 group showed elevated expression of complex I and reduced expression of complexes III, V, and V-type ATP synthase subunit genes, reduced expression of genes involved in phosphorylation dependent signaling along MAPK, Jak-STAT, JNK, and p38 MAP kinase pathways, signs of activated apoptosis and oxidative stress resembling phenotype of premature senescent fibroblasts. No specific functionally meaningful changes, except of signs of activated apoptosis, were detected in the N2 group. Evaluation of individual gene expression profiles confirmed already known ATP6/ATP8 defect in patients from the M group and indicated several candidate

  1. Antisense oligonucleotides in cancer.

    Science.gov (United States)

    Castanotto, Daniela; Stein, Cy A

    2014-11-01

    Over the past several dozen years, regardless of the substantial effort directed toward developing rational oligonucleotide strategies to silence gene expression, antisense oligonucleotide-based cancer therapy has not been successful. This review focuses on the most likely reasons for this lack of success, and on the barriers that still need to be overcome to make a clinical cancer treatment reality out of the promise of antisense therapy. Considerable progress has been made in the design and delivery of nucleic acid fragments. Chemical modifications have considerably improved oligonucleotide absorption, distribution and metabolism while at the same time reducing toxicity. Nevertheless, the delivery and the cellular uptake of these molecules are still not adequate to provide the desired therapeutic outcome. Recent therapeutic interventional phase III trials of antisense oligodeoxyribonucleotides for a cancer indication will be discussed, in addition to those studies that markedly improve the scientific understanding of the properties of these molecules. We still do not have a marketed antisense oligonucleotide for a cancer indication. This is because critical aspects of the cellular, tumor pharmacology and delivery properties of these agents are still not well understood.

  2. Hepatotoxic Potential of Therapeutic Oligonucleotides Can Be Predicted from Their Sequence and Modification Pattern

    Science.gov (United States)

    Hagedorn, Peter H.; Yakimov, Victor; Ottosen, Søren; Kammler, Susanne; Nielsen, Niels F.; Høg, Anja M.; Hedtjärn, Maj; Meldgaard, Michael; Møller, Marianne R.; Ørum, Henrik; Koch, Troels

    2013-01-01

    Antisense oligonucleotides that recruit RNase H and thereby cleave complementary messenger RNAs are being developed as therapeutics. Dose-dependent hepatic changes associated with hepatocyte necrosis and increases in serum alanine-aminotransferase levels have been observed after treatment with certain oligonucleotides. Although general mechanisms for drug-induced hepatic injury are known, the characteristics of oligonucleotides that determine their hepatotoxic potential are not well understood. Here, we present a comprehensive analysis of the hepatotoxic potential of locked nucleic acid-modified oligonucleotides in mice. We developed a random forests classifier, in which oligonucleotides are regarded as being composed of dinucleotide units, which distinguished between 206 oligonucleotides with high and low hepatotoxic potential with 80% accuracy as estimated by out-of-bag validation. In a validation set, 17 out of 23 oligonucleotides were correctly predicted (74% accuracy). In isolation, some dinucleotide units increase, and others decrease, the hepatotoxic potential of the oligonucleotides within which they are found. However, a complex interplay between all parts of an oligonucleotide can influence the hepatotoxic potential. Using the classifier, we demonstrate how an oligonucleotide with otherwise high hepatotoxic potential can be efficiently redesigned to abate hepatotoxic potential. These insights establish analysis of sequence and modification patterns as a powerful tool in the preclinical discovery process for oligonucleotide-based medicines. PMID:23952551

  3. An imputation approach for oligonucleotide microarrays.

    Directory of Open Access Journals (Sweden)

    Ming Li

    Full Text Available Oligonucleotide microarrays are commonly adopted for detecting and qualifying the abundance of molecules in biological samples. Analysis of microarray data starts with recording and interpreting hybridization signals from CEL images. However, many CEL images may be blemished by noises from various sources, observed as "bright spots", "dark clouds", and "shadowy circles", etc. It is crucial that these image defects are correctly identified and properly processed. Existing approaches mainly focus on detecting defect areas and removing affected intensities. In this article, we propose to use a mixed effect model for imputing the affected intensities. The proposed imputation procedure is a single-array-based approach which does not require any biological replicate or between-array normalization. We further examine its performance by using Affymetrix high-density SNP arrays. The results show that this imputation procedure significantly reduces genotyping error rates. We also discuss the necessary adjustments for its potential extension to other oligonucleotide microarrays, such as gene expression profiling. The R source code for the implementation of approach is freely available upon request.

  4. Particle-Based Microarrays of Oligonucleotides and Oligopeptides.

    Science.gov (United States)

    Nesterov-Mueller, Alexander; Maerkle, Frieder; Hahn, Lothar; Foertsch, Tobias; Schillo, Sebastian; Bykovskaya, Valentina; Sedlmayr, Martyna; Weber, Laura K; Ridder, Barbara; Soehindrijo, Miriam; Muenster, Bastian; Striffler, Jakob; Bischoff, F Ralf; Breitling, Frank; Loeffler, Felix F

    2014-10-28

    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.

  5. Particle-Based Microarrays of Oligonucleotides and Oligopeptides

    Directory of Open Access Journals (Sweden)

    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.

  6. Optimized in situ construction of oligomers on an array surface

    Science.gov (United States)

    Tolonen, Andrew C.; Albeanu, Dinu F.; Corbett, Julia F.; Handley, Heather; Henson, Charlotte; Malik, Pratap

    2002-01-01

    Oligonucleotide arrays are powerful tools to study changes in gene expression for whole genomes. These arrays can be synthesized by adapting photolithographic techniques used in microelectronics. Using this method, oligonucleotides are built base by base directly on the array surface by numerous cycles of photodeprotection and nucleotide addition. In this paper we examine strategies to reduce the number of synthesis cycles required to construct oligonucleotide arrays. By computer modeling oligonucleotide synthesis, we found that the number of required synthesis cycles could be significantly reduced by focusing upon how oligonucleotides are chosen from within genes and upon the order in which nucleotides are deposited on the array. The methods described here could provide a more efficient strategy to produce oligonucleotide arrays. PMID:12384609

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

    Science.gov (United States)

    Tripathy, Sushant

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

  8. A novel multifunctional oligonucleotide microarray for Toxoplasma gondii

    Directory of Open Access Journals (Sweden)

    Chen Feng

    2010-10-01

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

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

    Science.gov (United States)

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

    2016-01-01

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

  10. Human transcriptome array for high-throughput clinical studies.

    Science.gov (United States)

    Xu, Weihong; Seok, Junhee; Mindrinos, Michael N; Schweitzer, Anthony C; Jiang, Hui; Wilhelmy, Julie; Clark, Tyson A; Kapur, Karen; Xing, Yi; Faham, Malek; Storey, John D; Moldawer, Lyle L; Maier, Ronald V; Tompkins, Ronald G; Wong, Wing Hung; Davis, Ronald W; Xiao, Wenzhong

    2011-03-01

    A 6.9 million-feature oligonucleotide array of the human transcriptome [Glue Grant human transcriptome (GG-H array)] has been developed for high-throughput and cost-effective analyses in clinical studies. This array allows comprehensive examination of gene expression and genome-wide identification of alternative splicing as well as detection of coding SNPs and noncoding transcripts. The performance of the array was examined and compared with mRNA sequencing (RNA-Seq) results over multiple independent replicates of liver and muscle samples. Compared with RNA-Seq of 46 million uniquely mappable reads per replicate, the GG-H array is highly reproducible in estimating gene and exon abundance. Although both platforms detect similar expression changes at the gene level, the GG-H array is more sensitive at the exon level. Deeper sequencing is required to adequately cover low-abundance transcripts. The array has been implemented in a multicenter clinical program and has generated high-quality, reproducible data. Considering the clinical trial requirements of cost, sample availability, and throughput, the GG-H array has a wide range of applications. An emerging approach for large-scale clinical genomic studies is to first use RNA-Seq to the sufficient depth for the discovery of transcriptome elements relevant to the disease process followed by high-throughput and reliable screening of these elements on thousands of patient samples using custom-designed arrays.

  11. Radiolabeled oligonucleotides for antisense imaging

    Science.gov (United States)

    Iyer, Arun K; He, Jiang

    2011-01-01

    Oligonucleotides radiolabeled with isotopes emitting γ-rays (for SPECT imaging) or positrons (for PET imaging) can be useful for targeting messenger RNA (mRNA) thereby serving as non-invasive imaging tools for detection of gene expression in vivo (antisense imaging). Radiolabeled oligonucleotides may also be used for monitoring their in vivo fate, thereby helping us better understand the barriers to its delivery for antisense targeting. These developments have led to a new area of molecular imaging and targeting, utilizing radiolabeled antisense oligonucleotides. However, the success of antisense imaging relies heavily on overcoming the barriers for its targeted delivery in vivo. Furthermore, the low ability of the radiolabeled antisense oligonucleotide to subsequently internalize into the cell and hybridize with its target mRNA poses additional challenges in realizing its potentials. This review covers the advances in the antisense imaging probe development for PET and SPECT, with an emphasis on radiolabeling strategies, stability, delivery and in vivo targeting. PMID:21822406

  12. Peptide-LNA oligonucleotide conjugates

    DEFF Research Database (Denmark)

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

    2013-01-01

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

  13. Solid-phase synthesis of siRNA oligonucleotides.

    Science.gov (United States)

    Beaucage, Serge L

    2008-03-01

    Since the discovery of RNA interference (RNAi) as a means to silence the expression of specific genes, small interfering RNA (siRNA) oligonucleotides have been recognized as powerful tools for targeting therapeutically important mRNAs and eliciting their destruction. This discovery has created a high demand for synthetic oligoribonucleotides as potential therapeutics and has spurred a renaissance in the development of rapid, efficient methods for solid-phase RNA synthesis. The design and implementation of 2'-hydroxyl protecting groups that provide ribonucleoside phosphoramidites with coupling kinetics and coupling efficiencies comparable to those of deoxyribonucleoside phosphoramidites are key to the production of RNA oligonucleotides in sufficient quantity and purity for pharmaceutical applications. In this context, various siRNAs were chemically modified to identify the biophysical and biochemical parameters necessary for effective and stable RNAi-mediated gene-silencing activities.

  14. Discovery of common Asian copy number variants using integrated high-resolution array CGH and massively parallel DNA sequencing.

    Science.gov (United States)

    Park, Hansoo; Kim, Jong-Il; Ju, Young Seok; Gokcumen, Omer; Mills, Ryan E; Kim, Sheehyun; Lee, Seungbok; Suh, Dongwhan; Hong, Dongwan; Kang, Hyunseok Peter; Yoo, Yun Joo; Shin, Jong-Yeon; Kim, Hyun-Jin; Yavartanoo, Maryam; Chang, Young Wha; Ha, Jung-Sook; Chong, Wilson; Hwang, Ga-Ram; Darvishi, Katayoon; Kim, Hyeran; Yang, Song Ju; Yang, Kap-Seok; Kim, Hyungtae; Hurles, Matthew E; Scherer, Stephen W; Carter, Nigel P; Tyler-Smith, Chris; Lee, Charles; Seo, Jeong-Sun

    2010-05-01

    Copy number variants (CNVs) account for the majority of human genomic diversity in terms of base coverage. Here, we have developed and applied a new method to combine high-resolution array comparative genomic hybridization (CGH) data with whole-genome DNA sequencing data to obtain a comprehensive catalog of common CNVs in Asian individuals. The genomes of 30 individuals from three Asian populations (Korean, Chinese and Japanese) were interrogated with an ultra-high-resolution array CGH platform containing 24 million probes. Whole-genome sequencing data from a reference genome (NA10851, with 28.3x coverage) and two Asian genomes (AK1, with 27.8x coverage and AK2, with 32.0x coverage) were used to transform the relative copy number information obtained from array CGH experiments into absolute copy number values. We discovered 5,177 CNVs, of which 3,547 were putative Asian-specific CNVs. These common CNVs in Asian populations will be a useful resource for subsequent genetic studies in these populations, and the new method of calling absolute CNVs will be essential for applying CNV data to personalized medicine.

  15. Chemiluminescent detection of sequential DNA hybridizations to high-density, filter-arrayed cDNA libraries: a subtraction method for novel gene discovery.

    Science.gov (United States)

    Guiliano, D; Ganatra, M; Ware, J; Parrot, J; Daub, J; Moran, L; Brennecke, H; Foster, J M; Supali, T; Blaxter, M; Scott, A L; Williams, S A; Slatko, B E

    1999-07-01

    A chemiluminescent approach for sequential DNA hybridizations to high-density filter arrays of cDNAs, using a biotin-based random priming method followed by a streptavidin/alkaline phosphatase/CDP-Star detection protocol, is presented. The method has been applied to the Brugia malayi genome project, wherein cDNA libraries, cosmid and bacterial artificial chromosome (BAC) libraries have been gridded at high density onto nylon filters for subsequent analysis by hybridization. Individual probes and pools of rRNA probes, ribosomal protein probes and expressed sequence tag probes show correct specificity and high signal-to-noise ratios even after ten rounds of hybridization, detection, stripping of the probes from the membranes and rehybridization with additional probe sets. This approach provides a subtraction method that leads to a reduction in redundant DNA sequencing, thus increasing the rate of novel gene discovery. The method is also applicable for detecting target sequences, which are present in one or only a few copies per cell; it has proven useful for physical mapping of BAC and cosmid high-density filter arrays, wherein multiple probes have been hybridized at one time (multiplexed) and subsequently "deplexed" into individual components for specific probe localizations.

  16. Differential oligonucleotide activity in cell culture versus mouse models.

    Science.gov (United States)

    Wickstrom, E; Tyson, F L

    1997-01-01

    The usual course of drug discovery begins with the demonstration of compound activity in cells and, usually, a lower level of activity in animals. Successive rounds of drug design may result in a compound with sufficient activity in animals to justify clinical trials. The basic endpoints of therapeutic oligonucleotide experiments include target antigen reduction, target messenger reduction and inhibition of transformed cell proliferation or viral replication. However, one should expect oligonucleotides to exhibit pleiotropic behaviour, as do all other drugs. In an animal oligonucleotides will necessarily bind to and dissociate from all macromolecules encountered in the blood, in tissues, on cell surfaces and within cellular compartments. Contrary to expectations, oligonucleotides designed to be complementary to certain transcripts have sometimes been found moderately effective in cell-free extracts, more effective in cell culture and most effective in animal models. If greater potency against standard endpoints is reported in mouse models than was observed in cell culture, critical examination must consider alternate modes of action in animals that may not apply in cell culture. This counterintuitive paradox will be examined, based on studies of Ha-ras expression in bladder cancer, Ki-ras expression in pancreatic cancer, erbB2 expression in ovarian cancer and c-myc expression in B cell lymphoma.

  17. Discovery of novel antiviral agents directed against the influenza A virus nucleoprotein using photo-cross-linked chemical arrays

    Energy Technology Data Exchange (ETDEWEB)

    Hagiwara, Kyoji [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Kondoh, Yasumitsu [Chemical Biology Core Facility, RIKEN, Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Ueda, Atsushi; Yamada, Kazunori [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Medical Genome Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Goto, Hideo [Department of Microbiology and Immunology, Institute of Medical Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Watanabe, Toshiki [Department of Medical Genome Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan); Nakata, Tadashi [Department of Chemistry, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601 (Japan); Osada, Hiroyuki [Chemical Biology Core Facility, RIKEN, Advanced Science Institute, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Aida, Yoko, E-mail: aida@riken.jp [Viral Infectious Diseases Unit, RIKEN, 2-1 Hirosawa, Wako-shi, Saitama 351-0198 (Japan); Department of Medical Genome Sciences, The University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639 (Japan)

    2010-04-09

    The nucleoprotein (NP) of the influenza virus is expressed in the early stage of infection and plays important roles in numerous steps of viral replication. NP is relatively well conserved compared with viral surface spike proteins. This study experimentally demonstrates that NP is a novel target for the development of new antiviral drugs against the influenza virus. First, artificial analogs of mycalamide A in a chemical array bound specifically with high affinity to NP. Second, the compounds inhibited multiplication of the influenza virus. Furthermore, surface plasmon resonance imaging experiments demonstrated that the binding activity of each compound to NP correlated with its antiviral activity. Finally, it was shown that these compounds bound NP within the N-terminal 110-amino acid region but their binding abilities were dramatically reduced when the N-terminal 13-amino acid tail was deleted, suggesting that the compounds might bind to this region, which mediates the nuclear transport of NP and its binding to viral RNA. These data suggest that compound binding to the N-terminal 13-amino acid tail region may inhibit viral replication by inhibiting the functions of NP. Collectively, these results strongly suggest that chemical arrays are convenient tools for the screening of viral product inhibitors.

  18. Thermodynamics of Oligonucleotide Duplex Melting

    Science.gov (United States)

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-01-01

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

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

    Science.gov (United States)

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

    2014-01-01

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

  20. From understanding cellular function to novel drug discovery: the role of planar patch-clamp array chip technology

    Directory of Open Access Journals (Sweden)

    Christophe ePy

    2011-10-01

    Full Text Available All excitable cell functions rely upon ion channels that are embedded in their plasma membrane. Perturbations of ion channel structure or function result in pathologies ranging from cardiac dysfunction to neurodegenerative disorders. Consequently, to understand the functions of excitable cells and to remedy their pathophysiology, it is important to understand the ion channel functions under various experimental conditions – including exposure to novel drug targets. Glass pipette patch-clamp is the state of the art technique to monitor the intrinsic and synaptic properties of neurons. However, this technique is labor-intensive and has low data throughput. Planar patch-clamp chips, integrated into automated systems, offer high throughputs but are limited to isolated cells from suspensions, resulting in questionable models of true physiological function, and are unsuitable for studies involving neuronal communication. Multi-electrode arrays (MEA, in contrast, have the ability to monitor network activity by measuring local field potentials from multiple extracellular sites, but specific ion channel activity is challenging to extract from these multiplexed signals. Here we describe a novel planar patch-clamp chip technology that enables the simultaneous high resolution electrophysiological interrogation of individual neurons at multiple sites in synaptically connected neuronal networks, thereby combining the advantages of MEA and patch-clamp techniques. Each neuron can be probed through an aperture that connects to a dedicated subterranean microfluidic channel. Neurons growing in networks are aligned to the apertures by physisorbed or chemisorbed chemical cues. In this review, we describe the design and fabrication process of these chips, the approach to the chemical patterning for cell placement, and present physiological data from cultured neuronal cells.

  1. Improving signal intensities for genes with low-expression on oligonucleotide microarrays

    Directory of Open Access Journals (Sweden)

    Hu Limei

    2004-06-01

    Full Text Available Abstract Background DNA microarrays using long oligonucleotide probes are widely used to evaluate gene expression in biological samples. These oligonucleotides are pre-synthesized and sequence-optimized to represent specific genes with minimal cross-hybridization to homologous genes. Probe length and concentration are critical factors for signal sensitivity, particularly when genes with various expression levels are being tested. We evaluated the effects of oligonucleotide probe length and concentration on signal intensity measurements of the expression levels of genes in a target sample. Results Selected genes of various expression levels in a single cell line were hybridized to oligonucleotide arrays of four lengths and four concentrations of probes to determine how these critical parameters affected the intensity of the signal representing their expression. We found that oligonucleotides of longer length significantly increased the signals of genes with low-expression in the target. High-expressing gene signals were also boosted but to a lesser degree. Increasing the probe concentration, however, did not linearly increase the signal intensity for either low- or high-expressing genes. Conclusions We conclude that the longer the oligonuclotide probe the better the signal intensities of low expressing genes on oligonucleotide arrays.

  2. Discovery of hard-magnetic domains in two-dimensional arrays of soft-magnetic Fe{sub 3}O{sub 4} nanocubes

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Ji; Sun, Shuangshuang; Wang, Tiantian; Chen, Kezheng, E-mail: kchen@qust.edu.cn [Lab of Functional and Biomedical Nanomaterials, College of Materials Science and Engineering, Qingdao University of Science and Technology, Qingdao 266042 (China)

    2015-08-21

    In this study, abnormal hard-magnetic domains were discovered in Fe{sub 3}O{sub 4}@C composite material, in which well-ordered 16-nm-sized Fe{sub 3}O{sub 4} cubes were tightly embedded into carbon sheets of tens of nanometers thick. It was found that ca. 40 columns of Fe{sub 3}O{sub 4} nanocubes magnetically self-assembled into a single strip-type domain with perpendicular magnetic anisotropy. More strikingly, remarkable domain misalignments, which were very similar to common edge dislocations among atomic planes in crystal lattices, were clearly observed and termed as “domain dislocation” in this work. The hard-magnetic properties of Fe{sub 3}O{sub 4}@C material, including large coercivity of 2150 Oe, high M{sub R}/M{sub S} value of 0.9, and strong anisotropy energy of 3.772 × 10{sup 5} erg/cm{sup 3}, were further ascertained by carefully designed electromagnetic absorption contrast experiments. It is anticipated that the discovery of hard-magnetic domains and domain dislocations within 2-D arrays of soft-magnetic nanomaterials will shed new light on the development of high-density perpendicular magnetic recording industry.

  3. Conceptual "Heat-Driven" approach to the synthesis of DNA oligonucleotides on microarrays.

    Science.gov (United States)

    Grajkowski, A; Cieślak, J; Chmielewski, M K; Marchán, V; Phillips, L R; Wilk, A; Beaucage, S L

    2003-12-01

    The discovery of deoxyribonucleoside cyclic N-acylphosphoramidites, a novel class of phosphoramidite monomers for solid-phase oligonucleotide synthesis, has led to the development of a number of phosphate protecting groups that can be cleaved from DNA oligonucleotides under thermolytic neutral conditions. These include the 2-(N-formyl-N-methyl)aminoethyl, 4-oxopentyl, 3-(N-tert-butyl)carboxamido-1-propyl, 3-(2-pyridyl)-1-propyl, 2-[N-methyl-N-(2-pyridyl)]aminoethyl, and 4-methythiobutyl groups. When used for 5'-hydroxyl protection of nucleosides, the analogous 1-phenyl-2-[N-methyl-N-(2-pyridyl)]aminoethyloxycarbonyl group exhibited excellent thermolytic properties, which may permit an iterative "heat-driven" synthesis of DNA oligonucleotides on microarrays. In this regard, progress has been made toward the use of deoxyribonucleoside cyclic N-acylphosphoramidites in solid-phase oligonucleotide syntheses without nucleobase protection. Given that deoxyribonucleoside cyclic N-acylphosphoramidites produce oligonucleotides with heat-sensitive phosphate protecting groups, blocking the 5'-hydroxyl of these monomers with, for example, the thermolabile 1-phenyl-2-[N-methyl-N-(2-pyridyl)]aminoethyloxycarbonyl group may provide a convenient thermo-controlled method for the synthesis of oligonucleotides on microarrays.

  4. Ultrathin oligonucleotide layers for fluorescence-based DNA sensors

    Science.gov (United States)

    Furch, M.; Ueberfeld, J.; Hartmann, Andreas; Bock, Daniel; Seeger, Stefan

    1996-11-01

    Preliminary investigations into the design of an affinity sensor using evanescent wave technology concentrate upon the means of immobilization of the receptor molecules. In this work DNA served as the selective recognition element. The molecular principle of a sequence-selective biosensor for DNA is based on a sandwich-hybridization assay wherein the analyte, a single-stranded (ss)DNA, bound specifically to both an immobilized capture probe and a dye-labeled oligonucleotide in free solution. The efficiency of the capture array depends on the density of highly organized oligonucleotides on the waveguide surface and correlates therefore directly with the specificity and the sensitivity of the sensor. In the present approach using the Langmuir- Blodgett technique cinnamoylbutylether-cellulose monolayers were transferred onto optical fibers or planar waveguides. These films served as matrices for the immobilization of biotinylated oligonucleotides via streptavidin. For the first time streptavidin was immobilized by that manner. The specificity of the streptavidin layer or the following bounded nucleic acid molecules were controlled by an enzyme- linked immunosorbent assay (ELISA). Finally, this application has also shown to be suitable for the detection of Salmonella, which is an important pathogen associated with acute gastroenteritidis and food borne diseases.

  5. Sex determination of bovine preimplantation embryos by oligonucleotide microarray.

    Science.gov (United States)

    Yang, Hua; Zhong, Fagang; Yang, Yonglin; Wang, Xinhua; Liu, Shouren; Zhu, Bin

    2013-06-01

    The aim has been to set up a rapid and accurate microarray assay using sandwich mode for sex determination of bovine preimplantation embryos. Twelve sequence-specific oligonucleotide capture probes used to discriminate 12 samples were spotted onto the aldehyde-modified glass slides by Arrayer. The 2 recognition probes used to identify coding regions of the sex-determining region of the Y chromosome gene (SRY) and β-casein (CSN2) reference gene were coupled with biotin. The assay was optimized by using genomic DNA extracted from blood samples of known sex individuals. Polymerase chain reaction (PCR) was used to amplify the fragments in the HMG box region of SRY gene and CSN2 gene with sequence-specific primers. The sex of samples was identified by detecting both the SRY and CSN2 genes simultaneously in 2 reaction cells of microarrays, with the male having SRY and CSN2 signals and the female only CSN2. The sex of 20 bovine preimplantation embryos was determined by oligonucleotide microarray. The protocol was run with a blind test that showed a 100% (82/82) specificity and accuracy in sexing of leukocytes. The bovine embryos were transferred into 20 bovine recipients, with a pregnant rate of 40% (8/20). Three calves were born at term, and 5 fetuses were miscarried. Their sexes were fully in accordance with the embryonic sex predetermination predicted by oligonucleotide microarray. This suggests that the oligonucleotide microarray method of SRY gene analysis can be used in early sex prediction of bovine embryos in breeding programs.

  6. Adaptive resolution simulation of oligonucleotides

    Science.gov (United States)

    Netz, Paulo A.; Potestio, Raffaello; Kremer, Kurt

    2016-12-01

    Nucleic acids are characterized by a complex hierarchical structure and a variety of interaction mechanisms with other molecules. These features suggest the need of multiscale simulation methods in order to grasp the relevant physical properties of deoxyribonucleic acid (DNA) and RNA using in silico experiments. Here we report an implementation of a dual-resolution modeling of a DNA oligonucleotide in physiological conditions; in the presented setup only the nucleotide molecule and the solvent and ions in its proximity are described at the atomistic level; in contrast, the water molecules and ions far from the DNA are represented as computationally less expensive coarse-grained particles. Through the analysis of several structural and dynamical parameters, we show that this setup reliably reproduces the physical properties of the DNA molecule as observed in reference atomistic simulations. These results represent a first step towards a realistic multiscale modeling of nucleic acids and provide a quantitatively solid ground for their simulation using dual-resolution methods.

  7. The Chemistry and Biology of Oligonucleotide Conjugates

    Science.gov (United States)

    Juliano, R.L.; Ming, Xin; Nakagawa, Osamu

    2012-01-01

    CONSPECTUS Short DNA or RNA oligonucleotides have tremendous potential as therapeutic agents. Because of their ability to engage in Watson-Crick base pairing they can interact with messenger mRNA or pre-mRNA targets with high selectivity and thus offer the possibility of precise manipulation of gene expression. This possibility has engendered extensive efforts to develop oligonucleotides as drugs, with many candidates already in clinical trials. However, a major impediment to the maturation of oligonucleotide-based therapeutics is the fact that these relatively large and usually highly charged molecules have great difficulty crossing cellular membranes and thus in penetrating to their sites of action in the cytosol or nucleus. In this Account we first summarize some basic aspects of the biology of antisense and siRNA oligonucleotides and then discuss chemical conjugation as an approach to improving the intracellular delivery and therapeutic potential of these agents. Our emphasis will be on the pharmacological ramifications of oligonucleotide conjugates rather than the details of conjugation chemistry. One important approach has been conjugation with ligands designed to bind to particular receptors and thus provide specificity to the interaction of cells with oligonucleotides. Another approach has been to couple antisense or siRNA with agents such as cell penetrating peptides that are designed to provoke escape of the conjugate from intracellular vesicular compartments. Both of these approaches have enjoyed some success. However, there remains much to be learned before oligonucleotide conjugates can find an important place in human therapeutics. PMID:22353142

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

    Science.gov (United States)

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

    2010-01-01

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

  9. Injection site reactions after subcutaneous oligonucleotide therapy

    NARCIS (Netherlands)

    van Meer, L. (Leonie); M. Moerland (Matthijs); Gallagher, J. (Jolie); M.B.A. van Doorn (Martijn); E.P. Prens (Errol); A.F. Cohen; Rissmann, R. (Robert); J. Burggraaf (Jacobus)

    2016-01-01

    textabstractOligonucleotides (ONs) are short fragments of nucleic acids, currently being investigated as therapeutic agents. When administered subcutaneously (sc), ONs cause a specific local reaction originating around the injection site, such as erythema, itching, discomfort and pain, including

  10. Discovery of VHE emission towards the Carina arm region with the H.E.S.S. telescope array: HESS J1018-589

    Science.gov (United States)

    H. E. S. S. Collaboration; Abramowski, A.; Acero, F.; Aharonian, F.; Akhperjanian, A. G.; Anton, G.; Balzer, A.; Barnacka, A.; Becherini, Y.; Becker, J.; Bernlöh, K.; Birsin, E.; Biteau, J.; Bochow, A.; Boisson, C.; Bolmont, J.; Bordas, P.; Brucker, J.; Brun, F.; Brun, P.; Bulik, T.; Büsching, I.; Carrigan, S.; Casanova, S.; Cerruti, M.; Chadwick, P. M.; Charbonnier, A.; Chaves, R. C. G.; Cheesebrough, A.; Cologna, G.; Conrad, J.; Dalton, M.; Daniel, M. K.; Davids, I. D.; Degrange, B.; Deil, C.; Dickinson, H. J.; Djannati-Ataï, A.; Domainko, W.; Drury, L. O'C.; Dubus, G.; Dutson, K.; Dyks, J.; Dyrda, M.; Egberts, K.; Eger, P.; Espigat, P.; Fallon, L.; Fegan, S.; Feinstein, F.; Fernandes, M. V.; Fiasson, A.; Fontaine, G.; Förster, A.; Füßling, M.; Gallant, Y. A.; Gast, H.; Gérard, L.; Gerbig, D.; Giebels, B.; Glicenstein, J. F.; Glück, B.; Göring, D.; Häffner, S.; Hague, J. D.; Hahn, J.; Hampf, D.; Harris, J.; Hauser, M.; Heinz, S.; Heinzelmann, G.; Henri, G.; Hermann, G.; Hillert, A.; Hinton, J. A.; Hofmann, W.; Hofverberg, P.; Holler, M.; Horns, D.; Jacholkowska, A.; de Jager, O. C.; Jahn, C.; Jamrozy, M.; Jung, I.; Kastendieck, M. A.; Katarzyński, K.; Katz, U.; Kaufmann, S.; Keogh, D.; Khélifi, B.; Klochkov, D.; Klużniak, D.; Kneiske, T.; Komin, Nu.; Kosack, K.; Kossakowski, R.; Krayzel, F.; Laffon, H.; Lamanna, G.; Lenain, J.-P.; Lennarz, D.; Lohse, T.; Lopatin, A.; Lu, C.-C.; Marandon, V.; Marcowith, A.; Masbou, J.; Maxted, N.; Mayer, M.; McComb, T. J. L.; Medina, M. C.; Méhault, J.; Moderski, R.; Mohamed, M.; Moulin, E.; Naumann, C. L.; Naumann-Godo, M.; de Naurois, M.; Nedbal, D.; Nekrassov, D.; Nguyen, N.; Nicholas, B.; Niemiec, J.; Nolan, S. J.; Ohm, S.; de Oña Wilhelmi, E.; Opitz, B.; Ostrowski, M.; Oya, I.; Panter, M.; Paz Arribas, M.; Pekeur, N. W.; Pelletier, G.; Perez, J.; Petrucci, P.-O.; Peyaud, B.; Pita, S.; Pühlhofer, G.; Punch, M.; Quirrenbach, A.; Raue, M.; Rayner, S. M.; Reimer, A.; Reimer, O.; Renaud, M.; de los Reyes, R.; Rieger, F.; Ripken, J.; Rob, L.; Rosier-Lees, S.; Rowell, G.; Rudak, B.; Rulten, C. B.; Sahakian, V.; Sanchez, D. A.; Santangelo, A.; Schlickeiser, R.; Schulz, A.; Schwanke, U.; Schwarzburg, S.; Schwemmer, S.; Sheidaei, F.; Skilton, J. L.; Sol, H.; Spengler, G.; Stawarz, Ł.; Steenkamp, R.; Stegmann, C.; Stinzing, F.; Stycz, K.; Sushch, I.; Szostek, A.; Tavernet, J.-P.; Terrier, R.; Tluczykont, M.; Valerius, K.; van Eldik, C.; Vasileiadis, G.; Venter, C.; Viana, A.; Vincent, P.; Völk, H. J.; Volpe, F.; Vorobiov, S.; Vorster, M.; Wagner, S. J.; Ward, M.; White, R.; Wierzcholska, A.; Zacharias, M.; Zajczyk, A.; Zdziarski, A. A.; Zech, A.; Zechlin, H.-S.

    2012-05-01

    The Carina arm region, containing the supernova remnant SNR G284.3-1.8, the high-energy (HE; E > 100 MeV) binary 1FGL J1018.6-5856 and the energetic pulsar PSR J1016-5857 and its nebula, has been observed with the H.E.S.S. telescope array. The observational coverage of the region in very-high-energy (VHE; E > 0.1 TeV) γ-rays benefits from deep exposure (40 h) of the neighboring open cluster Westerlund 2. The observations have revealed a new extended region of VHE γ-ray emission. The new VHE source HESS J1018-589 shows a bright, point-like emission region positionally coincident with SNR G284.3-1.8 and 1FGL J1018.6-5856 and a diffuse extension towards the direction of PSR J1016-5857. A soft (Γ = 2.7 ± 0.5stat)photon index, with a differential flux at 1 TeV of N0 = (4.2 ± 1.1) × 10-13 TeV-1 cm-2 s-1 is found for the point-like source, whereas the total emission region including the diffuse emission region is well fit by a power-law function with spectral index Γ = 2.9 ± 0.4stat and differential flux at 1 TeV of N0 = (6.8 ± 1.6) × 10-13 TeV-1 cm-2 s-1. This H.E.S.S. detection motivated follow-up X-ray observations with the XMM-Newton satellite to investigate the origin of the VHE emission. The analysis of the XMM-Newton data resulted in the discovery of a bright, non-thermal point-like source (XMMU J101855.4-58564) with a photon index of Γ = 1.65 ± 0.08 in the center of SNR G284.3-1.8, and a thermal, extended emission region coincident with its bright northern filament. The characteristics of this thermal emission are used to estimate the plasma density in the region as n ≈ 0.5 cm-3 (2.9 kpc/d)2. The position of XMMU J101855.4-58564 is compatible with the position reported by the Fermi-LAT collaboration for the binary system 1FGL J1018.6-5856 and the variable Swift XRT source identified with it. The new X-ray data are used alongside archival multi-wavelength data to investigate the relationship between the VHE γ-ray emission from HESS J1018-589 and the

  11. Direct microcontact printing of oligonucleotides for biochip applications

    Directory of Open Access Journals (Sweden)

    Trévisiol E

    2005-07-01

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

  12. Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

    Directory of Open Access Journals (Sweden)

    Hongguang Sun

    2014-01-01

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

  13. Visible Genotype Sensor Array

    Directory of Open Access Journals (Sweden)

    Takashi Imai

    2008-04-01

    Full Text Available A visible sensor array system for simultaneous multiple SNP genotyping has been developed using a new plastic base with specific surface chemistry. Discrimination of SNP alleles is carried out by an allele-specific extension reaction using immobilized oligonucleotide primers. The 3’-ends of oligonucleotide primers are modified with a locked nucleic acid to enhance their efficiency in allelic discrimination. Biotin-dUTPs included in the reaction mixture are selectively incorporated into extending primer sequences and are utilized as tags for alkaline phosphatase-mediated precipitation of colored chemical substrates onto the surface of the plastic base. The visible precipitates allow immediate inspection of typing results by the naked eye and easy recording by a digital camera equipped on a commercial mobile phone. Up to four individuals can be analyzed on a single sensor array and multiple sensor arrays can be handled in a single operation. All of the reactions can be performed within one hour using conventional laboratory instruments. This visible genotype sensor array is suitable for “focused genomics” that follows “comprehensive genomics”.

  14. An oligonucleotide hybridization approach to DNA sequencing.

    Science.gov (United States)

    Khrapko, K R; Lysov YuP; Khorlyn, A A; Shick, V V; Florentiev, V L; Mirzabekov, A D

    1989-10-09

    We have proposed a DNA sequencing method based on hybridization of a DNA fragment to be sequenced with the complete set of fixed-length oligonucleotides (e.g., 4(8) = 65,536 possible 8-mers) immobilized individually as dots of a 2-D matrix [(1989) Dokl. Akad. Nauk SSSR 303, 1508-1511]. It was shown that the list of hybridizing octanucleotides is sufficient for the computer-assisted reconstruction of the structures for 80% of random-sequence fragments up to 200 bases long, based on the analysis of the octanucleotide overlapping. Here a refinement of the method and some experimental data are presented. We have performed hybridizations with oligonucleotides immobilized on a glass plate, and obtained their dissociation curves down to heptanucleotides. Other approaches, e.g., an additional hybridization of short oligonucleotides which continuously extend duplexes formed between the fragment and immobilized oligonucleotides, should considerably increase either the probability of unambiguous reconstruction, or the length of reconstructed sequences, or decrease the size of immobilized oligonucleotides.

  15. A review of statistical methods for preprocessing oligonucleotide microarrays.

    Science.gov (United States)

    Wu, Zhijin

    2009-12-01

    Microarrays have become an indispensable tool in biomedical research. This powerful technology not only makes it possible to quantify a large number of nucleic acid molecules simultaneously, but also produces data with many sources of noise. A number of preprocessing steps are therefore necessary to convert the raw data, usually in the form of hybridisation images, to measures of biological meaning that can be used in further statistical analysis. Preprocessing of oligonucleotide arrays includes image processing, background adjustment, data normalisation/transformation and sometimes summarisation when multiple probes are used to target one genomic unit. In this article, we review the issues encountered in each preprocessing step and introduce the statistical models and methods in preprocessing.

  16. Empirical evaluation of oligonucleotide probe selection for DNA microarrays.

    Directory of Open Access Journals (Sweden)

    Jennifer G Mulle

    Full Text Available DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs, and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.

  17. Empirical evaluation of oligonucleotide probe selection for DNA microarrays.

    Science.gov (United States)

    Mulle, Jennifer G; Patel, Viren C; Warren, Stephen T; Hegde, Madhuri R; Cutler, David J; Zwick, Michael E

    2010-03-29

    DNA-based microarrays are increasingly central to biomedical research. Selecting oligonucleotide sequences that will behave consistently across experiments is essential to the design, production and performance of DNA microarrays. Here our aim was to improve on probe design parameters by empirically and systematically evaluating probe performance in a multivariate context. We used experimental data from 19 array CGH hybridizations to assess the probe performance of 385,474 probes tiled in the Duchenne muscular dystrophy (DMD) region of the X chromosome. Our results demonstrate that probe melting temperature, single nucleotide polymorphisms (SNPs), and homocytosine motifs all have a strong effect on probe behavior. These findings, when incorporated into future microarray probe selection algorithms, may improve microarray performance for a wide variety of applications.

  18. Methidium intercalator inserted into synthetic oligonucleotides.

    Energy Technology Data Exchange (ETDEWEB)

    Timofeev, E. N.; Smirnov, I. P.; Haff, L. A.; Tishchenko, E. I.; Mirzabekov, A. D.; Florentiev, V. L.; Center for Mechanistic Biology and Biotechnology; Engelhardt Inst. of Molecular Biology; PerSeptive BioSystems Inc.

    1996-01-01

    A new methidium intercalator phosphoramidite has been synthesized. Methidium incorporation into an oligonucleotide during the synthesis was confirmed by UV and MALDI TOF MS data. UV melting experiments showed enhanced stability of a duplex, containing internal methidium. Methidium phosphoramidite has been synthesized and used for insertion of intercalator into the deoxyoligonucleotides.

  19. Chemosensitization by antisense oligonucleotides targeting MDM2.

    Science.gov (United States)

    Bianco, Roberto; Ciardiello, Fortunato; Tortora, Giampaolo

    2005-02-01

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

  20. Clinical potential of oligonucleotide-based therapeutics in the respiratory system.

    Science.gov (United States)

    Moschos, Sterghios A; Usher, Louise; Lindsay, Mark A

    2017-01-01

    The discovery of an ever-expanding plethora of coding and non-coding RNAs with nodal and causal roles in the regulation of lung physiology and disease is reinvigorating interest in the clinical utility of the oligonucleotide therapeutic class. This is strongly supported through recent advances in nucleic acids chemistry, synthetic oligonucleotide delivery and viral gene therapy that have succeeded in bringing to market at least three nucleic acid-based drugs. As a consequence, multiple new candidates such as RNA interference modulators, antisense, and splice switching compounds are now progressing through clinical evaluation. Here, manipulation of RNA for the treatment of lung disease is explored, with emphasis on robust pharmacological evidence aligned to the five pillars of drug development: exposure to the appropriate tissue, binding to the desired molecular target, evidence of the expected mode of action, activity in the relevant patient population and commercially viable value proposition.

  1. Guided Discoveries.

    Science.gov (United States)

    Ehrlich, Amos

    1991-01-01

    Presented are four mathematical discoveries made by students on an arithmetical function using the Fibonacci sequence. Discussed is the nature of the role of the teacher in directing the students' discovery activities. (KR)

  2. Volatility Discovery

    DEFF Research Database (Denmark)

    Dias, Gustavo Fruet; Scherrer, Cristina; Papailias, Fotis

    The price discovery literature investigates how homogenous securities traded on different markets incorporate information into prices. We take this literature one step further and investigate how these markets contribute to stochastic volatility (volatility discovery). We formally show...... that the realized measures from homogenous securities share a fractional stochastic trend, which is a combination of the price and volatility discovery measures. Furthermore, we show that volatility discovery is associated with the way that market participants process information arrival (market sensitivity...

  3. Mechanism of antisense oligonucleotide interaction with natural RNAs.

    Science.gov (United States)

    Serikov, R; Petyuk, V; Vorobijev, Y; Koval, V; Fedorova, O; Vlassov, V; Zenkova, M

    2011-08-01

    Oligonucleotides find several numbers of applications: as diagnostic probes, RT and PCR primers and antisense agents due to their ability of forming specific interactions with complementary nucleotide sequences within nucleic acids. These interactions are strongly affected by accessibility of the target sequence in the RNA structure. In the present work the mechanism of invasion of RNA structure by oligonucleotide was investigated using a model system: yeast tRNA(Phe) and oligonucleotides complementary to the 3'-part of this molecule. Kinetics of interaction of oligonucleotides with in vitro transcript of yeast tRNAPhe was studied using stopped-flow technique with fluorescence quenching detection, 5'-DABCYL labeled oligonucleotide was hybridized with 3'-fluorescein labeled tRNA(Phe). The results evidence for a four-step invasion process of the oligonucleotide-RNA complex formation. The process is initiated by formation of transition complexes with nucleotides in the T-loop and ACCA sequence. This complex formation is followed by RNA unfolding and formation of an extended heteroduplex with the oligonucleotide via strand displacement process. Computer modeling of oligonucleotide-tRNA(Phe) interaction revealed potential factors that could favor transition complexes formation and confirmed the proposed mechanism, showing the oligonucleotide to be a molecular "wedge". Our data evidence that oligonucleotide invasion into structured RNA is initiated by loop-single strand interactions, similar to the initial step of the antisense RNA-RNA interactions. The obtained results can be used for choosing efficient oligonucleotide probes.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  5. Dermal/transdermal delivery of small interfering RNA and antisense oligonucleotides- advances and hurdles.

    Science.gov (United States)

    Ita, Kevin

    2017-03-01

    A diverse array of nucleic acids has been studied by several researchers for the management of several diseases. Among these compounds, small interfering RNA and antisense oligonucleotides have attracted considerable attention. Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA while siRNAs, on the other hand, are double-stranded RNA molecules which can hybridize with a specific mRNA sequence and block the translation of numerous genes. One of the main obstacles in the dermal or transdermal delivery of these compounds is their low skin permeability. In this review, various techniques used to enhance the delivery of these molecules into or across the skin are described and in some cases, the correlation between enhanced dermal/transdermal delivery and therapeutic efficacy is highlighted. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  6. Antisense oligonucleotides in therapy for neurodegenerative disorders.

    Science.gov (United States)

    Evers, Melvin M; Toonen, Lodewijk J A; van Roon-Mom, Willeke M C

    2015-06-29

    Antisense oligonucleotides are synthetic single stranded strings of nucleic acids that bind to RNA and thereby alter or reduce expression of the target RNA. They can not only reduce expression of mutant proteins by breakdown of the targeted transcript, but also restore protein expression or modify proteins through interference with pre-mRNA splicing. There has been a recent revival of interest in the use of antisense oligonucleotides to treat several neurodegenerative disorders using different approaches to prevent disease onset or halt disease progression and the first clinical trials for spinal muscular atrophy and amyotrophic lateral sclerosis showing promising results. For these trials, intrathecal delivery is being used but direct infusion into the brain ventricles and several methods of passing the blood brain barrier after peripheral administration are also under investigation. Copyright © 2015. Published by Elsevier B.V.

  7. Electrochemical study of hepta–oligonucleotides

    Directory of Open Access Journals (Sweden)

    Zdenka Balcarova

    2010-12-01

    Full Text Available The study deals with the description and characterization of twohepta–oligonucleotides (DNA and RNA forming special structures.We studied their electrochemical behaviour by means of cyclicvoltammetry (CV and elimination voltammetry with linear scan(EVLS in combination with adsorptive stripping (AdS technique.Differences in electrochemical behaviour of hepta–deoxyribonucleotide and its RNA analog were discussed with regardto their different structures in solutions and their melting points.

  8. Abundant oligonucleotides common to most bacteria.

    Directory of Open Access Journals (Sweden)

    Colin F Davenport

    Full Text Available BACKGROUND: Bacteria show a bias in their genomic oligonucleotide composition far beyond that dictated by G+C content. Patterns of over- and underrepresented oligonucleotides carry a phylogenetic signal and are thus diagnostic for individual species. Patterns of short oligomers have been investigated by multiple groups in large numbers of bacteria genomes. However, global distributions of the most highly overrepresented mid-sized oligomers have not been assessed across all prokaryotes to date. We surveyed overrepresented mid-length oligomers across all prokaryotes and normalised for base composition and embedded oligomers using zero and second order Markov models. PRINCIPAL FINDINGS: Here we report a presumably ancient set of oligomers conserved and overrepresented in nearly all branches of prokaryotic life, including Archaea. These oligomers are either adenine rich homopurines with one to three guanine nucleosides, or homopyridimines with one to four cytosine nucleosides. They do not show a consistent preference for coding or non-coding regions or aggregate in any coding frame, implying a role in DNA structure and as polypeptide binding sites. Structural parameters indicate these oligonucleotides to be an extreme and rigid form of B-DNA prone to forming triple stranded helices under common physiological conditions. Moreover, the narrow minor grooves of these structures are recognised by DNA binding and nucleoid associated proteins such as HU. CONCLUSION: Homopurine and homopyrimidine oligomers exhibit distinct and unusual structural features and are present at high copy number in nearly all prokaryotic lineages. This fact suggests a non-neutral role of these oligonucleotides for bacterial genome organization that has been maintained throughout evolution.

  9. Synthesis and hybridization properties of inverse oligonucleotides.

    OpenAIRE

    Marangoni, M.; Van Aerschot, Arthur; Augustijns, Patrick; Rozenski, Jef; Herdewijn , Piet

    1997-01-01

    The synthesis of adenine and thymine cyclopentylethyl nucleosides is presented. This novel constrained monomeric building block is very difficult to incorporate into oligonucleotides. It was introduced in 13mer oligodeoxynucleotide sequences at a single position using H-phosphonate chemistry. Phosphoramidite chemistry completely failed in this particular case. The H-phosphonate building blocks were obtained starting from the corresponding phosphoramidites. Stability of duplexes with RNA and D...

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

    Science.gov (United States)

    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 the targeted RNA and is able to activate RNase H to cleave the RNA. The other oligonucleotide, which is complementary to the wild type allele of the targeted RNA, is able to inhibit RNase H cleavage. Five types of SNPs, C/G, G/C, G/A, A/G, and C/U, were analyzed within the sequence context of genes associated with neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, ALS (Amyotrophic Lateral Sclerosis), and Machado-Joseph disease. For most analyzed cases, the application of the tandem approach increased allele-selective RNA degradation 1.5-15 fold relative to the use of a single antisense oligonucleotide. The presented study proves that differentiation between single substitution is highly dependent on the nature of the SNP and surrounding nucleotides. These variables are crucial for determining the proper length of the inhibitor antisense oligonucleotide. In the tandem approach, the comparison of thermodynamic stability of the favorable duplexes WT RNA-inhibitor and Mut RNA-gapmer with the other possible duplexes allows for the evaluation of chances for the allele-selective degradation of RNA. A larger difference in thermodynamic stability between favorable duplexes and those that could possibly form, usually results in the better allele selectivity of RNA degradation.

  11. Molecular phylogenetics before sequences: oligonucleotide catalogs as k-mer spectra.

    Science.gov (United States)

    Ragan, Mark A; Bernard, Guillaume; Chan, Cheong Xin

    2014-01-01

    From 1971 to 1985, Carl Woese and colleagues generated oligonucleotide catalogs of 16S/18S rRNAs from more than 400 organisms. Using these incomplete and imperfect data, Carl and his colleagues developed unprecedented insights into the structure, function, and evolution of the large RNA components of the translational apparatus. They recognized a third domain of life, revealed the phylogenetic backbone of bacteria (and its limitations), delineated taxa, and explored the tempo and mode of microbial evolution. For these discoveries to have stood the test of time, oligonucleotide catalogs must carry significant phylogenetic signal; they thus bear re-examination in view of the current interest in alignment-free phylogenetics based on k-mers. Here we consider the aims, successes, and limitations of this early phase of molecular phylogenetics. We computationally generate oligonucleotide sets (e-catalogs) from 16S/18S rRNA sequences, calculate pairwise distances between them based on D 2 statistics, compute distance trees, and compare their performance against alignment-based and k-mer trees. Although the catalogs themselves were superseded by full-length sequences, this stage in the development of computational molecular biology remains instructive for us today.

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

    Directory of Open Access Journals (Sweden)

    Fairbanks Benjamin D

    2006-04-01

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

  13. Guanine-tethered antisense oligonucleotides as synthetic riboregulators.

    Science.gov (United States)

    Hagihara, Masaki

    2014-01-01

    Regulation of gene expression by short oligonucleotides (antisense oligonucleotides), which can modulate RNA structures and inhibit subsequent associations with the translation machinery, is a potential approach for gene therapy. This chapter describes an alternative antisense strategy using guanine-tethered antisense oligonucleotides (G-ASs) to introduce a DNA-RNA heteroquadruplex structure at a designated sequence on RNA targets. The feasibility of using G-ASs to modulate RNA conformation may allow control of RNA function by inducing biologically important quadruplex structures. This approach to manipulate quadruplex structures using G-ASs may expand the strategies for regulating RNA structures and the functions of short oligonucleotide riboregulators.

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

    Directory of Open Access Journals (Sweden)

    Mohammed Bakkali

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

  15. Lipid Oligonucleotide Conjugates as Responsive Material

    Science.gov (United States)

    2012-09-28

    U.S. Army Research Office P.O. Box 12211 Research Triangle Park, NC 27709-2211 15. SUBJECT TERMS Amphiphiles, oligonucleotides, lipids...peer-reviewed journals: (c) Presentations 1. Philippe Barthélémy, « Hybrid Lipids for Biomedical Applications », Targeting and Triggering Basic Research ...Steadel C. ; Pierre, N. ; Barthélémy, P. : Oligonucléotides amphiphile : Journée Scientifique de l’IFR 66, Talence, le 2 décembre 2008, France 29. Taib

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

    Directory of Open Access Journals (Sweden)

    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.

  17. In situ synthesis of DNA micro-arrays using typography technique

    Institute of Scientific and Technical Information of China (English)

    2007-01-01

    A novel typography technique was developed to in situ synthesize oligonucleotide arrays on glass slide,which has the celerity,high spatial resolution,lower cost,reliable operation,and high synthetic efficiency.The principle and process of the typography technique for fabricating gene-chips have been described in detail.A suit of poly(terafluoroethylene)devices for synthesizing oligonucleotide arrays were designed and prepared,and the fiber tubes with a number of nano-or micron-channels were em- ployed.The oligonucleotide arrays of 16 and 160 features with four different probes were synthesized using the typography technique.The four specific oligonucleotide probes including the matched and the mismatched by the fluorescent target sequence gave obviously different hybridization fluorescent signals.It was indicated that the gene-chip fabricated by the typography method could be used to rapidly screen single-nucleotide polymorphisms(SNP)and to detect mutations.

  18. In situ synthesis of DNA micro-arrays using typography technique

    Institute of Scientific and Technical Information of China (English)

    TANG JianXin; HE NongYue; LI Song

    2007-01-01

    A novel typography technique was developed to in situ synthesize oligonucleotide arrays on glass slide, which has the celerity, high spatial resolution, lower cost, reliable operation, and high synthetic efficiency. The principle and process of the typography technique for fabricating gene-chips have been described in detail. A suit of poly(terafluoroethylene) devices for synthesizing oligonucleotide arrays were designed and prepared, and the fiber tubes with a number of nano- or micron-channels were employed. The oligonucleotide arrays of 16 and 160 features with four different probes were synthesized using the typography technique. The four specific oligonucleotide probes including the matched and the mismatched by the fluorescent target sequence gave obviously different hybridization fluorescent signals. It was indicated that the gene-chip fabricated by the typography method could be used to rapidly screen single-nucleotide polymorphisms (SNP) and to detect mutations.

  19. Antisense Oligonucleotide Therapy in Diabetic Retinopathy

    Science.gov (United States)

    Hnik, Peter; Boyer, David S.; Grillone, Lisa R.; Clement, John G.; Henry, Scott P.; Green, Ellen A.

    2009-01-01

    Diabetic retinopathy is one of the leading causes of blindness in the United States and other parts of the world. Historically, laser photocoagulation and vitrectomy surgery have been used for the treatment of diabetic retinopathy, including diabetic macular edema. Both procedures have proven to be useful under certain conditions but have their limitations. New pathways and processes that promote diabetic retinopathy have been identified, and several new therapeutic approaches are under investigation. These new therapies may be beneficial in the treatment of diabetic retinopathy and include antivascular endothelial growth factor agents, corticosteroids, and therapies that may potentially target a number of additional diabetic retinopathy-related factors and processes, including antisense oligonucleotides. Second-generation antisense oligonucleotides, such as iCo-007, may offer a significant advantage in the treatment of diabetic retinopathy by downregulating the signal pathways of multiple growth factors that seem to play a critical role in the process of ocular angiogenesis and vascular leakage. Benefits of such molecules are expected to include the specificity of the kinase target and an extended half-life, resulting in less frequent intravitreal drug administration, resistance to molecule degradation, and a good safety profile. PMID:20144342

  20. Template switching between PNA and RNA oligonucleotides

    Science.gov (United States)

    Bohler, C.; Nielsen, P. E.; Orgel, L. E.; Miller, S. L. (Principal Investigator)

    1995-01-01

    The origin of the RNA world is not easily understood, as effective prebiotic syntheses of the components of RNA, the beta-ribofuranoside-5'-phosphates, are hard to envisage. Recognition of this difficulty has led to the proposal that other genetic systems, the components of which are more easily formed, may have preceded RNA. This raises the question of how transitions between one genetic system and another could occur. Peptide nucleic acid (PNA) resembles RNA in its ability to form double-helical complexes stabilized by Watson-Crick hydrogen bonding between adenine and thymine and between cytosine and guanine, but has a backbone that is held together by amide rather than by phosphodiester bonds. Oligonucleotides bases on RNA are known to act as templates that catalyse the non-enzymatic synthesis of their complements from activated mononucleotides, we now show that RNA oligonucleotides facilitate the synthesis of complementary PNA strands and vice versa. This suggests that a transition between different genetic systems can occur without loss of information.

  1. Oligonucleotides Containing Aminated 2′-Amino-LNA Nucleotides

    DEFF Research Database (Denmark)

    Lou, Chenguang; Samuelsen, Simone V.; Christensen, Niels Johan

    2017-01-01

    Mono- and diaminated 2′-amino-LNA monomers were synthesized and introduced into oligonucleotides. Each modification imparts significant stabilization of nucleic acid duplexes and triplexes, excellent sequence selectivity, and significant nuclease resistance. Molecular modeling suggested...... that structural stabilization occurs via intrastrand electrostatic attraction between the protonated amino groups of the aminated 2′-amino-LNA monomers and the host oligonucleotide backbone....

  2. Voltage-gated calcium channel and antisense oligonucleotides thereto

    Science.gov (United States)

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

    1998-01-01

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

  3. Pharmacokinetics, biodistribution and cell uptake of antisense oligonucleotides.

    Science.gov (United States)

    Geary, Richard S; Norris, Daniel; Yu, Rosie; Bennett, C Frank

    2015-06-29

    Pharmacokinetic properties of oligonucleotides are largely driven by chemistry of the backbone and thus are sequence independent within a chemical class. Tissue bioavailability (% of administered dose) is assisted by plasma protein binding that limits glomerular filtration and ultimate urinary excretion of oligonucleotides. The substitution of one non-bridging oxygen with the more hydrophobic sulfur atom (phosphorothioate) increases both plasma stability and plasma protein binding and thus, ultimately, tissue bioavailability. Additional modifications of the sugar at the 2' position, increase RNA binding affinity and significantly increase potency, tissue half-life and prolong RNA inhibitory activity. Oligonucleotides modified in this manner consistently exhibit the highest tissue bioavailability (>90%). Systemic biodistribution is broad, and organs typically with highest concentrations are liver and kidney followed by bone marrow, adipocytes, and lymph nodes. Cell uptake is predominantly mediated by endocytosis. Both size and charge for most oligonucleotides prevents distribution across the blood brain barrier. However, modified single-strand oligonucleotides administered by intrathecal injection into the CSF distribute broadly in the CNS. The majority of intracellular oligonucleotide distribution following systemic or local administration occurs rapidly in just a few hours following administration and is facilitated by rapid endocytotic uptake mechanisms. Further understanding of the intracellular trafficking of oligonucleotides may provide further enhancements in design and ultimate potency of antisense oligonucleotides in the future. Copyright © 2015. Published by Elsevier B.V.

  4. Enhanced fluorescence of silver nanoclusters stabilized with branched oligonucleotides.

    Science.gov (United States)

    Latorre, Alfonso; Lorca, Romina; Zamora, Félix; Somoza, Álvaro

    2013-05-28

    DNA stabilized silver nanoclusters (AgNCs) are promising optical materials, whose fluorescence properties can be tuned by the selection of the DNA sequence employed. In this work we have used modified oligonucleotides in the preparation of AgNCs. The fluorescent intensity obtained was 60 times higher than that achieved with standard oligonucleotides.

  5. Noncoding oligonucleotides: the belle of the ball in gene therapy.

    Science.gov (United States)

    Shum, Ka-To; Rossi, John J

    2015-01-01

    Gene therapy carries the promise of cures for many diseases based on manipulating the expression of a person's genes toward the therapeutic goal. The relevance of noncoding oligonucleotides to human disease is attracting widespread attention. Noncoding oligonucleotides are not only involved in gene regulation, but can also be modified into therapeutic tools. There are many strategies that leverage noncoding oligonucleotides for gene therapy, including small interfering RNAs, antisense oligonucleotides, aptamers, ribozymes, decoys, and bacteriophage phi 29 RNAs. In this chapter, we will provide a broad, comprehensive overview of gene therapies that use noncoding oligonucleotides for disease treatment. The mechanism and development of each therapeutic will be described, with a particular focus on its clinical development. Finally, we will discuss the challenges associated with developing nucleic acid therapeutics and the prospects for future success.

  6. Predicting oligonucleotide-directed mutagenesis failures in protein engineering.

    Science.gov (United States)

    Wassman, Christopher D; Tam, Phillip Y; Lathrop, Richard H; Weiss, Gregory A

    2004-01-01

    Protein engineering uses oligonucleotide-directed mutagenesis to modify DNA sequences through a two-step process of hybridization and enzymatic synthesis. Inefficient reactions confound attempts to introduce mutations, especially for the construction of vast combinatorial protein libraries. This paper applied computational approaches to the problem of inefficient mutagenesis. Several results implicated oligonucleotide annealing to non-target sites, termed 'cross-hybridization', as a significant contributor to mutagenesis reaction failures. Test oligonucleotides demonstrated control over reaction outcomes. A novel cross-hybridization score, quickly computable for any plasmid and oligonucleotide mixture, directly correlated with yields of deleterious mutagenesis side products. Cross-hybridization was confirmed conclusively by partial incorporation of an oligonucleotide at a predicted cross-hybridization site, and by modification of putative template secondary structure to control cross-hybridization. Even in low concentrations, cross-hybridizing species in mixtures poisoned reactions. These results provide a basis for improved mutagenesis efficiencies and increased diversities of cognate protein libraries.

  7. Large scale real-time PCR validation on gene expression measurements from two commercial long-oligonucleotide microarrays

    Directory of Open Access Journals (Sweden)

    Chan Frances

    2006-03-01

    Full Text Available Abstract Background DNA microarrays are rapidly becoming a fundamental tool in discovery-based genomic and biomedical research. However, the reliability of the microarray results is being challenged due to the existence of different technologies and non-standard methods of data analysis and interpretation. In the absence of a "gold standard"/"reference method" for the gene expression measurements, studies evaluating and comparing the performance of various microarray platforms have often yielded subjective and conflicting conclusions. To address this issue we have conducted a large scale TaqMan® Gene Expression Assay based real-time PCR experiment and used this data set as the reference to evaluate the performance of two representative commercial microarray platforms. Results In this study, we analyzed the gene expression profiles of three human tissues: brain, lung, liver and one universal human reference sample (UHR using two representative commercial long-oligonucleotide microarray platforms: (1 Applied Biosystems Human Genome Survey Microarrays (based on single-color detection; (2 Agilent Whole Human Genome Oligo Microarrays (based on two-color detection. 1,375 genes represented by both microarray platforms and spanning a wide dynamic range in gene expression levels, were selected for TaqMan® Gene Expression Assay based real-time PCR validation. For each platform, four technical replicates were performed on the same total RNA samples according to each manufacturer's standard protocols. For Agilent arrays, comparative hybridization was performed using incorporation of Cy5 for brain/lung/liver RNA and Cy3 for UHR RNA (common reference. Using the TaqMan® Gene Expression Assay based real-time PCR data set as the reference set, the performance of the two microarray platforms was evaluated focusing on the following criteria: (1 Sensitivity and accuracy in detection of expression; (2 Fold change correlation with real-time PCR data in pair

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

    Directory of Open Access Journals (Sweden)

    Jee Justin

    2008-12-01

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

  9. Cellular uptake and trafficking of antisense oligonucleotides.

    Science.gov (United States)

    Crooke, Stanley T; Wang, Shiyu; Vickers, Timothy A; Shen, Wen; Liang, Xue-Hai

    2017-03-01

    Antisense oligonucleotides (ASOs) modified with phosphorothioate (PS) linkages and different 2' modifications can be used either as drugs (e.g., to treat homozygous familial hypercholesterolemia and spinal muscular atrophy) or as research tools to alter gene expression. PS-ASOs can enter cells without additional modification or formulation and can be designed to mediate sequence-specific cleavage of different types of RNA (including mRNA and non-coding RNA) targeted by endogenous RNase H1. Although PS-ASOs function in both the cytoplasm and nucleus, localization to different subcellular regions can affect their therapeutic potency. Cellular uptake and intracellular distribution of PS ASOs are mediated by protein interactions. The main proteins involved in these processes have been identified, and intracellular sites in which PS ASOs are active, or inactive, cataloged.

  10. Conjugation of fluorescent proteins with DNA oligonucleotides.

    Science.gov (United States)

    Lapiene, Vidmantas; Kukolka, Florian; Kiko, Kathrin; Arndt, Andreas; Niemeyer, Christof M

    2010-05-19

    This work describes the synthesis of covalent ssDNA conjugates of six fluorescent proteins, ECFP, EGFP, E(2)GFP, mDsRed, Dronpa, and mCherry, which were cloned with an accessible C-terminal cystein residue to enable site-selective coupling using a heterobispecific cross-linker. The resulting conjugates revealed similar fluorescence emission intensity to the unconjugated proteins, and the functionality of the tethered oligonucleotide was proven by specific Watson-Crick base pairing to cDNA-modified gold nanoparticles. Fluorescence spectroscopy analysis indicated that the fluorescence of the FP is quenched by the gold particle, and the extent of quenching varied with the intrinsic spectroscopic properties of FP as well as with the configuration of surface attachment. Since this study demonstrates that biological fluorophores can be selectively incorporated into and optically coupled with nanoparticle-based devices, applications in DNA-based nanofabrication can be foreseen.

  11. Direct oligonucleotide-photosensitizer conjugates for photochemical delivery of antisense oligonucleotides.

    Science.gov (United States)

    Yuan, Ahu; Laing, Brian; Hu, Yiqiao; Ming, Xin

    2015-04-18

    Activation of photosensitizers in endosomes enables release of therapeutic macromolecules into the cytosol of the target cells for pharmacological actions. In this study, we demonstrate that direct conjugation of photosensitizers to oligonucleotides (ONs) allows spatial and temporal co-localization of the two modalities in the target cells, and thus leads to superior functional delivery of ONs. Further, light-activated delivery of an anticancer ON caused cancer cell killing via modulation of an oncogene and photodynamic therapy.

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

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

  14. Antisense Oligonucleotide-Based Therapy for Neuromuscular Disease.

    Science.gov (United States)

    Sardone, Valentina; Zhou, Haiyan; Muntoni, Francesco; Ferlini, Alessandra; Falzarano, Maria Sofia

    2017-04-05

    Neuromuscular disorders such as Duchenne Muscular Dystrophy and Spinal Muscular Atrophy are neurodegenerative genetic diseases characterized primarily by muscle weakness and wasting. Until recently there were no effective therapies for these conditions, but antisense oligonucleotides, a new class of synthetic single stranded molecules of nucleic acids, have demonstrated promising experimental results and are at different stages of regulatory approval. The antisense oligonucleotides can modulate the protein expression via targeting hnRNAs or mRNAs and inducing interference with splicing, mRNA degradation, or arrest of translation, finally, resulting in rescue or reduction of the target protein expression. Different classes of antisense oligonucleotides are being tested in several clinical trials, and limitations of their clinical efficacy and toxicity have been reported for some of these compounds, while more encouraging results have supported the development of others. New generation antisense oligonucleotides are also being tested in preclinical models together with specific delivery systems that could allow some of the limitations of current antisense oligonucleotides to be overcome, to improve the cell penetration, to achieve more robust target engagement, and hopefully also be associated with acceptable toxicity. This review article describes the chemical properties and molecular mechanisms of action of the antisense oligonucleotides and the therapeutic implications these compounds have in neuromuscular diseases. Current strategies and carrier systems available for the oligonucleotides delivery will be also described to provide an overview on the past, present and future of these appealing molecules.

  15. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    Science.gov (United States)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    Yue-Bei Luo

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

  18. Beyond Discovery

    DEFF Research Database (Denmark)

    Korsgaard, Steffen; Sassmannshausen, Sean Patrick

    2015-01-01

    as their central concepts and conceptualization of the entrepreneurial function. On this basis we discuss three central themes that cut across the four alternatives: process, uncertainty, and agency. These themes provide new foci for entrepreneurship research and can help to generate new research questions......In this chapter we explore four alternatives to the dominant discovery view of entrepreneurship; the development view, the construction view, the evolutionary view, and the Neo-Austrian view. We outline the main critique points of the discovery presented in these four alternatives, as well...

  19. Anti-fouling characteristics of surface-confined oligonucleotide strands bioconjugated on streptavidin platforms in the presence of nanomaterials.

    Science.gov (United States)

    Mir, Mònica; Cameron, Petra J; Zhong, Xinhua; Azzaroni, Omar; Alvarez, Marta; Knoll, Wolfgang

    2009-05-15

    This work describes our studies on the molecular design of interfacial architectures suitable for DNA sensing which could resist non-specific binding of nanomaterials commonly used as labels for amplifying biorecognition events. We observed that the non-specific binding of bio-nanomaterials to surface-confined oligonucleotide strands is highly dependent on the characteristics of the interfacial architecture. Thiolated double stranded oligonucleotide arrays assembled on Au surfaces evidence significant fouling in the presence of nanoparticles (NPs) at the nanomolar level. The non-specific interaction between the oligonucleotide strands and the nanomaterials can be sensitively minimized by introducing streptavidin (SAv) as an underlayer conjugated to the DNA arrays. The role of the SAv layer was attributed to the significant hydrophilic repulsion between the SAv-modified surface and the nanomaterials in close proximity to the interface, thus conferring outstanding anti-fouling characteristics to the interfacial architecture. These results provide a simple and straightforward strategy to overcome the limitations introduced by the non-specific binding of labels to achieve reliable detection of DNA-based biorecognition events.

  20. Optical Characterization of Oligonucleotide DNA Influenced by Magnetic Fields

    Directory of Open Access Journals (Sweden)

    Seyedeh Maryam Banihashemian

    2013-09-01

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

  1. DNA microarray synthesis by using PDMS molecular stamp (II) -- Oligonucleotide on-chip synthesis using PDMS stamp

    Institute of Scientific and Technical Information of China (English)

    2001-01-01

    Based on the standard phosphoramidites chemistry protocol, two oligonucleotides synthetic routes were studied by contact stamping reactants to a modified glass slide. Route A was a contact coupling reaction, in which a nucleoside monomer was transferred and coupled to reactive groups (OH) on a substrate by spreading the nucleoside activated with tetrazole on a polydimethylsiloxane (PDMS) stamp. Route B was a contact detritylation, in which one nucleoside was fixed on the desired synthesis regions where dimethoxytrityl (DMT) protecting groups on the 5′-hydroxyl of the support-bound nucleoside were removed by stamping trichloroacetic acid (TCA) distributed on features on a PDMS stamp. Experiments showed that the synthetic yield and the reaction speed of route A were higher than those of route B. It was shown that 20 mer oligonucleotide arrays immobilized on the glass slide were successfully synthesized using the PDMS stamps, and the coupling efficiency showed no difference between the PDMS stamping and the conventional synthesis methods.

  2. FGX : a frequentist gene expression index for Affymetrix arrays

    NARCIS (Netherlands)

    Purutçuoğlu, Vilda; Wit, Ernst

    2007-01-01

    We consider a new frequentist gene expression index for Affymetrix oligonucleotide DNA arrays, using a similar probe intensity model as suggested previously, called the Bayesian gene expression index (BGX). According to this model, the perfect match and mismatch values are assumed to be correlated a

  3. Multilocus sequence typing of Staphylococcus aureus with DNA array technology

    NARCIS (Netherlands)

    W.B. van Leeuwen (Willem); C. Jay (Corinne); S.V. Snijders (Susan); N. Durin (Nathalia); B. Lacroix (Bruno); H.A. Verbrugh (Henri); M.C. Enright (Mark); A. Troesch (Alain); A.F. van Belkum (Alex)

    2003-01-01

    textabstractA newly developed oligonucleotide array suited for multilocus sequence typing (MLST) of Staphylococcus aureus strains was analyzed with two strain collections in a two-center study. MLST allele identification for the first strain collection fully agreed with conventiona

  4. Optimized design and assessment of whole genome tiling arrays.

    NARCIS (Netherlands)

    Graf, S.; Nielsen, F.G.G.; Kurtz, S.; Huynen, M.A.; Birney, E.; Stunnenberg, H.G.; Flicek, P.

    2007-01-01

    MOTIVATION: Recent advances in microarray technologies have made it feasible to interrogate whole genomes with tiling arrays and this technique is rapidly becoming one of the most important high-throughput functional genomics assays. For large mammalian genomes, analyzing oligonucleotide tiling arra

  5. Effects of surface chemistry and size on iron oxide nanoparticle delivery of oligonucleotides

    Science.gov (United States)

    Shen, Christopher

    The discovery of RNA interference and the increasing understanding of disease genetics have created a new class of potential therapeutics based on oligonucleotides. This therapeutic class includes antisense molecules, small interfering RNA (siRNA), and microRNA modulators such as antagomirs (antisense directed against microRNA) and microRNA mimics, all of which function by altering gene expression at the translational level. While these molecules have the promise of treating a host of diseases from neurological disorders to cancer, a major hurdle is their inability to enter cells on their own, where they may render therapeutic effect. Nanotechnology is the engineering of materials at the nanometer scale and has gained significant interest for nucleic acid delivery due to its biologically relevant length-scale and amenability to multifunctionality. While a number of nanoparticle vehicles have shown promise for oligonucleotide delivery, there remains a lack of understanding of how nanoparticle coating and size affect these delivery processes. This dissertation seeks to elucidate some of these factors by evaluating oligonucleotide delivery efficiencies of a panel of iron oxide nanoparticles with varying cationic coatings and sizes. A panel of uniformly-sized nanoparticles was prepared with surface coatings comprised of various amine groups representing high and low pKas. A separate panel of nanoparticles with sizes of 40, 80, 150, and 200 nm but with the same cationic coating was also prepared. Results indicated that both nanoparticle surface coating and nanoparticle hydrodynamic size affect transfection efficiency. Specific particle coatings and sizes were identified that gave superior performance. The intracellular fate of iron oxide nanoparticles was also tracked by electron microscopy and suggests that they function via the proton sponge effect. The research presented in this dissertation may aid in the rational design of improved nanoparticle delivery vectors for

  6. Multilocus sequence typing of Staphylococcus aureus with DNA array technology

    OpenAIRE

    2003-01-01

    textabstractA newly developed oligonucleotide array suited for multilocus sequence typing (MLST) of Staphylococcus aureus strains was analyzed with two strain collections in a two-center study. MLST allele identification for the first strain collection fully agreed with conventional strain typing. Analysis of strains from the second collection revealed that chip-defined MLST was concordant with conventional MLST. Array-mediated MLST data were reproducible, exchangeable, and epidemiologically ...

  7. Hole hopping rates in single strand oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

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

    2014-08-31

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

  8. Oligonucleotide and Long Polymeric DNA Encoding

    Energy Technology Data Exchange (ETDEWEB)

    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.

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

    Directory of Open Access Journals (Sweden)

    Weizhong Tang

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

  10. Antisense oligonucleotide therapy for the treatment of C9ORF72 ALS/FTD diseases.

    Science.gov (United States)

    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.

  11. Analysis of mutations in oral poliovirus vaccine by hybridization with generic oligonucleotide microchips.

    Energy Technology Data Exchange (ETDEWEB)

    Proudnikov, D.; Kirillov, E.; Chumakov, K.; Donion, J.; Rezapkin, G.; Mirzabekov, A.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Center for Biologics Evaluation and Research

    2000-01-01

    This paper describes use of a new technology of hybridization with a micro-array of immobilized oligonucleotides for detection and quantification of neurovirulent mutants in Oral Poliovirus Vaccine (OPV). We used a micro-array consisting of three-dimensional gel-elements containing all possible hexamers (total of 4096 probes). Hybridization of fluorescently labelled viral cDNA samples with such microchips resulted in a pattern of spots that was registered and quantified by a computer-linked CCD camera, so that the sequence of the original cDNA could be deduced. The method could reliably identify single point mutations, since each of them affected fluorescence intensity of 12 micro-array elements. Micro-array hybridization of DNA mixtures with varying contents of point mutants demonstrated that the method can detect as little as 10% of revertants in a population of vaccine virus. This new technology should be useful for quality control of live viral vaccines, as well as for other applications requiring identification and quantification of point mutations.

  12. Targeting of single stranded oligonucleotides through metal-induced cyclization of short complementary strands : Targeting of single stranded oligonucleotides

    OpenAIRE

    Freville, Fabrice; Richard, Tristan; Bathany, Katell; Moreau, Serge

    2006-01-01

    International audience; A new strategy to cyclize a short synthetic oligonucleotide on a DNA or a RNA target strand is described. This one relies on a metal-mediated cyclization of short synthetic oligonucleotides conjugated with two chelating 2,2':6',2”-terpyridine moieties at their 3' and 5' ends. Cyclization following metal addition (Zn2+, Fe2+) was demonstrated using UV monitored thermal denaturation experiments, mass spectrometry analysis and gel shift assays. NMR experiments were used t...

  13. Development and validation of an oligonucleotide microarray to characterise ectomycorrhizal fungal communities

    Directory of Open Access Journals (Sweden)

    Reich Marlis

    2009-01-01

    Full Text Available Abstract Background In forest ecosystems, communities of ectomycorrhizal fungi (ECM are influenced by several biotic and abiotic factors. To understand their underlying dynamics, ECM communities have been surveyed with ribosomal DNA-based sequencing methods. However, most identification methods are both time-consuming and limited by the number of samples that can be treated in a realistic time frame. As a result of ongoing implementation, the array technique has gained throughput capacity in terms of the number of samples and the capacity for parallel identification of several species. Thus far, although phylochips (microarrays that are used to detect species have been mostly developed to trace bacterial communities or groups of specific fungi, no phylochip has been developed to carry oligonucleotides for several ectomycorrhizal species that belong to different genera. Results We have constructed a custom ribosomal DNA phylochip to identify ECM fungi. Specific oligonucleotide probes were targeted to the nuclear internal transcribed spacer (ITS regions from 95 fungal species belonging to 21 ECM fungal genera. The phylochip was first validated using PCR amplicons of reference species. Ninety-nine percent of the tested oligonucleotides generated positive hybridisation signals with their corresponding amplicons. Cross-hybridisation was mainly restricted at the genus level, particularly for Cortinarius and Lactarius species. The phylochip was subsequently tested with environmental samples that were composed of ECM fungal DNA from spruce and beech plantation fungal communities. The results were in concordance with the ITS sequencing of morphotypes and the ITS clone library sequencing results that were obtained using the same PCR products. Conclusion For the first time, we developed a custom phylochip that is specific for several ectomycorrhizal fungi. To overcome cross-hybridisation problems, specific filter and evaluation strategies that used spot

  14. Reversing Antisense Oligonucleotide Activity with a Sense Oligonucleotide Antidote: Proof of Concept Targeting Prothrombin.

    Science.gov (United States)

    Crosby, Jeff R; Zhao, Chenguang; Zhang, Hong; MacLeod, A Robert; Guo, Shuling; Monia, Brett P

    2015-12-01

    The tissue half-life of second-generation antisense oligonucleotide drugs (ASOs) is generally longer than traditional small molecule therapeutics. Thus, a strategy to reverse the activity of antisense drugs is warranted in certain settings. In this study, we describe a strategy employing the administration of a complementary sense oligonucleotide antidote (SOA). As a model system we have chosen to target the coagulation factor and antithrombotic drug target, prothrombin, to assess the feasibility of this approach. ASO targeting mouse prothrombin specifically suppressed >90% hepatic prothrombin mRNA levels and circulating prothrombin protein in mice. These effects were dose- and time-dependent, and as expected produced predictable increases in anticoagulation activity [prothrombin time/activated partial thromboplastin time (PT/aPTT)]. Treatment with prothrombin SOAs resulted in a dose-dependent reversal of ASO activity, as measured by a return in prothrombin mRNA levels and thrombin activity, and normalization of aPTT and PT. The antithrombotic activity of prothrombin ASOs was demonstrated in a FeCl3-induced thrombosis mouse model, and as predicted for this target, the doses required for antithrombotic activity were also associated with increased bleeding. Treatment with SOA was able to prevent prothrombin ASO-induced bleeding in a dose-dependent manner. These studies demonstrate for the first time the utility of SOAs to selectively and specifically reverse the intracellular effects of an antisense therapy.

  15. Antisense oligonucleotides for the treatment of dyslipidaemia.

    Science.gov (United States)

    Visser, Maartje E; Witztum, Joseph L; Stroes, Erik S G; Kastelein, John J P

    2012-06-01

    Antisense oligonucleotides (ASOs) are short synthetic analogues of natural nucleic acids designed to specifically bind to a target messenger RNA (mRNA) by Watson-Crick hybridization, inducing selective degradation of the mRNA or prohibiting translation of the selected mRNA into protein. Antisense technology has the ability to inhibit unique targets with high specificity and can be used to inhibit synthesis of a wide range of proteins that could influence lipoprotein levels and other targets. A number of different classes of antisense agents are under development. To date, mipomersen, a 2'-O-methoxyethyl phosphorothioate 20-mer ASO, is the most advanced ASO in clinical development. It is a second-generation ASO developed to inhibit the synthesis of apolipoprotein B (apoB)-100 in the liver. In Phase 3 clinical trials, mipomersen has been shown to significantly reduce plasma low-density lipoprotein cholesterol (LDL-c) as well as other atherogenic apoB containing lipoproteins such as lipoprotein (a) [Lp(a)] and small-dense LDL particles. Although concerns have been raised because of an increase in intrahepatic triglyceride content, preliminary data from long-term studies suggest that with continued treatment, liver fat levels tend to stabilize or decline. Further studies are needed to evaluate potential clinical relevance of these changes. Proprotein convertase subtilisin/kexin-9 (PCSK9) is another promising novel target for lowering LDL-c by ASOs. Both second-generation ASOs and ASOs using locked nucleic acid technology have been developed to inhibit PCSK9 and are under clinical development. Other targets currently being addressed include apoC-III and apo(a) or Lp(a). By directly inhibiting the synthesis of specific proteins, ASO technology offers a promising new approach to influence the metabolism of lipids and to control lipoprotein levels. Its application to a wide variety of potential targets can be expected if these agents prove to be clinically safe and

  16. Molecular Subtyping of Salmonella Typhimurium with Multiplex Oligonucleotide Ligation-PCR (MOL-PCR).

    Science.gov (United States)

    Wuyts, Véronique; Mattheus, Wesley; Roosens, Nancy H C; Marchal, Kathleen; Bertrand, Sophie; De Keersmaecker, Sigrid C J

    2017-01-01

    A multiplex oligonucleotide ligation-PCR (MOL-PCR) assay is a valuable high-throughput technique for the detection of bacteria and viruses, for characterization of pathogens and for diagnosis of genetic diseases, as it allows one to combine different types of molecular markers in a high-throughput multiplex assay. A MOL-PCR assay starts with a multiplex oligonucleotide ligation reaction for detection of the molecular marker, followed by a singleplex PCR for signal amplification and analysis of the MOL-PCR products on a Luminex platform. This last step occurs through a liquid bead suspension array in which the MOL-PCR products are hybridized to MagPlex-TAG beads.In this chapter, we describe the complete procedure for a MOL-PCR assay for subtyping of Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) and its monophasic variant S. 1,4[5],12:i:- from DNA isolation through heat lysis up to data interpretation through a Gödel Prime Product. The subtyping assay consists of 50 discriminative molecular markers and two internal positive control markers divided over three MOL-PCR assays.

  17. Strategies for the discovery of therapeutic Aptamers

    Science.gov (United States)

    Yang, Xianbin; Li, Na; Gorenstein, David G.

    2010-01-01

    Importance of the field Therapeutic aptamers are synthetic, structured oligonucleotides that bind to a very broad range of targets with high affinity and specificity. They are an emerging class of targeting ligand that show great promise for treating a number of diseases. A series of aptamers currently in various stages of clinical development highlights the potential of aptamers for therapeutic applications. Area covered in this review This review will cover in vitro selection of oligonucleotide ligands, called aptamers, from a combinatorial library using the Systematic Evolution of Ligands by Exponential Enrichment (SELEX) process as well as the other known strategies for finding aptamers against various targets. What the reader will gain Readers will gain an understanding of the highly useful strategies for successful aptamer discovery. They may also be able combine two or more of the presented strategies for their aptamer discovery projects. Take home message Although many processes are available for discovering aptamers, it is not trivial to discover an aptamer candidate that is ready to move toward pharmaceutical drug development. It is also apparent that there have been relatively few therapeutic advances and clinical trials undertaken due to the small number of companies that participate in aptamer development. PMID:21359096

  18. Discovery Mondays

    CERN Multimedia

    2003-01-01

    Many people don't realise quite how much is going on at CERN. Would you like to gain first-hand knowledge of CERN's scientific and technological activities and their many applications? Try out some experiments for yourself, or pick the brains of the people in charge? If so, then the «Lundis Découverte» or Discovery Mondays, will be right up your street. Starting on May 5th, on every first Monday of the month you will be introduced to a different facet of the Laboratory. CERN staff, non-scientists, and members of the general public, everyone is welcome. So tell your friends and neighbours and make sure you don't miss this opportunity to satisfy your curiosity and enjoy yourself at the same time. You won't have to listen to a lecture, as the idea is to have open exchange with the expert in question and for each subject to be illustrated with experiments and demonstrations. There's no need to book, as Microcosm, CERN's interactive museum, will be open non-stop from 7.30 p.m. to 9 p.m. On the first Discovery M...

  19. Characteristic archaebacterial 16S rRNA oligonucleotides

    Science.gov (United States)

    McGill, T. J.; Jurka, J.; Sobieski, J. M.; Pickett, M. H.; Woese, C. R.; Fox, G. E.

    1986-01-01

    A method of analyzing 16S rRNA catalog data has been developed in which groupings at various taxonomic levels can be characterized in terms of specific "signature" oligonucleotides. This approach provides an alternative means for evaluating higher order branching possibilities and can be used to assess the phylogenetic position of isolates that are poorly placed by the usual clustering procedures. This signature approach has been applied to forty archaebacterial catalogs and every oligonucleotide with significant signature value has been identified. Sets of specific oligonucleotides were identified for every major group on a dendrogram produced by cluster analysis procedures. Signatures that would establish between group relationships were also sought and found. In the case of the Methanobacteriaceae the clustering methods suggest a specific relationship to the Methanococcaceae. This inclusion is in fact supported by six strong signature oligonucleotides. However there are also significant numbers of signature oligonucleotides supporting a specific relationship of the Methanobacteriaceae to either the Halobacteriaceae or the Methanomicrobiaceae. Thus the placement of the Methanobacteriaceae is less certain than the usual dendrograms imply. The signature approach also was used to assess the phylogenetic position of Thermoplasma acidophilum which is found to be more closely related to the methanogen/halophile Division than to the sulfur dependent Division of the archaebacteria. This does not imply however that Thermoplasma acidophilum is properly regarded as being in the methanogen/halophile Division.

  20. Nano-topographic evaluation of highly disordered fractal-like structures of immobilized oligonucleotides using AFM

    Energy Technology Data Exchange (ETDEWEB)

    Sawant, P.D. [BioNanoEngineering Laboratory, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, Vic. 4122 (Australia)]. E-mail: psawant@swin.edu.au; Nicolau, D.V. [BioNanoEngineering Laboratory, Faculty of Engineering and Industrial Sciences, Swinburne University of Technology, Hawthorn, Vic. 4122 (Australia)

    2006-07-25

    In a recent trend of micro- and nano-array technologies, polymers are gaining preference over traditional substrates such as glass, silicates, etc. as a model biosurface to immobilize biomolecules. In present paper, we compared model polymeric surfaces such as cyclo olefin copolymer (COC) and polycarbonate (PC), with traditional surfaces such as silicone-wafer and mica. We used a 2D fractal dimension method, i.e. the perimeter-area relationship (PAR) to study the immobilization of 26 base pair oligonucleotide primer on surfaces which are imaged by AFM. Results revealed that the efficiency of the vertical immobilization is in the following order: COC > PC > mica > Si-wafer which can be contributed to chemical and nano-topographical heterogeneity. This study is useful for in-depth understanding of fundamental issues such as effects of manufacturing parameters and evaluation of surface nanotopographies for the development of high-density biochips.

  1. Improving oligonucleotide fingerprinting of rRNA genes by implementation of polony microarray technology

    Science.gov (United States)

    Ruegger, Paul M.; Bent, Elizabeth; Li, Wei; Jeske, Daniel R.; Cui, Xinping; Braun, Jonathan; Jiang, Tao; Borneman, James

    2012-01-01

    Improvements to oligonucleotide fingerprinting of rRNA genes (OFRG) were obtained by implementing polony microarray technology. OFRG is an array-based method for analyzing microbial community composition. Polonies are discrete clusters of DNA, produced by solid-phase PCR in hydrogels, and derived from individual, spatially isolated DNA molecules. The advantages of a polony-based OFRG method include higher throughput and reductions in the PCR-induced errors and compositional skew inherent in all other PCR-based community composition methods, including high throughput sequencing of rRNA genes. Given the similarities between polony microarrays and certain aspects of sequencing methods such as the Illumina platform, we suggest that if concepts presented in this study were implemented in high throughput sequencing protocols, a reduction of PCR-induced errors and compositional skew may be realized. PMID:22640891

  2. High-throughput flow cytometric screening of combinatorial chemistry bead libraries for proteomics and drug discovery

    Science.gov (United States)

    Leary, James F.; Reece, Lisa M.; Yang, Xian-Bin; Gorenstein, David

    2005-04-01

    For proteomics drug discovery applications, combinatorial microbead thioaptamer libraries (one thioaptamer sequence per bead) are being created by split synthesis method, creating a "proteomics library" of protein capture beads which can be analyzed by high-throughput screening methods in this case, flow cytometry and cell sorting. Thioaptamers, oligonucleotides with thiophosphate backbone substitutions, function like antibodies in terms of recognizing specific protein sequences but have a number of advantages over antibody libraries. These proteomics beads can then be analyzed by high-speed flow cytometry and sorted to single-bead level depending on relative fluorescence brightness of fluorescently-labeled proteins, or for a specific protein from all of the molecules of cell subpopulations being analyzed. The thioaptamer sequences on a given bead showing high affinity for that protein can then be sequenced. Alternatively, the protein-capturing beads can be analyzed by MALDI-TOF mass spectrometry for analysis of the bound proteins. The beads can be thought of as equivalent to single-element positions of a proteomics chip arrays but with the advantage of being able to much more rapidly analyze hundreds of millions of possible amino acid sequences/epitopes on the basis of thioaptamer sequence affinities to select single sequences of interest. Additionally, those beads can be manipulated and isolated at the single bead level by high-throughput flow cytometry/cell sorting for subsequent sequencing of the thioaptamer sequences.

  3. Investigations of oligonucleotide usage variance within and between prokaryotes

    DEFF Research Database (Denmark)

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

    2008-01-01

    Oligonucleotide usage in archaeal and bacterial genomes can be linked to a number of properties, including codon usage (trinucleotides), DNA base-stacking energy (dinucleotides), and DNA structural conformation (di-to tetranucleotides). We wanted to assess the statistical information potential...... 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 GC-rich and free-living genomes, and this variation was mainly located in non-coding regions. Bias (selectional pressure) in tetranucleotide usage correlated with GC content, and coding regions were more biased than non-coding regions. Non-coding regions were also found to be approximately 5.5% more...

  4. Delivery of RNAi-Based Oligonucleotides by Electropermeabilization

    Directory of Open Access Journals (Sweden)

    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.

  5. Retro-1 Analogues Differentially Affect Oligonucleotide Delivery and Toxin Trafficking.

    Science.gov (United States)

    Yang, Bing; Ming, Xin; Abdelkafi, Hajer; Pons, Valerie; Michau, Aurelien; Gillet, Daniel; Cintrat, Jean-Christophe; Barbier, Julien; Juliano, Rudy

    2016-11-21

    Retro-1 is a small molecule that displays two important biological activities: First, it blocks the actions of certain toxins by altering their intracellular trafficking. Second, it enhances the activity of oligonucleotides by releasing them from entrapment in endosomes. This raises the question of whether the two actions involve the same cellular target. Herein we report the effects of several Retro-1 analogues on both toxins and oligonucleotides. We found analogues that affect toxins but not oligonucleotides and vice-versa, while Retro-1 is the only compound that affects both. This indicates that the molecular target(s) involved in the two processes are distinct. © 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

  6. Oligonucleotide Therapy for Obstructive and Restrictive Respiratory Diseases

    Directory of Open Access Journals (Sweden)

    Wupeng Liao

    2017-01-01

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

  7. Oligonucleotide Therapy for Obstructive and Restrictive Respiratory Diseases.

    Science.gov (United States)

    Liao, Wupeng; Dong, Jinrui; Peh, Hong Yong; Tan, Lay Hong; Lim, Kah Suan; Li, Li; Wong, Wai-Shiu Fred

    2017-01-17

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

  8. A novel catechol-based universal support for oligonucleotide synthesis.

    Science.gov (United States)

    Anderson, Keith M; Jaquinod, Laurent; Jensen, Michael A; Ngo, Nam; Davis, Ronald W

    2007-12-21

    A novel universal support for deoxyribo- and ribonucleic acid synthesis has been developed. The support, constructed from 1,4-dimethoxycatechol, represents an improvement over existing universal supports because of its ability to cleave and deprotect under mild conditions in standard reagents. Because no nonvolatile additives are required for cleavage and deprotection, the synthesized oligonucleotides do not require purification prior to use in biochemical assays. Using reverse phase HPLC and electrospray mass spectroscopy, it was determined that oligonucleotides synthesized on the universal support (UL1) 3'-dephosphorylate quickly (9 h in 28-30% ammonium hydroxide (NH4OH) at 55 degrees C, 2 h in 28-30% NH4OH at 80 degrees C, or <1 h in ammonium hydroxide/methylamine (1:1) (AMA) at 80 degrees C). Oligonucleotides used as primers for the polymerase chain reaction (PCR) assay were found to perform identically to control primers, demonstrating full biological compatibility. In addition, a method was developed for sintering the universal support directly into a filter plug which can be pressure fit into the synthesis column of a commercial synthesizer. The universal support plugs allow the synthesis of high-quality oligonucleotides at least 120 nucleotides in length, with purity comparable to non-universal commercial supports and approximately 50% lower reagent consumption. The universal support plugs are routinely used to synthesize deoxyribo-, ribo-, 3'-modified, 5'-modified, and thioated oligonucleotides. The flexibility of the universal support and the efficiency of 3'-dephosphorylation are expected to increase the use of universal supports in oligonucleotide synthesis.

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    KRAS is mutated in >90% of pancreatic ductal adenocarcinomas. As its inactivation leads to tumour regression, mutant KRAS is considered an attractive target for anticancer drugs. In this study we report a new delivery strategy for a G4-decoy oligonucleotide that sequesters MAZ, a transcription...... factor essential for KRAS transcription. It is based on the use of palmitoyl-oleyl-phosphatidylcholine (POPC) liposomes functionalized with lipid-modified G4-decoy oligonucleotides and a lipid-modified cell penetrating TAT peptide. The potency of the strategy in pancreatic cancer cells is demonstrated...

  10. Inhibition of microRNA with antisense oligonucleotides.

    Science.gov (United States)

    Esau, Christine C

    2008-01-01

    Antisense inhibition of microRNA (miRNA) function has been an important tool for uncovering miRNA biology. Chemical modification of anti-miRNA oligonucleotides (AMOs) is necessary to improve affinity for target miRNA, stabilize the AMO to nuclease degradation, and to promote tissue uptake for in vivo delivery. Here I summarize the work done to evaluate the effectiveness of various chemically modified AMOs for use in cultured cells and rodent models, and outline important issues to consider when inhibiting miRNAs with antisense oligonucleotides.

  11. Synthesis of Peptide-Oligonucleotide Conjugates Using a Heterobifunctional Crosslinker

    Science.gov (United States)

    Williams, Berea A.R.; Chaput, John C.

    2010-01-01

    Peptide-oligonucleotide conjugates (POCs) are molecular chimeras composed of a nucleic acid moiety covalently attached to a polypeptide moiety. POCs have been used in numerous applications from therapeutics to nanotechnology, and most recently as combinatorial agents in the assembly of bivalent protein affinity reagents. This unit describes the synthesis and purification of POC molecules using the heterobifunctional crosslinking reagent succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), which enables amine-modified oligonucleotides to become covalently linked to cysteine-modified polypeptides. This solution-based protocol consists of a two-step synthesis followed by a single purification step. PMID:20827717

  12. Chemical phosphorylation of deoxyribonucleosides and thermolytic DNA oligonucleotides.

    Science.gov (United States)

    Ausín, Cristina; Grajkowski, Andrzej; Cieślak, Jacek; Beaucage, Serge L

    2006-10-01

    The phosphorylating reagent bis[S-(4,4'-dimethoxytrityl)-2-mercaptoethyl]-N,N-diisopropylphosphoramidite is prepared in three steps from commercial methyl thioglycolate and diisopropylphosphoramidous dichloride. The phosphorylating reagent has been used successfully in the solid-phase synthesis of deoxyribonucleoside 5'-/3'-phosphate or -thiophosphate monoesters and oligonucleotide 5'-phosphate/-thiophosphate monoesters. Bis[S-(4,4'-dimethoxytrityl)-2-mercaptoethyl]-N,N-diisopropylphosphoramidite has also been employed in the construction of a thermolytic dinucleotide prodrug model to evaluate the ability of the reagent to produce thermosentive oligonucleotide prodrugs under mild temperature conditions ( approximately 25 degrees C) for potential therapeutic applications.

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

    Science.gov (United States)

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

    2010-11-15

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

  14. Anti sense and sensibility : renal and skin effects of (antisense) oligonucleotides

    NARCIS (Netherlands)

    Meer, van L.

    2017-01-01

    This thesis describes the clinical investigation of a novel treatment strategy for type 2 diabetes mellitus (t2dm) using an antisense oligonucleotide(aon)to inhibit the sglt2 receptor. Furthermore it describes skin effects of oligonucleotides

  15. Delivery of antisense oligonucleotides using cholesterol-modified sense dendrimers and cationic lipids

    NARCIS (Netherlands)

    Chaltin, Patrick; Margineanu, Anca; Marchand, Damien; Aerschot, Arthur Van; Rozenski, Jef; Schryver, Frans De; Herrmann, Andreas; Müllen, Klaus; Juliano, Rudolph; Fisher, Michael H.; Kang, Hyunmin; Feyter, Steven De; Herdewijn, Piet

    2005-01-01

    Cholesterol modified mono-, di-, and tetrameric oligonucleotides were synthesized and hybridized with antisense oligonucleotides to study their incorporation in cationic liposomes together with the influence of this dendrimeric delivery system on biological activity. Electrostatic interactions seem

  16. Array-based techniques for fingerprinting medicinal herbs

    Directory of Open Access Journals (Sweden)

    Xue Charlie

    2011-05-01

    Full Text Available Abstract Poor quality control of medicinal herbs has led to instances of toxicity, poisoning and even deaths. The fundamental step in quality control of herbal medicine is accurate identification of herbs. Array-based techniques have recently been adapted to authenticate or identify herbal plants. This article reviews the current array-based techniques, eg oligonucleotides microarrays, gene-based probe microarrays, Suppression Subtractive Hybridization (SSH-based arrays, Diversity Array Technology (DArT and Subtracted Diversity Array (SDA. We further compare these techniques according to important parameters such as markers, polymorphism rates, restriction enzymes and sample type. The applicability of the array-based methods for fingerprinting depends on the availability of genomics and genetics of the species to be fingerprinted. For the species with few genome sequence information but high polymorphism rates, SDA techniques are particularly recommended because they require less labour and lower material cost.

  17. Effects of fluid flow on the oligonucleotide folding in single-walled carbon nanotubes.

    Science.gov (United States)

    Lim, M C G; Zhong, Z W

    2009-10-01

    This paper presents molecular-dynamics (MD) simulations of DNA oligonucleotide and water molecules translocating through carbon nanotube (CNT) channels. An induced pressure difference is applied to the system by pushing a layer of water molecules toward the flow direction to drive the oligonucleotide and other molecules. This MD simulation investigates the changes that occur in the conformation of the oligonucleotide due to water molecules in nanochannels while controlling the temperature and volume of the system in a canonical ensemble. The results show that the oligonucleotide in the (8,8)-(12,12) CNT channel forms a folded state at a lower pressure, whereas the oligonucleotide in the (10,10)-(14,14) CNT channel forms a folded state at a higher pressure instead. The van der Waals forces between the water molecules and the oligonucleotide suggest that the attraction between these two types of molecules results in the linear arrangements of the bases of the oligonucleotide. For a larger nanotube channel, the folding of the oligonucleotide is mainly dependent on the solvent (water molecules), whereas pressure, the size of the nanotube junction, and water molecules are the considering factors of the folding of the oligonucleotide at a smaller nanotube channel. For a folded oligonucleotide, the water distribution around the oligonucleotide is concentrated at a smaller range than that for the distribution around an unfolded oligonucleotide.

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

    DEFF Research Database (Denmark)

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

    2017-01-01

    by oligonucleotide duplex and triplex formation. POC synthesis was achieved by copper-free alkyne-azide cycloaddition between three oligonucleotides and a 23-mer peptide, which by itself exhibited multiple oligomeric states in solution. The oligonucleotide domain was designed to furnish a stable parallel triplex...

  19. Oligonucleotide-directed mutagenesis for precision gene editing.

    Science.gov (United States)

    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.

  20. Chromosome-specific painting in Cucumis species using bulked oligonucleotides

    Science.gov (United States)

    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 sing...

  1. Splice-switching antisense oligonucleotides as therapeutic drugs

    National Research Council Canada - National Science Library

    Havens, Mallory A; Hastings, Michelle L

    2016-01-01

    Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA-RNA base-pairing or protein-RNA...

  2. Antithrombotic effect of antisense factor XI oligonucleotide treatment in primates.

    Science.gov (United States)

    Crosby, Jeffrey R; Marzec, Ulla; Revenko, Alexey S; Zhao, Chenguang; Gao, Dacao; Matafonov, Anton; Gailani, David; MacLeod, A Robert; Tucker, Erik I; Gruber, Andras; Hanson, Stephen R; Monia, Brett P

    2013-07-01

    During coagulation, factor IX (FIX) is activated by 2 distinct mechanisms mediated by the active proteases of either FVIIa or FXIa. Both coagulation factors may contribute to thrombosis; FXI, however, plays only a limited role in the arrest of bleeding. Therefore, therapeutic targeting of FXI may produce an antithrombotic effect with relatively low hemostatic risk. We have reported that reducing FXI levels with FXI antisense oligonucleotides produces antithrombotic activity in mice, and that administration of FXI antisense oligonucleotides to primates decreases circulating FXI levels and activity in a dose-dependent and time-dependent manner. Here, we evaluated the relationship between FXI plasma levels and thrombogenicity in an established baboon model of thrombosis and hemostasis. In previous studies with this model, antibody-induced inhibition of FXI produced potent antithrombotic effects. In the present article, antisense oligonucleotides-mediated reduction of FXI plasma levels by ≥ 50% resulted in a demonstrable and sustained antithrombotic effect without an increased risk of bleeding. These results indicate that reducing FXI levels using antisense oligonucleotides is a promising alternative to direct FXI inhibition, and that targeting FXI may be potentially safer than conventional antithrombotic therapies that can markedly impair primary hemostasis.

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

    NARCIS (Netherlands)

    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

  4. Regioselective immobilization of short oligonucleotides to acrylic copolymer gels.

    Science.gov (United States)

    Timofeev, E; Kochetkova, S V; Mirzabekov, A D; Florentiev, V L

    1996-01-01

    Four types of polyacrylamide or polydimethyl-acrylamide gels for regioselective (by immobilization at the 3' end) of short oligonucleotides have been designed for use in manufacturing oligonucleotide microchips. Two of these supports contain amino or aldehyde groups in the gel, allowing coupling with oligonucleotides bearing aldehyde or amino groups, respectively, in the presence of a reducing agent. The aldehyde gel support showed a higher immobilization efficiency relative to the amino gel. Of all reducing agents tested, the best results were obtained with a pyridine-borane complex. The other supports are based on an acrylamide gel activated with glutaraldehyde or a hydroxyalkyl-functionalized gel treated with mesyl chloride. The use of dimethylacrylamide instead of acrylamide allows subsequent gel modifications in organic solvents. All the immobilization methods are easy and simple to perform, give high and reproducible yields, allow long durations of storage of the activated support, and provide high stability of attachment and low non-specific binding. Although these gel supports have been developed for preparing oligonucleotide microchips, they may be used for other purposes as well. PMID:8774893

  5. LNA 5'-phosphoramidites for 5'→3'-oligonucleotide synthesis

    DEFF Research Database (Denmark)

    Madsen, Andreas Stahl; Kumar, Santhosh T.; Wengel, Jesper

    2010-01-01

    Hereby we report an efficient synthesis of LNA thymine and LNA 5-methylcytosine 5′-phosphoramidites, allowing incorporation of LNA thymine and LNA 5-methylcytosine into oligonucleotides synthesized in the 5′→3′ direction. Key steps include regioselective enzymatic benzoylation of the 5′-hydroxy...

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

    NARCIS (Netherlands)

    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

  7. A strategy for detection of known and unknown SNP using a minimum number of oligonucleotides applicable in the clinical settings

    Directory of Open Access Journals (Sweden)

    Klein Harvey

    2003-08-01

    Full Text Available Abstract Detection of unknown single nucleotide polymorphism (SNP relies on large scale sequencing expeditions of genomic fragments or complex high-throughput chip technology. We describe a simplified strategy for fluorimetric detection of known and unknown SNP by proportional hybridization to oligonucleotide arrays based on optimization of the established principle of signal loss or gain that requires a drastically reduced number of matched or mismatched probes. The array consists of two sets of 18-mer oligonucleotide probes. One set includes overlapping oligos with 4-nucleotide tiling representing an arbitrarily selected "consensus" sequence (consensus-oligos, the other includes oligos specific for known SNP within the same genomic region (variant-oligos. Fluorescence-labeled DNA amplified from a homozygous source identical to the consensus represents the reference target and is co-hybridized with a differentially-labeled test sample. Lack of hybridization of the test sample to consensus- with simultaneous hybridization to variant-oligos designates a known allele. Lack of hybridization to consensus- and variant-oligos indicates a new allele. Detection of unknown variants in heterozygous samples depends upon fluorimetric analysis of signal intensity based on the principle that homozygous samples generate twice the amount of signal. This method can identify unknown SNP in heterozygous conditions with a sensitivity of 82% and specificity of 90%. This strategy should dramatically increase the efficiency of SNP detection throughout the human genome and will decrease the cost and complexity of applying genomic wide analysis in the context of clinical trials.

  8. Glycoclusters on oligonucleotide and PNA scaffolds: synthesis and applications.

    Science.gov (United States)

    Spinelli, Nicolas; Defrancq, Eric; Morvan, François

    2013-06-07

    Conjugation of oligonucleotides (ONs) to a variety of reporter groups has been the subject of intensive research during the last decade. Conjugation is indeed of great interest because it can be used not only to improve the existing ONs properties but also to impart new ones. In this context tremendous efforts have been made to conjugate carbohydrate moieties to ONs. Indeed carbohydrates play an important role in biological processes such as signal transduction and cell adhesion through the recognition with sugar-binding proteins (i.e. lectins) located on the surface of cells. For this reason, carbohydrate-oligonucleotide conjugates (COCs) have been first developed for improving the poor cellular uptake or tissue specific delivery of ONs through receptor-mediated endocytosis. Besides the targeted ONs delivery, carbohydrate-oligonucleotide conjugates (COCs) are also evaluated in the context of carbohydrate biochips in which surface coating with carbohydrates is achieved by using the DNA-directed immobilization strategy (DDI). Peptide nucleic acids (PNAs) have also been extensively investigated as a surrogate of DNA for diverse applications. Therefore attachment of carbohydrate moieties to this class of molecules has been studied. The aforementioned applications of COCs require mimicking of the natural processes, in which the weak individual protein-carbohydrate binding is overcome by using multivalent interactions. This tutorial review focuses on the recent advances in carbohydrate-oligonucleotide conjugates and describes the major synthetic approaches available. In addition, an overview of applications that have been developed using various scaffolds allowing multivalent interactions is provided. Finally recent results on the use of peptide nucleic acids as oligonucleotides surrogate are described.

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

    Science.gov (United States)

    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 non-canonical homologous hydrogen bonding. In the presence of magnesium ions, chimeric TFOs are able to form highly stable α,β-triplexes, as indicated by native gel-electrophoresis, on-array thermal denaturation and fluorescence-quenching experiments. CD spectra of chimeric triplexes exhibited features typically observed for anti-parallel purine triplexes with a GA or GT third strand. The high potential of chimeric α,β-TFOs in targeting double-stranded DNA was demonstrated in the EcoRI endonuclease protection assay. In this paper, we report, for the first time, the recognition of base pair inversions in a duplex by chimeric TFOs containing α-thymidine and α-deoxyguanosine. PMID:22641847

  10. High-throughput screening identifies small molecules that enhance the pharmacological effects of oligonucleotides

    Science.gov (United States)

    Yang, B.; Ming, X.; Cao, C.; Laing, B.; Yuan, A.; Porter, M. A.; Hull-Ryde, E. A.; Maddry, J.; Suto, M.; Janzen, W. P.; Juliano, R. L.

    2015-01-01

    The therapeutic use of antisense and siRNA oligonucleotides has been constrained by the limited ability of these membrane-impermeable molecules to reach their intracellular sites of action. We sought to address this problem using small organic molecules to enhance the effects of oligonucleotides by modulating their intracellular trafficking and release from endosomes. A high-throughput screen of multiple small molecule libraries yielded several hits that markedly potentiated the actions of splice switching oligonucleotides in cell culture. These compounds also enhanced the effects of antisense and siRNA oligonucleotides. The hit compounds preferentially caused release of fluorescent oligonucleotides from late endosomes rather than other intracellular compartments. Studies in a transgenic mouse model indicated that these compounds could enhance the in vivo effects of a splice-switching oligonucleotide without causing significant toxicity. These observations suggest that selected small molecule enhancers may eventually be of value in oligonucleotide-based therapeutics. PMID:25662226

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

    Directory of Open Access Journals (Sweden)

    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.

  12. Montmorillonite, oligonucleotides, RNA and origin of life

    Science.gov (United States)

    Ertem, Gozen

    2004-01-01

    isomers (Ertem and Ferris, 2000). Formation of phosphodiester bonds between mononucleotides by montmorillonite catalysis is a fascinating discovery, and a significant step forward in efforts to find out how the first RNA-like oligomers might have formed in the course of chemical evolution. However, as has been pointed out in several publications, these systems should be regarded as models rather than a literal representation of prebiotic chemistry (Orgel, 1998; Joyce and Orgel, 1999; Schwartz, 1999).

  13. Functionalization of magnetic gold/iron-oxide composite nanoparticles with oligonucleotides and magnetic separation of specific target

    Energy Technology Data Exchange (ETDEWEB)

    Kinoshita, Takuya [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)]. E-mail: t-kinoshita@mit.eng.osaka-u.ac.jp; Seino, Satoshi [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Mizukoshi, Yoshiteru [Faculty of Engineering, Nagasaki University, 1-14 Bunkyo-machi, Nagasaki 852-8521 (Japan); Nakagawa, Takashi [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan); Yamamoto, Takao A. [Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita, Osaka 565-0871 (Japan)

    2007-04-15

    Magnetic composite nanoparticles of gold and iron-oxide synthesized with gamma-rays or ultrasonics were functionalized with thiol-modified oligonucleotides. The amount of oligonucleotides bound to the functionalized nanoparticle probes via hybridization was quantified with fluorescently-labeled target oligonucleotides. Our composite nanoparticles magnetically separated the specific target oligonucleotides without the non-specific adsorption.

  14. Using Aptamers for Cancer Biomarker Discovery

    Directory of Open Access Journals (Sweden)

    Yun Min Chang

    2013-01-01

    Full Text Available Aptamers are single-stranded synthetic DNA- or RNA-based oligonucleotides that fold into various shapes to bind to a specific target, which includes proteins, metals, and molecules. Aptamers have high affinity and high specificity that are comparable to that of antibodies. They are obtained using iterative method, called (Systematic Evolution of Ligands by Exponential Enrichment SELEX and cell-based SELEX (cell-SELEX. Aptamers can be paired with recent advances in nanotechnology, microarray, microfluidics, and other technologies for applications in clinical medicine. One particular area that aptamers can shed a light on is biomarker discovery. Biomarkers are important in diagnosis and treatment of cancer. In this paper, we will describe ways in which aptamers can be used to discover biomarkers for cancer diagnosis and therapeutics.

  15. Fluorescence quenching of TMR by guanosine in oligonucleotides

    Institute of Scientific and Technical Information of China (English)

    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.

  16. Electrochemical uranyl cation biosensor with DNA oligonucleotides as receptor layer.

    Science.gov (United States)

    Jarczewska, Marta; Ziółkowski, Robert; Górski, Łukasz; Malinowska, Elżbieta

    2014-04-01

    The present study aims at the further development of the uranyl oligonucleotide-based voltammetric biosensor, which takes advantage of strong interaction between UO2(2+) and phosphate DNA backbone. Herein we report the optimization of working parameters of previously elaborated electrochemical DNA biosensor. It is shown that the sensor sensitivity is highly dependent on the oligonucleotide probe length and the incubation time of sensor in a sample solution. Consequently, the highest sensitivity was obtained for 10-nucleotide sequence and 60 min incubation time. The lower detection limit towards uranyl cation for developed biosensor was 30 nM. The influence of mixed monolayers and the possibility of developing a non-calibration device were also investigated. The selectivity of the proposed biosensor was significantly improved via elimination of adenine nucleobases from the DNA probe. Moreover, the regeneration procedure was elaborated and tested to prolong the use of the same biosensor for 4 subsequent determinations of UO2(2+).

  17. Palladium-Catalyzed Modification of Unprotected Nucleosides, Nucleotides, and Oligonucleotides

    Directory of Open Access Journals (Sweden)

    Kevin H. Shaughnessy

    2015-05-01

    Full Text Available Synthetic modification of nucleoside structures provides access to molecules of interest as pharmaceuticals, biochemical probes, and models to study diseases. Covalent modification of the purine and pyrimidine bases is an important strategy for the synthesis of these adducts. Palladium-catalyzed cross-coupling is a powerful method to attach groups to the base heterocycles through the formation of new carbon-carbon and carbon-heteroatom bonds. In this review, approaches to palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides are reviewed. Polar reaction media, such as water or polar aprotic solvents, allow reactions to be performed directly on the hydrophilic nucleosides and nucleotides without the need to use protecting groups. Homogeneous aqueous-phase coupling reactions catalyzed by palladium complexes of water-soluble ligands provide a general approach to the synthesis of modified nucleosides, nucleotides, and oligonucleotides.

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

    Institute of Scientific and Technical Information of China (English)

    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.

  19. Inhibition of HTLV-III by exogenous oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    Goodchild, J.; Zamecnik, P.C.

    1989-02-21

    A method is described of detecting the presence of HTLV-III virus in a sample by demonstrating inhibition of replication of the virus in cells which are normally killed by the HTLV-III virus after the cells have been (a) combined with the sample and an oligonucleotide complementary to at least one highly conserved region of the HTLV-III genome necessary for HTLV-III replication and capable of hybridizing with at least the highly conserved region, the highly conserved region of the HTLV-III genome being a nucleotide sequence present in the genomes of HTLV-III isolates and the oligonucleotide complementary to at least one highly conserved region of the HTLV-III genome necessary for HTLV-III replication being complementary to a region of the HTLV-III genome.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2013-05-15

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

  1. Cardiovascular and Metabolic Effects of ANGPTL3 Antisense Oligonucleotides.

    Science.gov (United States)

    Graham, Mark J; Lee, Richard G; Brandt, Teresa A; Tai, Li-Jung; Fu, Wuxia; Peralta, Raechel; Yu, Rosie; Hurh, Eunju; Paz, Erika; McEvoy, Bradley W; Baker, Brenda F; Pham, Nguyen C; Digenio, Andres; Hughes, Steven G; Geary, Richard S; Witztum, Joseph L; Crooke, Rosanne M; Tsimikas, Sotirios

    2017-07-20

    Epidemiologic and genomewide association studies have linked loss-of-function variants in ANGPTL3, encoding angiopoietin-like 3, with low levels of plasma lipoproteins. We evaluated antisense oligonucleotides (ASOs) targeting Angptl3 messenger RNA (mRNA) for effects on plasma lipid levels, triglyceride clearance, liver triglyceride content, insulin sensitivity, and atherosclerosis in mice. Subsequently, 44 human participants (with triglyceride levels of either 90 to 150 mg per deciliter [1.0 to 1.7 mmol per liter] or >150 mg per deciliter, depending on the dose group) were randomly assigned to receive subcutaneous injections of placebo or an antisense oligonucleotide targeting ANGPTL3 mRNA in a single dose (20, 40, or 80 mg) or multiple doses (10, 20, 40, or 60 mg per week for 6 weeks). The main end points were safety, side-effect profile, pharmacokinetic and pharmacodynamic measures, and changes in levels of lipids and lipoproteins. The treated mice had dose-dependent reductions in levels of hepatic Angptl3 mRNA, Angptl3 protein, triglycerides, and low-density lipoprotein (LDL) cholesterol, as well as reductions in liver triglyceride content and atherosclerosis progression and increases in insulin sensitivity. After 6 weeks of treatment, persons in the multiple-dose groups had reductions in levels of ANGPTL3 protein (reductions of 46.6 to 84.5% from baseline, Pantisense oligonucleotide and three who received placebo reported dizziness or headache. There were no serious adverse events. Oligonucleotides targeting mouse Angptl3 retarded the progression of atherosclerosis and reduced levels of atherogenic lipoproteins in mice. Use of the same strategy to target human ANGPTL3 reduced levels of atherogenic lipoproteins in humans. (Funded by Ionis Pharmaceuticals; ClinicalTrials.gov number, NCT02709850 .).

  2. Voltammetric behaviour of oligonucleotide lipoplexes adsorbed onto glassy carbon electrodes

    OpenAIRE

    Piedade, J. A. P.; M. Mano; Lima, M. C. Pedroso de; Oretskaya, T S; Oliveira-Brett, A. M.

    2004-01-01

    The voltammetric behaviour of oligonucleotide lipoplexes (ODN-lipoplexes) prepared from short oligodeoxynucleotides (ODN), with different base compositions, and liposomes of the cationic lipid DOTAP, was studied by differential pulse voltammetry with a glassy carbon mini-electrode. It was found that the ODN base composition influences the ODN-lipoplex voltammetric response. Differential pulse voltammograms for ODN-lipoplexes of the ODN adenosine nucleotides present two different features when...

  3. Thermodynamic treatment of oligonucleotide duplex–simplex equilibria

    Science.gov (United States)

    Owczarzy, Richard; Dunietz, Isard; Behlke, Mark A.; Klotz, Irving M.; Walder, Joseph A.

    2003-01-01

    Thermodynamic formulations have been devised to obtain ΔG° values directly from spectroscopic data at a fixed common temperature in nucleic acid duplex–simplex melting curves. In addition, the dependence of melting on salt concentration has been expressed in terms of a stepwise stoichiometric representation, which leads to a specific equation for the partition of the added sodium ions between the different oligonucleotide forms. PMID:14657395

  4. Anti-tumor activity of splice-switching oligonucleotides

    OpenAIRE

    Bauman, John A; Li, Shyh-Dar; Yang, Angela; Huang, Leaf; Kole, Ryszard

    2010-01-01

    Alternative splicing has emerged as an important target for molecular therapies. Splice-switching oligonucleotides (SSOs) modulate alternative splicing by hybridizing to pre-mRNA sequences involved in splicing and blocking access to the transcript by splicing factors. Recently, the efficacy of SSOs has been established in various animal disease models; however, the application of SSOs against cancer targets has been hindered by poor in vivo delivery of antisense therapeutics to tumor cells. T...

  5. Triplex-forming oligonucleotide target sequences in the human genome

    OpenAIRE

    Goñi, J Ramon; de la Cruz, Xavier; Orozco, Modesto

    2004-01-01

    The existence of sequences in the human genome which can be a target for triplex formation, and accordingly are candidates for anti-gene therapies, has been studied by using bioinformatics tools. It was found that the population of triplex-forming oligonucleotide target sequences (TTS) is much more abundant than that expected from simple random models. The population of TTS is large in all the genome, without major differences between chromosomes. A wide analysis along annotated regions of th...

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

    Directory of Open Access Journals (Sweden)

    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.

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

    Science.gov (United States)

    Musumeci, Domenica; Riccardi, Claudia; Montesarchio, Daniela

    2015-09-22

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

  8. Recursive construction of perfect DNA molecules from imperfect oligonucleotides.

    Science.gov (United States)

    Linshiz, Gregory; Yehezkel, Tuval Ben; Kaplan, Shai; Gronau, Ilan; Ravid, Sivan; Adar, Rivka; Shapiro, Ehud

    2008-01-01

    Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology and synthetic biology. Here, we show for the first time how recursion can be used to address this challenge and demonstrate a recursive procedure that constructs error-free DNA molecules and their libraries from error-prone oligonucleotides. Divide and Conquer (D&C), the quintessential recursive problem-solving technique, is applied in silico to divide the target DNA sequence into overlapping oligonucleotides short enough to be synthesized directly, albeit with errors; error-prone oligonucleotides are recursively combined in vitro, forming error-prone DNA molecules; error-free fragments of these molecules are then identified, extracted and used as new, typically longer and more accurate, inputs to another iteration of the recursive construction procedure; the entire process repeats until an error-free target molecule is formed. Our recursive construction procedure surpasses existing methods for de novo DNA synthesis in speed, precision, amenability to automation, ease of combining synthetic and natural DNA fragments, and ability to construct designer DNA libraries. It thus provides a novel and robust foundation for the design and construction of synthetic biological molecules and organisms.

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

    Science.gov (United States)

    Moreno, Pedro; Pêgo, Ana

    2014-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Pedro Miguel Duarte Moreno

    2014-10-01

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

  11. DNA/RNA heteroduplex oligonucleotide for highly efficient gene silencing

    Science.gov (United States)

    Nishina, Kazutaka; Piao, Wenying; Yoshida-Tanaka, Kie; Sujino, Yumiko; Nishina, Tomoko; Yamamoto, Tsuyoshi; Nitta, Keiko; Yoshioka, Kotaro; Kuwahara, Hiroya; Yasuhara, Hidenori; Baba, Takeshi; Ono, Fumiko; Miyata, Kanjiro; Miyake, Koichi; Seth, Punit P.; Low, Audrey; Yoshida, Masayuki; Bennett, C. Frank; Kataoka, Kazunori; Mizusawa, Hidehiro; Obika, Satoshi; Yokota, Takanori

    2015-01-01

    Antisense oligonucleotides (ASOs) are recognized therapeutic agents for the modulation of specific genes at the post-transcriptional level. Similar to any medical drugs, there are opportunities to improve their efficacy and safety. Here we develop a short DNA/RNA heteroduplex oligonucleotide (HDO) with a structure different from double-stranded RNA used for short interfering RNA and single-stranded DNA used for ASO. A DNA/locked nucleotide acid gapmer duplex with an α-tocopherol-conjugated complementary RNA (Toc-HDO) is significantly more potent at reducing the expression of the targeted mRNA in liver compared with the parent single-stranded gapmer ASO. Toc-HDO also improves the phenotype in disease models more effectively. In addition, the high potency of Toc-HDO results in a reduction of liver dysfunction observed in the parent ASO at a similar silencing effect. HDO technology offers a novel concept of therapeutic oligonucleotides, and the development of this molecular design opens a new therapeutic field. PMID:26258894

  12. Targeting several CAG expansion diseases by a single antisense oligonucleotide.

    Directory of Open Access Journals (Sweden)

    Melvin M Evers

    Full Text Available To date there are 9 known diseases caused by an expanded polyglutamine repeat, with the most prevalent being Huntington's disease. Huntington's disease is a progressive autosomal dominant neurodegenerative disorder for which currently no therapy is available. It is caused by a CAG repeat expansion in the HTT gene, which results in an expansion of a glutamine stretch at the N-terminal end of the huntingtin protein. This polyglutamine expansion plays a central role in the disease and results in the accumulation of cytoplasmic and nuclear aggregates. Here, we make use of modified 2'-O-methyl phosphorothioate (CUGn triplet-repeat antisense oligonucleotides to effectively reduce mutant huntingtin transcript and protein levels in patient-derived Huntington's disease fibroblasts and lymphoblasts. The most effective antisense oligonucleotide, (CUG(7, also reduced mutant ataxin-1 and ataxin-3 mRNA levels in spinocerebellar ataxia 1 and 3, respectively, and atrophin-1 in dentatorubral-pallidoluysian atrophy patient derived fibroblasts. This antisense oligonucleotide is not only a promising therapeutic tool to reduce mutant huntingtin levels in Huntington's disease but our results in spinocerebellar ataxia and dentatorubral-pallidoluysian atrophy cells suggest that this could also be applicable to other polyglutamine expansion disorders as well.

  13. Characterization of self-assembled DNA concatemers from synthetic oligonucleotides

    Directory of Open Access Journals (Sweden)

    Lu Sun

    2014-08-01

    Full Text Available Studies of DNA–ligand interaction on a single molecule level provide opportunities to understand individual behavior of molecules. Construction of DNA molecules with repetitive copies of the same segments of sequences linked in series could be helpful for enhancing the interaction possibility for sequence-specific binding ligand to DNA. Here we report on the use of synthetic oligonucleotides to self-assembly into duplex DNA concatemeric molecules. Two strands of synthetic oligonucleotides used here were designed with 50-mer in length and the sequences are semi-complimentary so to hybridize spontaneously into concatemers of double stranded DNA. In order to optimize the length of the concatemers the oligonucleotides were incubated at different oligomer concentrations, ionic strengths and temperatures for different durations. Increasing the salt concentration to 200 mM NaCl was found to be the major optimizing factor because at this enhanced ionic strength the concatemers formed most quickly and the other parameters had no detectable effect. The size and shape of formed DNA concatemers were studied by gel electrophoresis in agarose, polyacrylamide gels and by AFM. Our results show that linear DNA constructs up to several hundred base pairs were formed and could be separated from a substantial fraction of non-linear constructs.

  14. De Novo Identification of Single Nucleotide Mutations in Caenorhabditis elegans Using Array Comparative Genomic Hybridization

    Science.gov (United States)

    Maydan, Jason S.; Okada, H. Mark; Flibotte, Stephane; Edgley, Mark L.; Moerman, Donald G.

    2009-01-01

    Array comparative genomic hybridization (aCGH) has been used primarily to detect copy-number variants between two genomes. Here we report using aCGH to detect single nucleotide mutations on oligonucleotide microarrays with overlapping 50-mer probes. This technique represents a powerful method for rapidly detecting novel homozygous single nucleotide mutations in any organism with a sequenced reference genome. PMID:19189945

  15. Synthesis of triazole-linked oligonucleotides with high affinity to DNA complements and an analysis of their compatibility with biosystems.

    Science.gov (United States)

    Varizhuk, Anna M; Kaluzhny, Dmitry N; Novikov, Roman A; Chizhov, Alexandr O; Smirnov, Igor P; Chuvilin, Andrey N; Tatarinova, Olga N; Fisunov, Gleb Y; Pozmogova, Galina E; Florentiev, Vladimir L

    2013-06-21

    New oligonucleotide analogues with triazole internucleotide linkages were synthesized, and their hybridization properties were studied. The analogues demonstrated DNA binding affinities similar to those of unmodified oligonucleotides. The modification was shown to protect the oligonucleotides from nuclease hydrolysis. The modified oligonucleotides were tested as PCR primers. Modifications remote from the 3'-terminus were tolerated by polymerases. Our results suggest that these new oligonucleotide analogues are among the most promising triazole DNA mimics characterized to date.

  16. [Sequencing by hybridization methods to generate large arrays of oligonucleotides]. Final technical report

    Energy Technology Data Exchange (ETDEWEB)

    NONE

    1996-12-31

    The subject of this project is to address a pressing need for custom DNA microarrays (chips) which can be easily and at low cost formatted and revised for research. In this sense, the term custom means chips for which there is need for limited quantities (less than hundreds) of any particular chip design which contains a large number of different, users defined sequences. Of the three principal approaches to fabricate DNA microarrays, the two which have been commercialized (a and b below) are not particularly suited to research purposes because of the significant time and costs required, once a result is obtained, to utilize that result in the design of a new and better chip: (a) the photodeprotection scheme used by Affymetrix; and (b) the spotting of pre-synthesized oligos or c-DNA onto surfaces.

  17. Universal strategies for the DNA-encoding of libraries of small molecules using the chemical ligation of oligonucleotide tags.

    Science.gov (United States)

    Litovchick, Alexander; Clark, Matthew A; Keefe, Anthony D

    2014-01-01

    The affinity-mediated selection of large libraries of DNA-encoded small molecules is increasingly being used to initiate drug discovery programs. We present universal methods for the encoding of such libraries using the chemical ligation of oligonucleotides. These methods may be used to record the chemical history of individual library members during combinatorial synthesis processes. We demonstrate three different chemical ligation methods as examples of information recording processes (writing) for such libraries and two different cDNA-generation methods as examples of information retrieval processes (reading) from such libraries. The example writing methods include uncatalyzed and Cu(I)-catalyzed alkyne-azide cycloadditions and a novel photochemical thymidine-psoralen cycloaddition. The first reading method "relay primer-dependent bypass" utilizes a relay primer that hybridizes across a chemical ligation junction embedded in a fixed-sequence and is extended at its 3'-terminus prior to ligation to adjacent oligonucleotides. The second reading method "repeat-dependent bypass" utilizes chemical ligation junctions that are flanked by repeated sequences. The upstream repeat is copied prior to a rearrangement event during which the 3'-terminus of the cDNA hybridizes to the downstream repeat and polymerization continues. In principle these reading methods may be used with any ligation chemistry and offer universal strategies for the encoding (writing) and interpretation (reading) of DNA-encoded chemical libraries.

  18. Oligonucleotide-Based Therapy for FTD/ALS Caused by the C9orf72 Repeat Expansion: A Perspective

    Directory of Open Access Journals (Sweden)

    Stephanie A. Fernandes

    2013-01-01

    Full Text Available Amyotrophic lateral sclerosis (ALS is a progressive and lethal disease of motor neuron degeneration, leading to paralysis of voluntary muscles and death by respiratory failure within five years of onset. Frontotemporal dementia (FTD is characterised by degeneration of frontal and temporal lobes, leading to changes in personality, behaviour, and language, culminating in death within 5–10 years. Both of these diseases form a clinical, pathological, and genetic continuum of diseases, and this link has become clearer recently with the discovery of a hexanucleotide repeat expansion in the C9orf72 gene that causes the FTD/ALS spectrum, that is, c9FTD/ALS. Two basic mechanisms have been proposed as being potentially responsible for c9FTD/ALS: loss-of-function of the protein encoded by this gene (associated with aberrant DNA methylation and gain of function through the formation of RNA foci or protein aggregates. These diseases currently lack any cure or effective treatment. Antisense oligonucleotides (ASOs are modified nucleic acids that are able to silence targeted mRNAs or perform splice modulation, and the fact that they have proved efficient in repeat expansion diseases including myotonic dystrophy type 1 makes them ideal candidates for c9FTD/ALS therapy. Here, we discuss potential mechanisms and challenges for developing oligonucleotide-based therapy for c9FTD/ALS.

  19. Oligonucleotide-Based Therapy for FTD/ALS Caused by the C9orf72 Repeat Expansion: A Perspective.

    Science.gov (United States)

    Fernandes, Stephanie A; Douglas, Andrew G L; Varela, Miguel A; Wood, Matthew J A; Aoki, Yoshitsugu

    2013-01-01

    Amyotrophic lateral sclerosis (ALS) is a progressive and lethal disease of motor neuron degeneration, leading to paralysis of voluntary muscles and death by respiratory failure within five years of onset. Frontotemporal dementia (FTD) is characterised by degeneration of frontal and temporal lobes, leading to changes in personality, behaviour, and language, culminating in death within 5-10 years. Both of these diseases form a clinical, pathological, and genetic continuum of diseases, and this link has become clearer recently with the discovery of a hexanucleotide repeat expansion in the C9orf72 gene that causes the FTD/ALS spectrum, that is, c9FTD/ALS. Two basic mechanisms have been proposed as being potentially responsible for c9FTD/ALS: loss-of-function of the protein encoded by this gene (associated with aberrant DNA methylation) and gain of function through the formation of RNA foci or protein aggregates. These diseases currently lack any cure or effective treatment. Antisense oligonucleotides (ASOs) are modified nucleic acids that are able to silence targeted mRNAs or perform splice modulation, and the fact that they have proved efficient in repeat expansion diseases including myotonic dystrophy type 1 makes them ideal candidates for c9FTD/ALS therapy. Here, we discuss potential mechanisms and challenges for developing oligonucleotide-based therapy for c9FTD/ALS.

  20. A study of oligonucleotide occurrence distributions in DNA coding segments.

    Science.gov (United States)

    Castrignanò, T; Colosimo, A; Morante, S; Parisi, V; Rossi, G C

    1997-02-21

    In this paper we present a general strategy designed to study the occurrence frequency distributions of oligonucleotides in DNA coding segments and to deal with the problem of detecting possible patterns of genomic compositional inhomogeneities and disuniformities. Identifying specific tendencies or peculiar deviations in the distributions of the effective occurrence frequencies of oligonucleotides, with respect to what can be a priori expected, is of the greatest importance in biology. Differences between expected and actual distributions may in fact suggest or confirm the existence of specific biological mechanisms related to them. Similarly, a marked deviation in the occurrence frequency of an oligonucleotide may suggest that it belongs to the class of so-called "DNA signal (target) sequences". The approach we have elaborated is innovative in various aspects. Firstly, the analysis of the genomic data is carried out in the light of the observation that the distribution of the four nucleotides along the coding regions of the genoma is biased by the existence of a well-defined "reading frame". Secondly, the "experimental" numbers found by counting the occurrences of the various oligonucleotide sequences are appropriately corrected for the many kinds of mistakes and redundancies present in the available genetic Data Bases. A methodologically significant further improvement of our approach over the existing searching strategies is represented by the fact that, in order to decide whether or not the (corrected) "experimental" value of the occurrence frequency of a given oligonucleotide is within statistical expectations, a measure of the strength of the selective pressure, having acted on it in the course of the evolution, is assigned to the sequence, in a way that takes into account both the value of the "experimental" occurrence frequency of the sequence and the magnitude of the probability that this number might be the result of statistical fluctuations. If the

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2004-03-17

    Desulfovibrio vulgaris Hildenborough has been the focus of biochemical and physiological studies in the laboratory, and the metabolic versatility of this organism has been largely recognized, particularly the reduction of sulfate, fumarate, iron, uranium and chromium. In addition, a Desulfovibrio sp. has been shown to utilize uranium as the sole electron acceptor. D. vulgaris is a d-Proteobacterium with a genome size of 3.6 Mb and 3584 ORFs. The whole-genome microarrays of D. vulgaris have been constructed using 70mer oligonucleotides. All ORFs in the genome were represented with 3471 (97.1%) unique probes and 103 (2.9%) non-specific probes that may have cross-hybridization with other ORFs. In preparation for use of the experimental microarrays, artificial probes and targets were designed to assess specificity and sensitivity and identify optimal hybridization conditions for oligonucleotide microarrays. The results indicated that for 50mer and 70mer oligonucleotide arrays, hybridization at 45 C to 50 C, washing at 37 C and a wash time of 2.5 to 5 minutes obtained specific and strong hybridization signals. In order to evaluate the performance of the experimental microarrays, growth conditions were selected that were expected to give significant hybridization differences for different sets of genes. The initial evaluations were performed using D. vulgaris cells grown at logarithmic and stationary phases. Transcriptional analysis of D. vulgaris cells sampled during logarithmic phase growth indicated that 25% of annotated ORFs were up-regulated and 3% of annotated ORFs were downregulated compared to stationary phase cells. The up-regulated genes included ORFs predicted to be involved with acyl chain biosynthesis, amino acid ABC transporter, translational initiation factors, and ribosomal proteins. In the stationary phase growth cells, the two most up-regulated ORFs (70-fold) were annotated as a carboxynorspermidine decarboxylase and a 2C-methyl-D-erythritol-2

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

    Science.gov (United States)

    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.

  3. Filter arrays

    Energy Technology Data Exchange (ETDEWEB)

    Page, Ralph H.; Doty, Patrick F.

    2017-08-01

    The various technologies presented herein relate to a tiled filter array that can be used in connection with performance of spatial sampling of optical signals. The filter array comprises filter tiles, wherein a first plurality of filter tiles are formed from a first material, the first material being configured such that only photons having wavelengths in a first wavelength band pass therethrough. A second plurality of filter tiles is formed from a second material, the second material being configured such that only photons having wavelengths in a second wavelength band pass therethrough. The first plurality of filter tiles and the second plurality of filter tiles can be interspersed to form the filter array comprising an alternating arrangement of first filter tiles and second filter tiles.

  4. Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1

    Directory of Open Access Journals (Sweden)

    Tobias Bonifert

    2016-01-01

    Full Text Available Inherited optic neuropathies (ION present an important cause of blindness in the European working-age population. Recently we reported the discovery of four independent families with deep intronic mutations in the main inherited optic neuropathies gene OPA1. These deep intronic mutations cause mis-splicing of the OPA1 pre-messenger-RNA transcripts by creating cryptic acceptor splice sites. As a rescue strategy we sought to prevent mis-splicing of the mutant pre-messenger-RNA by applying 2′O-methyl-antisense oligonucleotides (AONs with a full-length phosphorothioate backbone that target the cryptic acceptor splice sites and the predicted novel branch point created by the deep intronic mutations, respectively. Transfection of patient-derived primary fibroblasts with these AONs induced correct splicing of the mutant pre-messenger-RNA in a time and concentration dependent mode of action, as detected by pyrosequencing of informative heterozygous variants. The treatment showed strong rescue effects (≃55% using the cryptic acceptor splice sites targeting AON and moderate rescue (≃16% using the branch point targeting AON. The highest efficacy of Splice correction could be observed 4 days after treatment however, significant effects were still seen 14 days post-transfection. Western blot analysis revealed increased amounts of OPA1 protein with maximum amounts at ≃3 days post-treatment. In summary, we provide the first mutation-specific in vitro rescue strategy for OPA1 deficiency using synthetic AONs.

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

    DEFF Research Database (Denmark)

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

    2012-01-01

    but reduced specificity when incorporating locked nucleic acid monomers, whereas the opposite was observed when introducing unlocked nucleic acid monomers. Our data suggest that phosphorothioate anti-microRNA oligonucleotides yield a greater activity than their phosphodiester counterparts and that a moderate...... truncation of the anti-microRNA oligonucleotide improves specificity without significantly losing activity. These results provide useful insights for design of anti-microRNA oligonucleotides to achieve both high activity as well as efficient mismatch discrimination....

  6. Synthesis of triazole-nucleoside phosphoramidites and their use in solid-phase oligonucleotide synthesis.

    Science.gov (United States)

    Peel, Brandon J; Efthymiou, Tim C; Desaulniers, Jean-Paul

    2014-12-19

    Triazole-backbone oligonucleotides are macromolecules that have one or more triazole units that are acting as a backbone mimic. Triazoles within the backbone have been used within oligonucleotides for a variety of applications. This unit describes the preparation and synthesis of two triazole-nucleoside phosphoramidites [uracil-triazole-uracil (UtU) and cytosine-triazole-uracil (CtU)] based on a PNA-like scaffold, and their incorporation within oligonucleotides.

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

    DEFF Research Database (Denmark)

    1997-01-01

    The present invention relates to a new functionalized achiral linker reagent for incorporating multiple primary amino groups or reporter groups into oligonucleotides following the phosphoramidite methodology. It is possible to substitute any ribodeoxynucleotide, deoxynucleotide, or nucleotide wit......, to a method for preparing a labelled oligonucleotide, and to the use of the labelled oligonucleotide as hybridisation probe, in polymerase chain reactions (PCR), in nucleic acid sequencing, in cloning recombinant DNA and $i(in vitro) mutagenesis....

  8. Hemopoiesis-stimulating activity of immobilized oligonucleotides and hyaluronidase during cytostatic-induced myelosuppression.

    Science.gov (United States)

    Dygai, A M; Skurikhin, E G; Pershina, O V; Zhdanov, V V; Khmelevskaya, A M; Andreeva, T V; Poponina, A M; Zjuzkov, G N; Udut, E V; Khrichkova, T Ju; Simanina, E V; Miroshnichenko, L A; Stavrova, L A; Tchaikovsky, A S; Markova, T S; Gurto, R V; Brjushinina, O S; Slepichev, V A

    2011-03-01

    The hemopoiesis-stimulating effect of combined treatment with immobilized oligonucleotides and hyaluronidase preparations was studied during cytostatic-induced myelosuppression caused by cyclophosphamide administration. Immobilized hyaluronidase was shown to increase the efficiency of correction of changes in the erythroid and granulocytic hemopoietic stems with immobilized oligonucleotides. This potentiation of the effect of immobilized oligonucleotides by immobilized hyaluronidase was related to an increase in functional activity of committed hemopoietic precursors.

  9. Usability of Discovery Portals

    NARCIS (Netherlands)

    Bulens, J.D.; Vullings, L.A.E.; Houtkamp, J.M.; Vanmeulebrouk, B.

    2013-01-01

    As INSPIRE progresses to be implemented in the EU, many new discovery portals are built to facilitate finding spatial data. Currently the structure of the discovery portals is determined by the way spatial data experts like to work. However, we argue that the main target group for discovery portals

  10. Usability of Discovery Portals

    NARCIS (Netherlands)

    Bulens, J.D.; Vullings, L.A.E.; Houtkamp, J.M.; Vanmeulebrouk, B.

    2013-01-01

    As INSPIRE progresses to be implemented in the EU, many new discovery portals are built to facilitate finding spatial data. Currently the structure of the discovery portals is determined by the way spatial data experts like to work. However, we argue that the main target group for discovery portals

  11. Coherent magnetic semiconductor nanodot arrays

    Directory of Open Access Journals (Sweden)

    Xiu Faxian

    2011-01-01

    Full Text Available Abstract In searching appropriate candidates of magnetic semiconductors compatible with mainstream Si technology for future spintronic devices, extensive attention has been focused on Mn-doped Ge magnetic semiconductors. Up to now, lack of reliable methods to obtain high-quality MnGe nanostructures with a desired shape and a good controllability has been a barrier to make these materials practically applicable for spintronic devices. Here, we report, for the first time, an innovative growth approach to produce self-assembled and coherent magnetic MnGe nanodot arrays with an excellent reproducibility. Magnetotransport experiments reveal that the nanodot arrays possess giant magneto-resistance associated with geometrical effects. The discovery of the MnGe nanodot arrays paves the way towards next-generation high-density magnetic memories and spintronic devices with low-power dissipation.

  12. Efficient assembly of very short oligonucleotides using T4 DNA Ligase

    Directory of Open Access Journals (Sweden)

    Holt Robert A

    2010-11-01

    Full Text Available Abstract Background In principle, a pre-constructed library of all possible short oligonucleotides could be used to construct many distinct gene sequences. In order to assess the feasibility of such an approach, we characterized T4 DNA Ligase activity on short oligonucleotide substrates and defined conditions suitable for assembly of a plurality of oligonucleotides. Findings Ligation by T4 DNA Ligase was found to be dependent on the formation of a double stranded DNA duplex of at least five base pairs surrounding the site of ligation. However, ligations could be performed effectively with overhangs smaller than five base pairs and oligonucleotides as small as octamers, in the presence of a second, complementary oligonucleotide. We demonstrate the feasibility of simultaneous oligonucleotide phosphorylation and ligation and, as a proof of principle for DNA synthesis through the assembly of short oligonucleotides, we performed a hierarchical ligation procedure whereby octamers were combined to construct a target 128-bp segment of the beta-actin gene. Conclusions Oligonucleotides as short as 8 nucleotides can be efficiently assembled using T4 DNA Ligase. Thus, the construction of synthetic genes, without the need for custom oligonucleotide synthesis, appears feasible.

  13. Determination of optimal sites of antisense oligonucleotide cleavage within TNFα mRNA

    Science.gov (United States)

    Lloyd, B. H.; Giles, R. V.; Spiller, D. G.; Grzybowski, J.; Tidd, D. M.; Sibson, D. R.

    2001-01-01

    Antisense oligonucleotides provide a powerful tool in order to determine the consequences of the reduced expression of a selected target gene and may include target validation and therapeutic applications. Methods of predicting optimum antisense sites are not always effective. We have compared the efficacy of antisense oligonucleotides, which were selected in vitro using random combinatorial oligonucleotide libraries of differing length and complexity, upon putative target sites within TNFα mRNA. The relationship of specific target site accessibility and oligonucleotide efficacy with respect to these parameters proved to be complex. Modification of the length of the recognition sequence of the oligonucleotide library illustrated that independent target sites demonstrated a preference for antisense oligonucleotides of a defined and independent optimal length. The efficacy of antisense oligonucleotide sequences selected in vitro paralleled that observed in phorbol 12-myristate 13-acetate (PMA)-activated U937 cells. The application of methylphosphonate:phosphodiester chimaeric oligonucleotides to U937 cells reduced mRNA levels to up to 19.8% that of the untreated cell population. This approach provides a predictive means to profile any mRNA of known sequence with respect to the identification and optimisation of sites accessible to antisense oligonucleotide activity. PMID:11522838

  14. Label-free detection of hybridization of oligonucleotides by oblique-incidence reflectivity difference method

    Institute of Scientific and Technical Information of China (English)

    2010-01-01

    The microarrays of 20-base oligonucleotide with different concentrations are detected before and after hybridization by the oblique-incidence reflectivity difference (OI-RD) method. The experimental results prove that OI-RD is a label-free method which can not only distinguish the concentration difference of oligonucleotides before and after the hybridization but also detect the hybridization of short oligonucleotides. At present the OI-RD method can detect 0.39 μmol/L 20-base oligonucleotide or less. These results suggest that the OI-RD method is a promising and potential technique for label-free detection of biological microarrays.

  15. Comparison of seven methods for producing Affymetrix expression scores based on False Discovery Rates in disease profiling data

    Directory of Open Access Journals (Sweden)

    Gruber Stephen B

    2005-02-01

    Full Text Available Abstract Background A critical step in processing oligonucleotide microarray data is combining the information in multiple probes to produce a single number that best captures the expression level of a RNA transcript. Several systematic studies comparing multiple methods for array processing have used tightly controlled calibration data sets as the basis for comparison. Here we compare performances for seven processing methods using two data sets originally collected for disease profiling studies. An emphasis is placed on understanding sensitivity for detecting differentially expressed genes in terms of two key statistical determinants: test statistic variability for non-differentially expressed genes, and test statistic size for truly differentially expressed genes. Results In the two data sets considered here, up to seven-fold variation across the processing methods was found in the number of genes detected at a given false discovery rate (FDR. The best performing methods called up to 90% of the same genes differentially expressed, had less variable test statistics under randomization, and had a greater number of large test statistics in the experimental data. Poor performance of one method was directly tied to a tendency to produce highly variable test statistic values under randomization. Based on an overall measure of performance, two of the seven methods (Dchip and a trimmed mean approach are superior in the two data sets considered here. Two other methods (MAS5 and GCRMA-EB are inferior, while results for the other three methods are mixed. Conclusions Choice of processing method has a major impact on differential expression analysis of microarray data. Previously reported performance analyses using tightly controlled calibration data sets are not highly consistent with results reported here using data from human tissue samples. Performance of array processing methods in disease profiling and other realistic biological studies should be

  16. Chemically modified oligonucleotides with efficient RNase H response

    DEFF Research Database (Denmark)

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

    2008-01-01

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

  17. Splice-switching antisense oligonucleotides as therapeutic drugs

    OpenAIRE

    Havens, Mallory A.; Hastings, Michelle L.

    2016-01-01

    Splice-switching oligonucleotides (SSOs) are short, synthetic, antisense, modified nucleic acids that base-pair with a pre-mRNA and disrupt the normal splicing repertoire of the transcript by blocking the RNA–RNA base-pairing or protein–RNA binding interactions that occur between components of the splicing machinery and the pre-mRNA. Splicing of pre-mRNA is required for the proper expression of the vast majority of protein-coding genes, and thus, targeting the process offers a means to manipu...

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

    DEFF Research Database (Denmark)

    Taskova, Maria; Uhd, Jesper; Miotke, Laura

    2017-01-01

    followed by click assembly and analysis of the read sequence by various techniques. As we demonstrate in this paper, using our new approach, a viral RNA sequence can be detected in less than 2 h without the need for cDNA synthesis or any other enzymatic reactions and with a sensitivity of ... opportunities in transcriptome analysis, virology, and other fields. Herein, we report for the first time a "click" chemistry approach to oligonucleotide probe elongation as a novel approach to specifically detect a viral sequence. We hybridized a library of short, terminally labeled probes to Ebola virus RNA...

  19. Identification of upper respiratory tract pathogens using electrochemical detection on an oligonucleotide microarray.

    Directory of Open Access Journals (Sweden)

    Michael J Lodes

    Full Text Available Bacterial and viral upper respiratory infections (URI produce highly variable clinical symptoms that cannot be used to identify the etiologic agent. Proper treatment, however, depends on correct identification of the pathogen involved as antibiotics provide little or no benefit with viral infections. Here we describe a rapid and sensitive genotyping assay and microarray for URI identification using standard amplification and hybridization techniques, with electrochemical detection (ECD on a semiconductor-based oligonucleotide microarray. The assay was developed to detect four bacterial pathogens (Bordetella pertussis, Streptococcus pyogenes, Chlamydia pneumoniae and Mycoplasma pneumoniae and 9 viral pathogens (adenovirus 4, coronavirus OC43, 229E and HK, influenza A and B, parainfluenza types 1, 2, and 3 and respiratory syncytial virus. This new platform forms the basis for a fully automated diagnostics system that is very flexible and can be customized to suit different or additional pathogens. Multiple probes on a flexible platform allow one to test probes empirically and then select highly reactive probes for further iterative evaluation. Because ECD uses an enzymatic reaction to create electrical signals that can be read directly from the array, there is no need for image analysis or for expensive and delicate optical scanning equipment. We show assay sensitivity and specificity that are excellent for a multiplexed format.

  20. Strong position-dependent effects of sequence mismatches on signal ratios measured using long oligonucleotide microarrays

    Directory of Open Access Journals (Sweden)

    Hulme Helen

    2008-07-01

    Full Text Available Abstract Background Microarrays are an important and widely used tool. Applications include capturing genomic DNA for high-throughput sequencing in addition to the traditional monitoring of gene expression and identifying DNA copy number variations. Sequence mismatches between probe and target strands are known to affect the stability of the probe-target duplex, and hence the strength of the observed signals from microarrays. Results We describe a large-scale investigation of microarray hybridisations to murine probes with known sequence mismatches, demonstrating that the effect of mismatches is strongly position-dependent and for small numbers of sequence mismatches is correlated with the maximum length of perfectly matched probe-target duplex. Length of perfect match explained 43% of the variance in log2 signal ratios between probes with one and two mismatches. The correlation with maximum length of perfect match does not conform to expectations based on considering the effect of mismatches purely in terms of reducing the binding energy. However, it can be explained qualitatively by considering the entropic contribution to duplex stability from configurations of differing perfect match length. Conclusion The results of this study have implications in terms of array design and analysis. They highlight the significant effect that short sequence mismatches can have upon microarray hybridisation intensities even for long oligonucleotide probes. All microarray data presented in this study are available from the GEO database 1, under accession number [GEO: GSE9669

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

    Directory of Open Access Journals (Sweden)

    Ping Gong

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

  2. An overview of sugar-modified oligonucleotides for antisense therapeutics.

    Science.gov (United States)

    Prakash, Thazha P

    2011-09-01

    Among the multitude of chemical modifications that have been described over the past two decades, oligonucleotide analogs that are modified at the 2'-position of the furanose sugar have been especially useful for improving the drug-like properties of antisense oligonucleotides (ASOs). These modifications bias the sugar pucker towards the 3'-endo-conformation and improve ASO affinity for its biological target (i.e., mRNA). In addition, antisense drugs incorporating 2'-modified nucleotides exhibit enhanced metabolic stability, and improved pharmacokinetic and toxicological properties. Further conformational restriction of the 2'-substituent to the 4'-position of the furanose ring yielded the 2',4'-bridged nucleic acid (BNA) analogs. ASOs containing BNA modifications showed unprecedented increase in binding affinity for target RNA, while also improved nuclease resistance, in vitro and in vivo potency. Several ASO drug candidates containing 2'-modified nucleotides have entered clinical trials and continue to make progress in the clinic for a variety of therapeutic indications. 2011 Verlag Helvetica Chimica Acta AG, Zürich.

  3. The use of oligonucleotide probes for meningococcal serotype characterization

    Directory of Open Access Journals (Sweden)

    SACCHI Claudio Tavares

    1998-01-01

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

  4. Photolithographic Synthesis of High-Density DNA and RNA Arrays on Flexible, Transparent, and Easily Subdivided Plastic Substrates.

    Science.gov (United States)

    Holden, Matthew T; Carter, Matthew C D; Wu, Cheng-Hsien; Wolfer, Jamison; Codner, Eric; Sussman, Michael R; Lynn, David M; Smith, Lloyd M

    2015-11-17

    The photolithographic fabrication of high-density DNA and RNA arrays on flexible and transparent plastic substrates is reported. The substrates are thin sheets of poly(ethylene terephthalate) (PET) coated with cross-linked polymer multilayers that present hydroxyl groups suitable for conventional phosphoramidite-based nucleic acid synthesis. We demonstrate that by modifying array synthesis procedures to accommodate the physical and chemical properties of these materials, it is possible to synthesize plastic-backed oligonucleotide arrays with feature sizes as small as 14 μm × 14 μm and feature densities in excess of 125 000/cm(2), similar to specifications attainable using rigid substrates such as glass or glassy carbon. These plastic-backed arrays are tolerant to a wide range of hybridization temperatures, and improved synthetic procedures are described that enable the fabrication of arrays with sequences up to 50 nucleotides in length. These arrays hybridize with S/N ratios comparable to those fabricated on otherwise identical arrays prepared on glass or glassy carbon. This platform supports the enzymatic synthesis of RNA arrays and proof-of-concept experiments are presented showing that the arrays can be readily subdivided into smaller arrays (or "millichips") using common laboratory-scale laser cutting tools. These results expand the utility of oligonucleotide arrays fabricated on plastic substrates and open the door to new applications for these important bioanalytical tools.

  5. The MOX/SUC precursor strategies: robust ways to construct functionalized oligonucleotides.

    Science.gov (United States)

    Polushin, N

    2001-01-01

    The use of phosphoramidites bearing one or more methoxyoxalamido (MOX) or succinimido (SUC) reactive groups for construction of functionalized oligonucleotides is described. The efficiency of the new precursor strategy was demonstrated in the synthesis of oligonucleotide containing up to 16 imidazole residues.

  6. Multicellular Tumor Spheroids as a Model for Assessing Delivery of Oligonucleotides in Three Dimensions

    Science.gov (United States)

    Carver, Kyle; Ming, Xin; Juliano, Rudolph L

    2014-01-01

    Oligonucleotides have shown promise in selectively manipulating gene expression in vitro, but that success has not translated to the clinic for cancer therapy. A potential reason for this is that cells behave differently in monolayer than in the three-dimensional tumor, resulting in limited penetration and distribution of oligonucleotides in the tumor. This may be especially true when oligonucleotides are associated with nanocarriers such as lipoplexes and polyplexes, commonly used delivery vehicles for oligonucleotides. The multicellular tumor spheroid (MCTS), a three-dimensional model that closely resembles small avascular tumors and micrometastases, has been utilized as an intermediate between monolayer culture and in vivo studies for the screening of small-molecule drugs. However, spheroids have been little used for the study of various oligonucleotide delivery formulations. Here, we have evaluated the uptake and efficacy of splice-switching antisense oligonucleotides using various delivery modalities in two- and three-dimensional culture models. We find that the size of the delivery agent dramatically influences penetration into the spheroid and thus the biological effect of the oligonucleotides. We hypothesize that the MCTS model will prove to be a useful tool in the future development of oligonucleotide delivery formulations. PMID:24618852

  7. Nucleobase azide-ethynylribose click chemistry contributes to stabilizing oligonucleotide duplexes and stem-loop structures.

    Science.gov (United States)

    Kitamura, Yoshiaki; Asakura, Ryo; Terazawa, Koki; Shibata, Aya; Ikeda, Masato; Kitade, Yukio

    2017-06-15

    The formation of 1,4-disubstituted 1,2,3-triazoles through copper-catalyzed azide-alkyne cycloaddition (CuAAC) in oligonucleotides bearing 1-deoxy-1-ethynyl-β-d-ribofuranose (R(E)) can have a positive impact on the stability of oligonucleotide duplexes and stem-loop structures. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

    Institute of Scientific and Technical Information of China (English)

    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.

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

    Science.gov (United States)

    Golova, Julia B.; Chernov, Boris K.

    2010-04-27

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

  10. Detection of oligonucleotide hybridization on a single microparticle by time-resolved fluorometry: hybridization assays on polymer particles obtained by direct solid phase assembly of the oligonucleotide probes.

    Science.gov (United States)

    Hakala, H; Heinonen, P; Iitiä, A; Lönnberg, H

    1997-01-01

    Oligodeoxyribonucleotides were assembled by conventional phosphoramidite chemistry on uniformly sized (50 microns) porous glycidyl methacrylate/ethylene dimethacrylate (SINTEF) and compact polystyrene (Dynosphere) particles, the aminoalkyl side chains of which were further derivatized with DMTrO-acetyl groups. The linker was completely resistant toward ammonolytic deprotection of the base moieties. The quality of oligonucleotides was assessed by repeating the synthesis on the same particles derivatized with a cleavable ester linker. The ability of the oligonucleotide-coated particles to bind complementary sequences via hybridization was examined by following the attachment of oligonucleotides bearing a photoluminescent europium(III) chelate to the particles. The fluorescence emission was measured directly on a single particle. The effects of the following factors on the kinetics and efficiency of hybridization were studied: number of particles in a given volume of the assay solution, loading of oligonucleotide on the particle, concentration of the target oligonucleotide in solution, length of the hybridizing sequence, presence of noncomplementary sequences, and ionic strength. The fluorescence signal measured on a single particle after hybridization was observed to be proportional to the concentration of the target oligonucleotide in solution over a concentration range of 5 orders of magnitude.

  11. Computational drug discovery

    Institute of Scientific and Technical Information of China (English)

    Si-sheng OU-YANG; Jun-yan LU; Xiang-qian KONG; Zhong-jie LIANG; Cheng LUO; Hualiang JIANG

    2012-01-01

    Computational drug discovery is an effective strategy for accelerating and economizing drug discovery and development process.Because of the dramatic increase in the availability of biological macromolecule and small molecule information,the applicability of computational drug discovery has been extended and broadly applied to nearly every stage in the drug discovery and development workflow,including target identification and validation,lead discovery and optimization and preclinical tests.Over the past decades,computational drug discovery methods such as molecular docking,pharmacophore modeling and mapping,de novo design,molecular similarity calculation and sequence-based virtual screening have been greatly improved.In this review,we present an overview of these important computational methods,platforms and successful applications in this field.

  12. Cellular Uptake and Intracellular Trafficking of Antisense and siRNA Oligonucleotides

    Science.gov (United States)

    Juliano, RL; Ming, Xin; Nakagawa, Osamu

    2012-01-01

    Significant progress is being made concerning the development of oligonucleotides as therapeutic agents. Studies with antisense, siRNA, and other forms of oligonucleotides have shown promise in cellular and animal models and in some clinical studies. Nonetheless our understanding of how oligonucleotides function in cells and tissues is really quite limited. One major issue concerns the modes of uptake and intracellular trafficking of oligonucleotides, whether as ‘free’ molecules, or linked to various delivery moieties such as nanoparticles or targeting ligands. In this review we examine the recent literature on oligonucleotide internalization and subcellular trafficking in the context of current insights into the basic machinery for endocytosis and intracellular vesicular traffic. PMID:21992697

  13. Affinity hydrogels for controlled protein release using nucleic acid aptamers and complementary oligonucleotides.

    Science.gov (United States)

    Soontornworajit, Boonchoy; Zhou, Jing; Snipes, Matthew P; Battig, Mark R; Wang, Yong

    2011-10-01

    Biomaterials for the precise control of protein release are important to the development of new strategies for treating human diseases. This study aimed to fundamentally understand aptamer--protein dissociation triggered by complementary oligonucleotides, and to apply this understanding to develop affinity hydrogels for controlled protein release. The results showed that the oligonucleotide tails of the aptamers played a critical role in inducing intermolecular hybridization and triggering aptamer--protein dissociation. In addition, the attachment of the oligonucleotide tails to the aptamers and the increase of hybridizing length could produce a synergistic effect on the dissociation of bound proteins from their aptamers. More importantly, pegylated complementary oligonucleotides could successfully trigger protein release from the aptamer-functionalized hydrogels at multiple time points. Based on these results, it is believed that aptamer-functionalized hydrogels and complementary oligonucleotides hold great potential of controlling the release of protein drugs to treat human diseases.

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

    Directory of Open Access Journals (Sweden)

    Magness Charles L

    2007-01-01

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

  15. High resolution microarray comparative genomic hybridisation analysis using spotted oligonucleotides.

    NARCIS (Netherlands)

    Carvalho, B; Ouwerkerk, E; Meijer, G.A.; Ylstra, B.

    2004-01-01

    BACKGROUND: Currently, comparative genomic hybridisation array (array CGH) is the method of choice for studying genome wide DNA copy number changes. To date, either amplified representations of bacterial artificial chromosomes (BACs)/phage artificial chromosomes (PACs) or cDNAs have been spotted as

  16. Reliable knowledge discovery

    CERN Document Server

    Dai, Honghua; Smirnov, Evgueni

    2012-01-01

    Reliable Knowledge Discovery focuses on theory, methods, and techniques for RKDD, a new sub-field of KDD. It studies the theory and methods to assure the reliability and trustworthiness of discovered knowledge and to maintain the stability and consistency of knowledge discovery processes. RKDD has a broad spectrum of applications, especially in critical domains like medicine, finance, and military. Reliable Knowledge Discovery also presents methods and techniques for designing robust knowledge-discovery processes. Approaches to assessing the reliability of the discovered knowledge are introduc

  17. A male newborn with VACTERL association and Fanconi anemia with a FANCB deletion detected by array comparative genomic hybridization (aCGH).

    Science.gov (United States)

    Umaña, Luis A; Magoulas, Pilar; Bi, Weimin; Bacino, Carlos A

    2011-12-01

    We report on a male newborn with multiple congenital abnormalities consistent with the diagnosis of VACTERL association (vertebral, anal, cardiac, tracheo-esophageal fistula, renal, and limb anomalies), who had Fanconi anemia (complementation group B) recognized by the detection of a deletion in chromosome Xp22.2 using an oligonucleotide array. The diagnosis of Fanconi anemia was confirmed by increased chromosomal breakage abnormalities observed in cultured cells that were treated with cross-linking agents. This is the first report in the literature of Fanconi anemia complementation group B detected by oligonucleotide array testing postnatally.

  18. Recommendations for safety pharmacology evaluations of oligonucleotide-based therapeutics.

    Science.gov (United States)

    Berman, Cindy L; Cannon, Keri; Cui, Yi; Kornbrust, Douglas J; Lagrutta, Armando; Sun, Sunny Z; Tepper, Jeff; Waldron, Gareth; Younis, Husam S

    2014-08-01

    This document was prepared by the Safety Pharmacology Subcommittee of the Oligonucleotide Safety Working Group (OSWG), a group of industry and regulatory scientists involved in the development and regulation of therapeutic oligonucleotides. The mission of the Subcommittee was to develop scientific recommendations for the industry regarding the appropriate scope and strategies for safety pharmacology evaluations of oligonucleotides (ONs). These recommendations are the consensus opinion of the Subcommittee and do not necessarily reflect the current expectations of regulatory authorities. 1) Safety pharmacology testing, as described in the International Conference on Harmonisation (ICH) S7 guidance, is as applicable to ONs as it is to small molecule drugs and biotherapeutics. 2) Study design considerations for ONs are similar to those for other classes of drugs. In general, as with other therapeutics, studies should evaluate the drug product administered via the clinical route. Species selection should ideally consider relevance of the model with regard to the endpoints of interest, pharmacological responsiveness, and continuity with the nonclinical development program. 3) Evaluation of potential effects in the core battery (cardiovascular, central nervous, and respiratory systems) is recommended. In general: a. In vitro human ether-a-go-go-related gene (hERG) testing does not provide any specific value and is not warranted. b. Emphasis should be placed on in vivo evaluation of cardiovascular function, typically in nonhuman primates (NHPs). c. Due to the low level of concern, neurologic and respiratory function can be assessed concurrently with cardiovascular safety pharmacology evaluation in NHPs, within repeat-dose toxicity studies, or as stand-alone studies. In the latter case, rodents are most commonly used. 4) Other dedicated safety pharmacology studies, beyond the core battery, may have limited value for ONs. Although ONs can accumulate in the kidney and liver

  19. Global Arrays

    Energy Technology Data Exchange (ETDEWEB)

    Krishnamoorthy, Sriram; Daily, Jeffrey A.; Vishnu, Abhinav; Palmer, Bruce J.

    2015-11-01

    Global Arrays (GA) is a distributed-memory programming model that allows for shared-memory-style programming combined with one-sided communication, to create a set of tools that combine high performance with ease-of-use. GA exposes a relatively straightforward programming abstraction, while supporting fully-distributed data structures, locality of reference, and high-performance communication. GA was originally formulated in the early 1990’s to provide a communication layer for the Northwest Chemistry (NWChem) suite of chemistry modeling codes that was being developed concurrently.

  20. Cyberinfrastructure for Atmospheric Discovery

    Science.gov (United States)

    Wilhelmson, R.; Moore, C. W.

    2004-12-01

    Each year across the United States, floods, tornadoes, hail, strong winds, lightning, hurricanes, and winter storms cause hundreds of deaths, routinely disrupt transportation and commerce, and result in billions of dollars in annual economic losses . MEAD and LEAD are two recent efforts aimed at developing the cyberinfrastructure for studying and forecasting these events through collection, integration, and analysis of observational data coupled with numerical simulation, data mining, and visualization. MEAD (Modeling Environment for Atmospheric Discovery) has been funded for two years as an NCSA (National Center for Supercomputing Applications) Alliance Expedition. The goal of this expedition has been the development/adaptation of cyberinfrastructure that will enable research simulations, datamining, machine learning and visualization of hurricanes and storms utilizing the high performance computing environments including the TeraGrid. Portal grid and web infrastructure are being tested that will enable launching of hundreds of individual WRF (Weather Research and Forecasting) simulations. In a similar way, multiple Regional Ocean Modeling System (ROMS) or WRF/ROMS simulations can be carried out. Metadata and the resulting large volumes of data will then be made available for further study and for educational purposes using analysis, mining, and visualization services. Initial coupling of the ROMS and WRF codes has been completed and parallel I/O is being implemented for these models. Management of these activities (services) are being enabled through Grid workflow technologies (e.g. OGCE). LEAD (Linked Environments for Atmospheric Discovery) is a recently funded 5-year, large NSF ITR grant that involves 9 institutions who are developing a comprehensive national cyberinfrastructure in mesoscale meteorology, particularly one that can interoperate with others being developed. LEAD is addressing the fundamental information technology (IT) research challenges needed

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

    DEFF Research Database (Denmark)

    Madsen, Andreas S; Wengel, Jesper

    2012-01-01

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

  2. Antisense Oligonucleotide-Mediated Transcript Knockdown in Zebrafish.

    Directory of Open Access Journals (Sweden)

    Andrea Pauli

    Full Text Available Antisense oligonucleotides (ASOs are synthetic, single-strand RNA-DNA hybrids that induce catalytic degradation of complementary cellular RNAs via RNase H. ASOs are widely used as gene knockdown reagents in tissue culture and in Xenopus and mouse model systems. To test their effectiveness in zebrafish, we targeted 20 developmental genes and compared the morphological changes with mutant and morpholino (MO-induced phenotypes. ASO-mediated transcript knockdown reproduced the published loss-of-function phenotypes for oep, chordin, dnd, ctnnb2, bmp7a, alk8, smad2 and smad5 in a dosage-sensitive manner. ASOs knocked down both maternal and zygotic transcripts, as well as the long noncoding RNA (lncRNA MALAT1. ASOs were only effective within a narrow concentration range and were toxic at higher concentrations. Despite this drawback, quantitation of knockdown efficiency and the ability to degrade lncRNAs make ASOs a useful knockdown reagent in zebrafish.

  3. Antisense Oligonucleotides: Translation from Mouse Models to Human Neurodegenerative Diseases.

    Science.gov (United States)

    Schoch, Kathleen M; Miller, Timothy M

    2017-06-21

    Multiple neurodegenerative diseases are characterized by single-protein dysfunction and aggregation. Treatment strategies for these diseases have often targeted downstream pathways to ameliorate consequences of protein dysfunction; however, targeting the source of that dysfunction, the affected protein itself, seems most judicious to achieve a highly effective therapeutic outcome. Antisense oligonucleotides (ASOs) are small sequences of DNA able to target RNA transcripts, resulting in reduced or modified protein expression. ASOs are ideal candidates for the treatment of neurodegenerative diseases, given numerous advancements made to their chemical modifications and delivery methods. Successes achieved in both animal models and human clinical trials have proven ASOs both safe and effective. With proper considerations in mind regarding the human applicability of ASOs, we anticipate ongoing in vivo research and clinical trial development of ASOs for the treatment of neurodegenerative diseases. Copyright © 2017 Elsevier Inc. All rights reserved.

  4. Antisense oligonucleotide targeting midkine suppresses in vivo angiogenesis

    Institute of Scientific and Technical Information of China (English)

    Li-Cheng Dai; Xiang Wang; Xing Yao; Yong-Liang Lu; Jin-Liang Ping; Jian-Fang He

    2007-01-01

    AIM: To evaluate the effect of antisense oligonucleotide targeting midkine (MK-AS) on angiogenesis in chick chorioallantoic membrane (CAM) andin situ human hepatocellular carcinoma (HCC).METHODS: An in situ human hepatocellular carcinoma (HCC) model and CAM assay were used in this experiment. The effect of MK-AS on angiogenesis was evaluated by cell proliferation assay and hematoxylineosin (HE) staining.RESULTS: MK-AS significantly inhibited human umbilical vein endothelial cells (HUVEC) and in situ human HCC growth. At the same time, MK-AS suppressed the angiogenesis both in human hepatocellular carcinoma cell line (HEPG2)-induced CAM and in situ human HCC tissues.CONCLUSION: MK-AS is an effective antiangiogenesis agent in vivo.

  5. Tetramerization of an RNA oligonucleotide containing a GGGG sequence.

    Science.gov (United States)

    Kim, J; Cheong, C; Moore, P B

    1991-05-23

    Poly rG can form four-stranded helices. The Hoogsteen-paired quartets of G residues on which such structures depend are so stable that they will form in 5'-GMP solutions, provided that Na+ or K+ are present (see for example, refs 2-4). Telomeric DNA sequences, which are G-rich, adopt four-stranded antiparallel G-quartet conformations in vitro, and parallel tetramerization of G-rich sequences may be involved in meiosis. Here we show that RNAs containing short runs of Gs can also tetramerize. A 19-base oligonucleotide derived from the 5S RNA of Escherichia coli (strand III), 5'GCCGAUGGUAGUGUGGGGU3', forms a K(+)-stabilized tetrameric aggregate that depends on the G residues at its 3' end. This complex is so stable that it would be surprising if similar structures do not occur in nature.

  6. Design and development of thermolytic DNA oligonucleotide prodrugs.

    Science.gov (United States)

    Grajkowski, Andrzej; Pedras-Vasconcelos, Joao; Ausín, Cristina; Verthelyi, Daniela; Beaucage, Serge L

    2005-11-01

    Deoxyribonucleoside phosphoramidites functionalized with the thermolytic 2-(N-formyl-N-methyl)aminoethyl group for phosphorus protection (1a-d) have been prepared and employed in the solid-phase synthesis of CpG ODN fma1555. Given that this modified oligonucleotide can be converted to the immunomodulatory CpG ODN 1555 under neutral conditions at 37 degrees C, its biologic activity was demonstrated in vivo by studies showing that intraperitoneal administration of CpG ODN fma1555 in mice resulted in the activation of cytokine-secreting splenocytes. Furthermore, administration of CpG ODN fma1555 to mice that were challenged intradermally in the ear with live L. major metacyclic promastigotes, reduced the severity of Leishmania skin lesions over time to an extent similar to that obtained with CpG ODN 1555. In another infectious model experiment, CpG ODN fma1555 protected newborn mice from death (65% survival) when administered 3 days before infection with the aggressive Tacaribe (TCRV) virus. A comparable immunoprotection was obtained by treatment of TCRV-infected mice with CpG ODN 1555 administered on the same day of infection (45% survival). However, when TCRV-infected mice were treated with CpG ODN fma1555 on the day of infection, they died as a consequence of the relatively slow conversion of the oligonucleotide prodrug to the bioactive CpG ODN 1555. Co-administration of both CpG ODN 1555 and CpG ODN fma1555 to mice 3 days prior to TCRV infection or on the day of infection provided protection from death (45-65% survival) and thus widened the immunoprotection window against TCRV-infection.

  7. Academic Drug Discovery Centres

    DEFF Research Database (Denmark)

    Kirkegaard, Henriette Schultz; Valentin, Finn

    2014-01-01

    Academic drug discovery centres (ADDCs) are seen as one of the solutions to fill the innovation gap in early drug discovery, which has proven challenging for previous organisational models. Prior studies of ADDCs have identified the need to analyse them from the angle of their economic...... their performance....

  8. Higgs Discovery Movie

    CERN Multimedia

    2014-01-01

    The ATLAS & CMS Experiments Celebrate the 2nd Anniversary of the Discovery of the Higgs boson. Here, are some images of the path from LHC startup to Nobel Prize, featuring a musical composition by Roger Zare, performed by the Donald Sinta Quartet, called “LHC”. Happy Discovery Day!

  9. Friends' Discovery Camp

    Science.gov (United States)

    Seymour, Seth

    2008-01-01

    This article features Friends' Discovery Camp, a program that allows children with and without autism spectrum disorder to learn and play together. In Friends' Discovery Camp, campers take part in sensory-rich experiences, ranging from hands-on activities and performing arts to science experiments and stories teaching social skills. Now in its 7th…

  10. Serendipity and Scientific Discovery.

    Science.gov (United States)

    Rosenman, Martin F.

    1988-01-01

    The discovery of penicillin is cited in a discussion of the role of serendipity as it relates to scientific discovery. The importance of sagacity as a personality trait is noted. Successful researchers have questioning minds, are willing to view data from several perspectives, and recognize and appreciate the unexpected. (JW)

  11. Empirical Discovery in Linguistics

    CERN Document Server

    Pericliev, V

    1995-01-01

    A discovery system for detecting correspondences in data is described, based on the familiar induction methods of J. S. Mill. Given a set of observations, the system induces the ``causally'' related facts in these observations. Its application to empirical linguistic discovery is described.

  12. Service discovery at home

    NARCIS (Netherlands)

    Sundramoorthy, V.; Scholten, Johan; Jansen, P.G.; Hartel, Pieter H.

    2003-01-01

    Service discovery is a fairly new field that kicked off since the advent of ubiquitous computing and has been found essential in the making of intelligent networks by implementing automated discovery and remote control between devices. This paper provides an overview and comparison of several promin

  13. Service Discovery At Home

    NARCIS (Netherlands)

    Sundramoorthy, V.; Scholten, Johan; Jansen, P.G.; Hartel, Pieter H.

    2003-01-01

    Service discovery is a fady new field that kicked off since the advent of ubiquitous computing and has been found essential in the making of intelligent networks by implementing automated discovery and remote control between deviies. This paper provides an ovewiew and comparison of several prominent

  14. Service discovery at home

    NARCIS (Netherlands)

    Sundramoorthy, V.; Scholten, Johan; Jansen, P.G.; Hartel, Pieter H.

    2003-01-01

    Service discovery is a fairly new field that kicked off since the advent of ubiquitous computing and has been found essential in the making of intelligent networks by implementing automated discovery and remote control between devices. This paper provides an overview and comparison of several

  15. Service Discovery At Home

    NARCIS (Netherlands)

    Sundramoorthy, V.; Scholten, Johan; Jansen, P.G.; Hartel, Pieter H.

    Service discovery is a fady new field that kicked off since the advent of ubiquitous computing and has been found essential in the making of intelligent networks by implementing automated discovery and remote control between deviies. This paper provides an ovewiew and comparison of several prominent

  16. Service discovery at home

    NARCIS (Netherlands)

    Sundramoorthy, Vasughi; Scholten, Hans; Jansen, Pierre; Hartel, Pieter

    2003-01-01

    Service discovery is a fairly new field that kicked off since the advent of ubiquitous computing and has been found essential in the making of intelligent networks by implementing automated discovery and remote control between devices. This paper provides an overview and comparison of several promin

  17. Sample phenotype clusters in high-density oligonucleotide microarray data sets are revealed using Isomap, a nonlinear algorithm

    Directory of Open Access Journals (Sweden)

    Malyj Wasyl

    2005-08-01

    Full Text Available Abstract Background Life processes are determined by the organism's genetic profile and multiple environmental variables. However the interaction between these factors is inherently non-linear 1. Microarray data is one representation of the nonlinear interactions among genes and genes and environmental factors. Still most microarray studies use linear methods for the interpretation of nonlinear data. In this study, we apply Isomap, a nonlinear method of dimensionality reduction, to analyze three independent large Affymetrix high-density oligonucleotide microarray data sets. Results Isomap discovered low-dimensional structures embedded in the Affymetrix microarray data sets. These structures correspond to and help to interpret biological phenomena present in the data. This analysis provides examples of temporal, spatial, and functional processes revealed by the Isomap algorithm. In a spinal cord injury data set, Isomap discovers the three main modalities of the experiment – location and severity of the injury and the time elapsed after the injury. In a multiple tissue data set, Isomap discovers a low-dimensional structure that corresponds to anatomical locations of the source tissues. This model is capable of describing low- and high-resolution differences in the same model, such as kidney-vs.-brain and differences between the nuclei of the amygdala, respectively. In a high-throughput drug screening data set, Isomap discovers the monocytic and granulocytic differentiation of myeloid cells and maps several chemical compounds on the two-dimensional model. Conclusion Visualization of Isomap models provides useful tools for exploratory analysis of microarray data sets. In most instances, Isomap models explain more of the variance present in the microarray data than PCA or MDS. Finally, Isomap is a promising new algorithm for class discovery and class prediction in high-density oligonucleotide data sets.

  18. "Eureka, Eureka!" Discoveries in Science

    Science.gov (United States)

    Agarwal, Pankaj

    2011-01-01

    Accidental discoveries have been of significant value in the progress of science. Although accidental discoveries are more common in pharmacology and chemistry, other branches of science have also benefited from such discoveries. While most discoveries are the result of persistent research, famous accidental discoveries provide a fascinating…

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

    Directory of Open Access Journals (Sweden)

    Kai Hoehlig

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

  20. Polymerase-Endonuclease Amplification Reaction (PEAR) for Large-Scale Enzymatic Production of Antisense Oligonucleotides

    Science.gov (United States)

    Wang, Xiaolong; Gou, Deming; Xu, Shuang-yong

    2010-01-01

    Antisense oligonucleotides targeting microRNAs or their mRNA targets prove to be powerful tools for molecular biology research and may eventually emerge as new therapeutic agents. Synthetic oligonucleotides are often contaminated with highly homologous failure sequences. Synthesis of a certain oligonucleotide is difficult to scale up because it requires expensive equipment, hazardous chemicals and a tedious purification process. Here we report a novel thermocyclic reaction, polymerase-endonuclease amplification reaction (PEAR), for the amplification of oligonucleotides. A target oligonucleotide and a tandem repeated antisense probe are subjected to repeated cycles of denaturing, annealing, elongation and cleaving, in which thermostable DNA polymerase elongation and strand slipping generate duplex tandem repeats, and thermostable endonuclease (PspGI) cleavage releases monomeric duplex oligonucleotides. Each round of PEAR achieves over 100-fold amplification. The product can be used in one more round of PEAR directly, and the process can be further repeated. In addition to avoiding dangerous materials and improved product purity, this reaction is easy to scale up and amenable to full automation. PEAR has the potential to be a useful tool for large-scale production of antisense oligonucleotide drugs. PMID:20062528

  1. Stereospecificity of Oligonucleotide Interactions Revisited: No Evidence for Heterochiral Hybridization and Ribozyme/DNAzyme Activity

    Science.gov (United States)

    Hoehlig, Kai; Bethge, Lucas; Klussmann, Sven

    2015-01-01

    A major challenge for the application of RNA- or DNA-oligonucleotides in biotechnology and molecular medicine is their susceptibility to abundant nucleases. One intriguing possibility to tackle this problem is the use of mirror-image (l-)oligonucleotides. For aptamers, this concept has successfully been applied to even develop therapeutic agents, so-called Spiegelmers. However, for technologies depending on RNA/RNA or RNA/DNA hybridization, like antisense or RNA interference, it has not been possible to use mirror-image oligonucleotides because Watson-Crick base pairing of complementary strands is (thought to be) stereospecific. Many scientists consider this a general principle if not a dogma. A recent publication proposing heterochiral Watson-Crick base pairing and sequence-specific hydrolysis of natural RNA by mirror-image ribozymes or DNAzymes (and vice versa) prompted us to systematically revisit the stereospecificity of oligonucleotides hybridization and catalytic activity. Using hyperchromicity measurements we demonstrate that hybridization only occurs among homochiral anti-parallel complementary oligonucleotide strands. As expected, achiral PNA hybridizes to RNA and DNA irrespective of their chirality. In functional assays we could not confirm an alleged heterochiral hydrolytic activity of ribozymes or DNAzymes. Our results confirm a strict stereospecificity of oligonucleotide hybridization and clearly argue against the possibility to use mirror-image oligonucleotides for gene silencing or antisense applications. PMID:25679211

  2. Release of DNA oligonucleotides and their conjugates from controlled-pore glass under thermolytic conditions.

    Science.gov (United States)

    Grajkowski, Andrzej; Cieślak, Jacek; Norris, Scott; Freedberg, Darón I; Kauffman, Jon S; Duff, Robert J; Beaucage, Serge L

    2008-12-01

    The sequential functionalization of long-chain alkylamine controlled-pore glass (CPG) with a 3-hydroxypropyl-(2-cyanoethyl)thiophosphoryl linker and a dinucleoside phosphorotetrazolide leads to a uniquely engineered support for solid-phase synthesis. Unlike conventional succinylated-CPG supports, this support is designed to allow oligonucleotide deprotection and elimination of deprotection side-products to proceed without release of the oligonucleotide. When needed, the DNA oligonucleotide can be thermolytically released in 2 hr under essentially neutral conditions. The modified CPG support has been successfully employed in the synthesis of both native and fully phosphorothioated DNA 20-mers. On the basis of reversed-phase HPLC and electrophoretic analyses, the purity of the released oligonucleotides is comparable to that of identical oligonucleotides synthesized from succinylated-CPG supports, in terms of both shorter-than-full-length oligonucleotide contaminants and overall yields. The detailed preparation of DNA oligonucleotides conjugated with exemplary reporter or functional groups, either at the 3'-terminus or at both 3'- and 5'-termini, is also described.

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

    Directory of Open Access Journals (Sweden)

    Xiaolong Wang

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

  4. A Brassica exon array for whole-transcript gene expression profiling.

    Directory of Open Access Journals (Sweden)

    Christopher G Love

    Full Text Available Affymetrix GeneChip® arrays are used widely to study transcriptional changes in response to developmental and environmental stimuli. GeneChip® arrays comprise multiple 25-mer oligonucleotide probes per gene and retain certain advantages over direct sequencing. For plants, there are several public GeneChip® arrays whose probes are localised primarily in 3' exons. Plant whole-transcript (WT GeneChip® arrays are not yet publicly available, although WT resolution is needed to study complex crop genomes such as Brassica, which are typified by segmental duplications containing paralogous genes and/or allopolyploidy. Available sequence data were sampled from the Brassica A and C genomes, and 142,997 gene models identified. The assembled gene models were then used to establish a comprehensive public WT exon array for transcriptomics studies. The Affymetrix GeneChip® Brassica Exon 1.0 ST Array is a 5 µM feature size array, containing 2.4 million 25-base oligonucleotide probes representing 135,201 gene models, with 15 probes per gene distributed among exons. Discrimination of the gene models was based on an E-value cut-off of 1E(-5, with ≤98% sequence identity. The 135 k Brassica Exon Array was validated by quantifying transcriptome differences between leaf and root tissue from a reference Brassica rapa line (R-o-18, and categorisation by Gene Ontologies (GO based on gene orthology with Arabidopsis thaliana. Technical validation involved comparison of the exon array with a 60-mer array platform using the same starting RNA samples. The 135 k Brassica Exon Array is a robust platform. All data relating to the array design and probe identities are available in the public domain and are curated within the BrassEnsembl genome viewer at http://www.brassica.info/BrassEnsembl/index.html.

  5. Parallel Syntheses of Peptides on Teflon-Patterned Paper Arrays (SyntArrays).

    Science.gov (United States)

    Deiss, Frédérique; Yang, Yang; Derda, Ratmir

    2016-01-01

    Screening of peptides to find the ligands that bind to specific targets is an important step in drug discovery. These high-throughput screens require large number of structural variants of peptides to be synthesized and tested. This chapter describes the generation of arrays of peptides on Teflon-patterned sheets of paper. First, the protocol describes the patterning of paper with a Teflon solution to produce arrays with solvophobic barriers that are able to confine organic solvents. Next, we describe the parallel syntheses of 96 peptides on Teflon-patterned arrays using the SPOT synthesis method.

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

    Science.gov (United States)

    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.

  7. Chimeric RNA Oligonucleotides with Triazole and Phosphate Linkages: Synthesis and RNA Interference.

    Science.gov (United States)

    Fujino, Tomoko; Kogashi, Kanako; Okada, Koudai; Mattarella, Martin; Suzuki, Takeru; Yasumoto, Kenichi; Sogawa, Kazuhiro; Isobe, Hiroyuki

    2015-12-01

    Chimeric RNA oligonucleotides with an artificial triazole linker were synthesized using solution-phase click chemistry and solid-phase automated synthesis. Scalable synthesis methods for jointing units for the chimeric structure have been developed, and after click-coupling of the jointing units with triazole linkers, a series of chimeric oligonucleotides was prepared by utilizing the well-established phosphoramidite method for the elongation. The series of chimeric 21-mer oligonucleotides that possessed the triazole linker at different strands and positions allowed for a screening study of the RNA interference to clarify the preference of the triazole modifications in small-interfering RNA molecules.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2016-11-15

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

  9. The Greatest Mathematical Discovery?

    Energy Technology Data Exchange (ETDEWEB)

    Bailey, David H.; Borwein, Jonathan M.

    2010-05-12

    What mathematical discovery more than 1500 years ago: (1) Is one of the greatest, if not the greatest, single discovery in the field of mathematics? (2) Involved three subtle ideas that eluded the greatest minds of antiquity, even geniuses such as Archimedes? (3) Was fiercely resisted in Europe for hundreds of years after its discovery? (4) Even today, in historical treatments of mathematics, is often dismissed with scant mention, or else is ascribed to the wrong source? Answer: Our modern system of positional decimal notation with zero, together with the basic arithmetic computational schemes, which were discovered in India about 500 CE.

  10. Major copy proportion analysis of tumor samples using SNP arrays

    Directory of Open Access Journals (Sweden)

    Li Cheng

    2008-04-01

    Full Text Available Abstract Background Single nucleotide polymorphisms (SNPs are the most common genetic variations in the human genome and are useful as genomic markers. Oligonucleotide SNP microarrays have been developed for high-throughput genotyping of up to 900,000 human SNPs and have been used widely in linkage and cancer genomics studies. We have previously used Hidden Markov Models (HMM to analyze SNP array data for inferring copy numbers and loss-of-heterozygosity (LOH from paired normal and tumor samples and unpaired tumor samples. Results We proposed and implemented major copy proportion (MCP analysis of oligonucleotide SNP array data. A HMM was constructed to infer unobserved MCP states from observed allele-specific signals through emission and transition distributions. We used 10 K, 100 K and 250 K SNP array datasets to compare MCP analysis with LOH and copy number analysis, and showed that MCP performs better than LOH analysis for allelic-imbalanced chromosome regions and normal contaminated samples. The major and minor copy alleles can also be inferred from allelic-imbalanced regions by MCP analysis. Conclusion MCP extends tumor LOH analysis to allelic imbalance analysis and supplies complementary information to total copy numbers. MCP analysis of mixing normal and tumor samples suggests the utility of MCP analysis of normal-contaminated tumor samples. The described analysis and visualization methods are readily available in the user-friendly dChip software.

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

    Energy Technology Data Exchange (ETDEWEB)

    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

  12. Microfluidic gene arrays for rapid genomic profiling

    Science.gov (United States)

    West, Jay A.; Hukari, Kyle W.; Hux, Gary A.; Shepodd, Timothy J.

    2004-12-01

    Genomic analysis tools have recently become an indispensable tool for the evaluation of gene expression in a variety of experiment protocols. Two of the main drawbacks to this technology are the labor and time intensive process for sample preparation and the relatively long times required for target/probe hybridization. In order to overcome these two technological barriers we have developed a microfluidic chip to perform on chip sample purification and labeling, integrated with a high density genearray. Sample purification was performed using a porous polymer monolithic material functionalized with an oligo dT nucleotide sequence for the isolation of high purity mRNA. These purified mRNA"s can then rapidly labeled using a covalent fluorescent molecule which forms a selective covalent bond at the N7 position of guanine residues. These labeled mRNA"s can then released from the polymer monolith to allow for direct hybridization with oligonucletide probes deposited in microfluidic channel. To allow for rapid target/probe hybridization high density microarray were printed in microchannels. The channels can accommodate array densities as high as 4000 probes. When oligonucleotide deposition is complete, these channels are sealed using a polymer film which forms a pressure tight seal to allow sample reagent flow to the arrayed probes. This process will allow for real time target to probe hybridization monitoring using a top mounted CCD fiber bundle combination. Using this process we have been able to perform a multi-step sample preparation to labeled target/probe hybridization in less than 30 minutes. These results demonstrate the capability to perform rapid genomic screening on a high density microfluidic microarray of oligonucleotides.

  13. Discovery and Innovation: Submissions

    African Journals Online (AJOL)

    Discovery and Innovation publishes articles and reports in a wide range of ... with the social sciences, particularly as they relate to major areas of concern in Africa. ... The article should begin with an Introduction, stating the hypothesis, defining ...

  14. The art of discovery

    Directory of Open Access Journals (Sweden)

    Susie J. Lee

    2009-06-01

    Full Text Available "The Art of Discovery" discusses an ambitious educational program taught by the artist which incorporated locative media, contemporary art, site specificity, and creative work as a proposal for the integration of art, technology and science.

  15. The Learning Discovery

    Science.gov (United States)

    Prout, Joan

    1975-01-01

    The learning discovery of youngsters is a do-it-yourself teaching method for clerical, administrative, and accountant trainees at the Bankside House headquarters of the Central Electricity Generating Board's South Eastern Region, London. (Author)

  16. Leadership and Discovery

    CERN Document Server

    Goethals, George R

    2009-01-01

    This book, a collection of essays from scholars across disciplines, explores leadership of discovery, probing the guided and collaborative exploration and interpretation of the experience of our inner thoughts and feelings, and of our external worlds

  17. Discovery and Innovation

    African Journals Online (AJOL)

    Discovery and Innovation is a journal of the African Academy of Sciences (AAS) and ... World, emphasizing the progress in scientific research and issues that impinge on these two areas as well as circumscribe science-driven development.

  18. Platforms for antibiotic discovery.

    Science.gov (United States)

    Lewis, Kim

    2013-05-01

    The spread of resistant bacteria, leading to untreatable infections, is a major public health threat but the pace of antibiotic discovery to combat these pathogens has slowed down. Most antibiotics were originally isolated by screening soil-derived actinomycetes during the golden era of antibiotic discovery in the 1940s to 1960s. However, diminishing returns from this discovery platform led to its collapse, and efforts to create a new platform based on target-focused screening of large libraries of synthetic compounds failed, in part owing to the lack of penetration of such compounds through the bacterial envelope. This article considers strategies to re-establish viable platforms for antibiotic discovery. These include investigating untapped natural product sources such as uncultured bacteria, establishing rules of compound penetration to enable the development of synthetic antibiotics, developing species-specific antibiotics and identifying prodrugs that have the potential to eradicate dormant persisters, which are often responsible for hard-to-treat infections.

  19. Antibiotic drug discovery.

    Science.gov (United States)

    Wohlleben, Wolfgang; Mast, Yvonne; Stegmann, Evi; Ziemert, Nadine

    2016-09-01

    Due to the threat posed by the increase of highly resistant pathogenic bacteria, there is an urgent need for new antibiotics; all the more so since in the last 20 years, the approval for new antibacterial agents had decreased. The field of natural product discovery has undergone a tremendous development over the past few years. This has been the consequence of several new and revolutionizing drug discovery and development techniques, which is initiating a 'New Age of Antibiotic Discovery'. In this review, we concentrate on the most significant discovery approaches during the last and present years and comment on the challenges facing the community in the coming years. © 2016 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

  20. Fateful discovery almost forgotten

    CERN Multimedia

    1989-01-01

    "The discovery of the fission of uranium exactly half a century ago is at risk of passing unremarked because of the general ambivalence towards the consequences of this development. Can that be wise?" (4 pages)

  1. Attachment of DNA to microfabricated arrays with self-assembled monolayer

    Energy Technology Data Exchange (ETDEWEB)

    Zhang Guojun; Tanii, Takashi; Miyake, Takeo; Funatsu, Takashi; Ohdomari, Iwao

    2004-10-01

    A novel approach of fabricating sub-10-{mu}m patterns on silicon surfaces by electron beam (EB) lithography for attachment of oligonucleotides was described. The shape of the microfabricated arrays was observed to be regular by optical microscopy. An octadecyltrimethoxysilane (ODS) monolayer was deposited on the regions outside the patterned areas to minimize the nonspecific binding of biomolecules. Cy 5-labeled target DNA was hybridized to both complementary and noncomplementary oligonucleotides that were covalently anchored to micropatterns. As a result, the micropatterns where specific binding occurred show strong signals, whereas no signals are observed in the case of nonspecific binding. These data indicate that miniature micro- and nano-arrays will find applications in biochips and biosensors.

  2. Conjugation with receptor-targeted histidine-rich peptides enhances the pharmacological effectiveness of antisense oligonucleotides.

    Science.gov (United States)

    Nakagawa, Osamu; Ming, Xin; Carver, Kyle; Juliano, Rudy

    2014-01-15

    Ineffective delivery to intracellular sites of action is one of the key limitations to the use of antisense and siRNA oligonucleotides as therapeutic agents. Here, we describe molecular scale antisense oligonucleotide conjugates that bind selectively to a cell surface receptor, are internalized, and then partially escape from nonproductive endosomal locations to reach their sites of action in the nucleus. Peptides that include bombesin sequences for receptor targeting and a run of histidine residues for endosomal disruption were covalently linked to a splice switching antisense oligonucleotide. The conjugates were tested for their ability to correct splicing and up-regulate expression of a luciferase reporter in prostate cancer cells that express the bombesin receptor. We found that trivalent conjugates that included both the targeting sequence and several histidine residues were substantially more effective than conjugates containing only the bombesin or histidine moieties. This demonstrates the potential of creating molecular scale oligonucleotide conjugates with both targeting and endosome escape capabilities.

  3. Chemically robust fluoroalkyl phthalocyanine-oligonucleotide bioconjugates and their GRP78 oncogene photocleavage activity.

    Science.gov (United States)

    Patel, Pradeepkumar; Patel, Hemantbhai H; Borland, Emily; Gorun, Sergiu M; Sabatino, David

    2014-06-18

    The first representative of functionalized fluoroalkyl phthalocyanines, F48H7(COOH)PcZn, is reported. The complex generates (1)O2 affording long-lasting photooxidation of an external substrate without self-decomposition. The carboxylic group couples with an antisense oligonucleotide targeting GRP78 oncogenes, resulting in the F48H7PcZn-cancer targeting oligonucleotide (CTO). The bioconjugated fluorophthalocyanine effectively hybridizes complementary GRP78 DNA and mRNA sequences. Piperidine cleavage assays reveal desired photochemical oligonucleotide oxidative degradation for both F48H7PcZn-CTO:DNA and F48H7PcZn-CTO:mRNA hybrids. This new materials strategy could be extended to other functional fluorinated phthalocyanines-antisense oligonucleotide combinations for long-lasting oncogene-targeting photodynamic therapy.

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

    Science.gov (United States)

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

    2016-04-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Nucleoside, nucleotide and oligonucleotide based amphiphiles: a successful marriage of nucleic acids with lipids.

    Science.gov (United States)

    Gissot, Arnaud; Camplo, Michel; Grinstaff, Mark W; Barthélémy, Philippe

    2008-04-21

    Amphiphilic molecules based on nucleosides, nucleotides and oligonucleotides are finding more and more biotechnological applications. This Perspective highlights their synthesis, supramolecular organization as well as their applications in the field of biotechnology.

  7. Cancer Stem Cell Biomarker Discovery Using Antibody Array Technology.

    Science.gov (United States)

    Burgess, Rob; Huang, Ruo-Pan

    2016-01-01

    Cancer is a complex disease involving hundreds of pathways and numerous levels of disease progression. In addition, there is a growing body of evidence that the origins and growth rates of specific types of cancer may involve "cancer stem cells," which are defined as "cells within a tumor that possess the capacity to self-renew and to cause the development of heterogeneous lineages of cancer cells that comprise the tumor.(1)" Many types of cancer are now thought to harbor cancer stem cells. These cells themselves are thought to be unique in comparison to other cells types present within the tumor and to exhibit characteristics that allow for the promotion of tumorigenesis and in some cases metastasis. In addition, it is speculated that each type of cancer stem cell exhibits a unique set of molecular and biochemical markers. These markers, alone or in combination, may act as a signature for defining not only the type of cancer but also the progressive state. These biomarkers may also double as signaling entities which act autonomously or upon neighboring cancer stem cells or other cells within the local microenvironment to promote tumorigenesis. This review describes the heterogeneic properties of cancer stem cells and outlines the identification and application of biomarkers and signaling molecules defining these cells as they relate to different forms of cancer. Other examples of biomarkers and signaling molecules expressed by neighboring cells in the local tumor microenvironment are also discussed. In addition, biochemical signatures for cancer stem cell autocrine/paracrine signaling, local site recruitment, tumorigenic potential, and conversion to a stem-like phenotype are described.

  8. Discovery Driven Growth

    DEFF Research Database (Denmark)

    Bukh, Per Nikolaj

    2009-01-01

    Anmeldelse af Discovery Driven Growh : A breakthrough process to reduce risk and seize opportunity, af Rita G. McGrath & Ian C. MacMillan, Boston: Harvard Business Press. Udgivelsesdato: 14 august......Anmeldelse af Discovery Driven Growh : A breakthrough process to reduce risk and seize opportunity, af Rita G. McGrath & Ian C. MacMillan, Boston: Harvard Business Press. Udgivelsesdato: 14 august...

  9. Chemoinformatics and Drug Discovery

    Directory of Open Access Journals (Sweden)

    Arnold Hagler

    2002-08-01

    Full Text Available This article reviews current achievements in the field of chemoinformatics and their impact on modern drug discovery processes. The main data mining approaches used in cheminformatics, such as descriptor computations, structural similarity matrices, and classification algorithms, are outlined. The applications of cheminformatics in drug discovery, such as compound selection, virtual library generation, virtual high throughput screening, HTS data mining, and in silico ADMET are discussed. At the conclusion, future directions of chemoinformatics are suggested.

  10. Sheath liquid effects in capillary high-performance liquid chromatography-electrospray mass spectrometry of oligonucleotides.

    Science.gov (United States)

    Huber, C G; Krajete, A

    2000-02-18

    Fused-silica capillary columns of 200 microm inner diameter were packed with micropellicular, octadecylated, 2.3 microm poly(styrene-divinylbenzene) particles and applied to the separation of oligonucleotides by ion-pair reversed-phase high-performance liquid chromatography. Oligonucleotides were eluted at 50 degrees C with gradients of 3-13% acetonitrile in 50 mM triethylammonium bicarbonate. Addition of sheath liquid to the column effluent allowed the detection of oligonucleotides by electrospray ionization mass spectrometry using full-scan data acquisition with a detectability comparable to that obtained with UV detection. The signal-to-noise ratios with different sheath liquids increased in the order isopropanololigonucleotides longer than 20 nucleotide units whereas no significant effect was observed with shorter oligonucleotides. Organic acids and bases in the sheath liquid generally deteriorated the signal-to-noise ratios in the chromatograms and mass spectra mainly because of increased background noise. Only a few charge states were observed in the mass spectra of oligonucleotides because of charge state reduction due to the presence of carbonic acid in the eluent. With triethylammonium hydrogencarbonate as chromatographic eluent and acetonitrile as sheath liquid, very few cation adducts of oligonucleotides were observed in the mass spectra. However, the presence of small amounts of monopotassium adducts enabled the calculation of the charge state of multiply charged ions. With acetonitrile as sheath liquid, 710 amol of a 16-mer oligonucleotide were detected using selected ion monitoring data acquisition with a signal-to-noise ratio of 3:1. Finally, capillary ion-pair reversed-phase high-performance liquid chromatography-electrospray ionization mass spectrometry was

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

    Science.gov (United States)

    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.

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

    Directory of Open Access Journals (Sweden)

    Tanaka Makoto

    2000-12-01

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

  13. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

    Science.gov (United States)

    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.

  14. Enzymic synthesis of oligonucleotides containing methylphosphonate internucleotide linkages.

    Science.gov (United States)

    Higuchi, H; Endo, T; Kaji, A

    1990-09-18

    Thymidine 5'-O-(pyrophosphoryl methylphosphonate) (dTTP alpha CH3) has been chemically synthesized by condensation of thymidine 5'-O-(methylphosphonate) with pyrophosphate. This novel nucleotide, which contained an alpha-phosphorus atom as methylphosphonate, was used as a substrate of terminal deoxynucleotidyltransferase (TDTase) in the presence of oligonucleotide (5'-GCTGTATCGTCAAGGCACTC-3') as an initiator. The reaction products were separated into two components by reverse-phase high-performance liquid chromatography (RP-HPLC). These products were, after purification, digested with nuclease P1 and alkaline phosphatase followed by separation of digested products by RP-HPLC. The result showed the presence of one of the isomers of 2'-deoxycytidyl-3'-methylphosphonyl-5'-thymidine (dCpCH3T) and 2'-deoxycytidyl-3'-methylphosphonyl-5'-thymidyl-3'-methyl phosphonyl-5'-thymidin e (dCpCH3TpCH3T), respectively. Fast atom bombardment mass spectrometry of these products further supported identification of the dinucleotide and the trinucleotide. These results indicated that dTTP alpha CH3 was used as a substrate of TDTase, resulting in methylphosphonate linkages. Produced oligomers were resistant to hydrolysis by snake venom phosphodiesterase I.

  15. Advancements of antisense oligonucleotides in treatment of breast cancer

    Institute of Scientific and Technical Information of China (English)

    YANGShuan-Ping; SONGSan-Tai; 等

    2003-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Tamar Canello

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

  17. Nanoexplosive gene therapy using triplex-forming oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

    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.

  18. Efficient in vivo delivery of antisense oligonucleotide to choroid plexus.

    Science.gov (United States)

    Piao, Wenying; Nishina, Kazutaka; Yoshida-Tanaka, Kie; Kuwahara, Hiroya; Nishina, Tomoko; Sakata, Mina; Mizusawa, Hidehiro; Yokota, Takanori

    2013-03-01

    The choroid plexus (CP) is present on the ventricular walls of the brain, produces cerebrospinal fluid (CSF), contains many blood vessels, and is a major functional component of the blood-CSF barrier. The CP is an important site in the pathophysiology of various neurological diseases, including Alzheimer's disease and meningeal amyloidosis. We performed gene silencing in the CP in vivo by using an antisense oligonucleotide (ASO). A short ASO of length 12 nucleotides was intravenously injected into rats. The ASO was not delivered to neurons or glia in the central nervous system, but was successfully delivered into the CP, and resulted in a significant reduction of endogenous target gene expression in epithelial cells within the CP. Although the mechanism of uptake of the ASO by the CP was not elucidated, the ASO bound to albumin in vivo, and the distribution of ASO delivery was similar to that of albumin delivery. These findings suggest that we inhibited target gene expression in the epithelial cells of the CP via albumin-ASO conjugates. This strategy should be useful for investigations of the function of CP, and for the development of new gene-silencing therapies for diseases with pathophysiology related to the CP.

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

    Science.gov (United States)

    Canello, Tamar; Ovadia, Haim; 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.

  20. Chimeric Antisense Oligonucleotide Conjugated to α-Tocopherol

    Directory of Open Access Journals (Sweden)

    Tomoko Nishina

    2015-01-01

    Full Text Available We developed an efficient system for delivering short interfering RNA (siRNA to the liver by using α-tocopherol conjugation. The α-tocopherol–conjugated siRNA was effective and safe for RNA interference–mediated gene silencing in vivo. In contrast, when the 13-mer LNA (locked nucleic acid-DNA gapmer antisense oligonucleotide (ASO was directly conjugated with α-tocopherol it showed markedly reduced silencing activity in mouse liver. Here, therefore, we tried to extend the 5′-end of the ASO sequence by using 5′-α-tocopherol–conjugated 4- to 7-mers of unlocked nucleic acid (UNA as a “second wing.” Intravenous injection of mice with this α-tocopherol–conjugated chimeric ASO achieved more potent silencing than ASO alone in the liver, suggesting increased delivery of the ASO to the liver. Within the cells, the UNA wing was cleaved or degraded and α-tocopherol was released from the 13-mer gapmer ASO, resulting in activation of the gapmer. The α-tocopherol–conjugated chimeric ASO showed high efficacy, with hepatic tropism, and was effective and safe for gene silencing in vivo. We have thus identified a new, effective LNA-DNA gapmer structure in which drug delivery system (DDS molecules are bound to ASO with UNA sequences.

  1. Kinetic Hairpin Oligonucleotide Blockers for Selective Amplification of Rare Mutations

    Science.gov (United States)

    Jia, Yanwei; Sanchez, J. Aquiles; Wangh, Lawrence J.

    2014-01-01

    Detection of rare mutant alleles in an excess of wild type alleles is increasingly important in cancer diagnosis. Several methods for selective amplification of a mutant allele via the polymerase chain reaction (PCR) have been reported, but each of these methods has its own limitations. A common problem is that Taq DNA polymerase errors early during amplification generate false positive mutations which also accumulate exponentially. In this paper, we described a novel method using hairpin oligonucleotide blockers that can selectively inhibit the amplification of wild type DNA during LATE-PCR amplification. LATE-PCR generates double-stranded DNA exponentially followed by linear amplification of single-stranded DNA. The efficiency of the blocker is optimized by adjusting the LATE-PCR temperature cycling profile. We also demonstrate that it is possible to minimize false positive signals caused by Taq DNA polymerase errors by using a mismatched excess primer plus a modified PCR profile to preferentially enrich for mutant target sequences prior to the start of the exponential phase of LATE-PCR amplification. In combination these procedures permit amplification of specific KRAS mutations in the presence of more than 10,000 fold excess of wild type DNA without false positive signals. PMID:25082368

  2. The development of bioactive triple helix-forming oligonucleotides.

    Science.gov (United States)

    Seidman, Michael M; Puri, Nitin; Majumdar, Alokes; Cuenoud, Bernard; Miller, Paul S; Alam, Rowshon

    2005-11-01

    We are developing triple helix-forming oligonucleotides (TFOs) as gene targeting reagents in mammalian cells. We have described psoralen-conjugated TFOs containing 2'-O-methyl (2'OMe) and 2'-O-aminoethoxy (AE) ribose substitutions. TFOs with a cluster of 3-4 AE residues, with all other sugars as 2'OMe, were bioactive in a gene knockout assay in mammalian cells. In contrast, TFOs with one or two clustered, or three dispersed, AE residues were inactive. Thermal stability analysis of the triplexes indicated that there were only incremental differences between the active and inactive TFOs. However the active and inactive TFOs could be distinguished by their association kinetics. The bioactive TFOs showed markedly greater on-rates than the inactive TFOs. It appears that the on-rate is a better predictor of TFO bioactivity than thermal stability. Our data are consistent with a model in which a cluster of 3-4 AE residues stabilizes the nucleation event that precedes formation of a complete triplex. It is likely that triplexes in cells are much less stable than triplexes in vitro probably as a result of elution by chromatin-associated translocases and helicases. Consequently the biologic assay will favor TFOs that can bind and rebind genomic targets quickly.

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

    Directory of Open Access Journals (Sweden)

    Moya Andrés

    2004-12-01

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

  4. Advantages of ion-exchange chromatography for oligonucleotide analysis.

    Science.gov (United States)

    Cook, Ken; Thayer, Jim

    2011-05-01

    The rapid development of therapeutic oligonucleotides (ONs) has created a need for in-depth characterization of ONs, beyond previous requirements. The natural migration to LC-MS requires the use of chromatography with MS-compatible eluents to introduce the large, highly charged biopolymers into the mass spectrometer. Most frequently this employs ion-pair reversed-phase liquid chromatography, which may leave gaps in the characterization, but these can be filled with the use of high-resolution ion-exchange chromatography. Several classes of isobaric isomers are among the impurities that will require further separation prior to MS analysis. This review shows how the use of ion exchange as an additional orthogonal analytical method can be used as standalone or interfaced with MS to achieve the highest possible analytical coverage in the characterization and quantification of impurities present in single- and double-stranded ON formulations. Some of these techniques have been in use for some time and the importance of others is just being recognized.

  5. Porous silicon-cell penetrating peptide hybrid nanocarrier for intracellular delivery of oligonucleotides.

    Science.gov (United States)

    Rytkönen, Jussi; Arukuusk, Piret; Xu, Wujun; Kurrikoff, Kaido; Langel, Ulo; Lehto, Vesa-Pekka; Närvänen, Ale

    2014-02-01

    The largest obstacle to the use of oligonucleotides as therapeutic agents is the delivery of these large and negatively charged biomolecules through cell membranes into intracellular space. Mesoporous silicon (PSi) is widely recognized as a potential material for drug delivery purposes due to its several beneficial features like large surface area and pore volume, high loading capacity, biocompatibility, and biodegradability. In the present study, PSi nanoparticles stabilized by thermal oxidation or thermal carbonization and subsequently modified by grafting aminosilanes on the surface are utilized as an oligonucleotide carrier. Splice correcting oligonucleotides (SCOs), a model oligonucleotide drug, were loaded into the positively charged PSi nanoparticles with a loading degree as high as 14.3% (w/w). Rapid loading was achieved by electrostatic interactions, with the loading efficiencies reaching 100% within 5 min. The nanoparticles were shown to deliver and release SCOs, in its biologically active form, inside cells when formulated together with cell penetrating peptides (CPP). The biological effect was monitored with splice correction assay and confocal microscopy utilizing HeLa pLuc 705 cells. Furthermore, the use of PSi carrier platform in oligonucleotide delivery did not reduce the cell viability. Additionally, the SCO-CPP complexes formed in the pores of the carrier were stabilized against proteolytic digestion. The advantageous properties of protecting and releasing the cargo and the possibility to further functionalize the carrier surface make the hybrid nanoparticles a potential system for oligonucleotide delivery.

  6. Investigation of the structural organization of cationic nanoemulsion/antisense oligonucleotide complexes.

    Science.gov (United States)

    Bruxel, Fernanda; Vilela, José Mario Carneiro; Andrade, Margareth Spangler; Malachias, Ângelo; Perez, Carlos A; Magalhães-Paniago, Rogério; Oliveira, Mônica Cristina; Teixeira, Helder F

    2013-12-01

    Atomic force microscopy image analysis and energy dispersive X-ray diffraction experiments were used to investigate the structural organization of cationic nanoemulsion/oligonucleotide complexes. Oligonucleotides targeting topoisomerase II gene were adsorbed on cationic nanoemulsions obtained by means of spontaneous emulsification procedure. Topographical analysis by atomic force microscopy allowed the observation of the nanoemulsion/oligonucleotide complexes through three-dimensional high-resolution images. Flattening of the oil droplets was observed, which was reduced in the complexes obtained at high amount of adsorbed oligonucleotides. In such conditions, complexes exhibit droplet size in the 600nm range. The oligonucleotides molecules were detected on the surface of the droplets, preventing their fusion during aggregation. A lamellar structure organization was identified by energy dispersive X-ray diffraction experiments. The presence of the nucleic acid molecules led to a disorganization of the lipid arrangement and an expansion in the lattice spacing, which was proportional to the amount of oligonucleotides added. Copyright © 2013 Elsevier B.V. All rights reserved.

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

    Science.gov (United States)

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

    2016-10-04

    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. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  8. The small molecule Retro-1 enhances the pharmacological actions of antisense and splice switching oligonucleotides.

    Science.gov (United States)

    Ming, Xin; Carver, Kyle; Fisher, Michael; Noel, Romain; Cintrat, Jean-Christophe; Gillet, Daniel; Barbier, Julien; Cao, Canhong; Bauman, John; Juliano, Rudolph L

    2013-04-01

    The attainment of strong pharmacological effects with oligonucleotides is hampered by inefficient access of these molecules to their sites of action in the cytosol or nucleus. Attempts to address this problem with lipid or polymeric delivery systems have been only partially successful. Here, we describe a novel alternative approach involving the use of a non-toxic small molecule to enhance the pharmacological effects of oligonucleotides. The compound Retro-1 was discovered in a screen for small molecules that reduce the actions of bacterial toxins and has been shown to block the retrograde trafficking pathway. We demonstrate that Retro-1 can also substantially enhance the effectiveness of antisense and splice switching oligonucleotides in cell culture. This effect occurs at the level of intracellular trafficking or processing and is correlated with increased oligonucleotide accumulation in the nucleus but does not involve the perturbation of lysosomal compartments. We also show that Retro-1 can alter the effectiveness of splice switching oligonucleotides in the in vivo setting. These observations indicate that it is possible to enhance the pharmacological actions of oligonucleotides using non-toxic and non-lysosomotropic small molecule adjuncts.

  9. Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus

    Institute of Scientific and Technical Information of China (English)

    Yong-Zhong Wang; Guo-Xiang Wu; Li-Bo Luo; Min Chen; Li-Hua Ruan

    2007-01-01

    AIM: To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B.METHODS: Mixture of samples with different genotypes and clinical serum samples from 126 chronic hepatitis B patients was tested for hepatitis B virus genotypes by oligonucleotide chip, real-time PCR and sequencing of PCR products, respectively. Clinical performances, time required and costs of the three assays were evaluated.RESULTS: Oligonucleotide chips and real-time PCR detected 1% and 0.1% genotypes, respectively, in mixed samples. Of the 126 clinical samples from patients with chronic hepatitis B, genotype B was detected in 41(33%), 41 (33%) and 45 (36%) samples, and genotype C in 76 (60%), 76 (60%) and 81 (64%) samples, by oligonucleotide chip, real-time PCR and sequencing,respectively. Oligonucleotide chip and real-time PCR detected mixed genotypes B and C in 9 samples. Realtime PCR was the rapidest and cheapest among the three assays.CONCLUSION: Oligonucleotide chip and real-time PCR are able to detect mixed genotypes, while sequencing only detects the dominant genotype in clinical samples.

  10. Toolbox for Antibiotics Discovery from Microorganisms.

    Science.gov (United States)

    Fisch, Katja M; Schäberle, Till F

    2016-09-01

    Microorganisms produce a vast array of biologically active metabolites. Such compounds are applied by humans to positively influence their health and, therefore, natural products serve as drug leads for pharmaceutical and medicinal chemistry. In this minireview, tools for the discovery and the production of potential drug leads are explained. A snapshot is provided, starting from the isolation of new producer strains, across genomic mining of (meta)genomes to identify biosynthetic gene clusters corresponding to natural products, toward heterologous expression to produce potential drug leads. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  11. New Discoveries from the PALFA Survey

    Science.gov (United States)

    Kaspi, Victoria M.; PALFA Consortium

    2012-01-01

    The Pulsar Arecibo L-Band Feed Array (PALFA) survey is a deep 1.4 GHz search for radio pulsars in the Galactic plane. The primary goal of the survey is to find long-period and millisecond pulsars (MSPs) at large distances and high dispersion measures to which previous Arecibo pulsar surveys were not sensitive. One particular science motivation is to find rotationally stable MSPs that can be used in pulsar timing arrays for detecting nanohertz gravitational waves. PALFA has recently made the transition to using the new Mock Spectrometers, which offer a wider bandwidth and better sensitivity than was previously available. To date, PALFA has discovered over 70 new pulsars, including many millisecond pulsars (MSPs). Here, we highlight some of the most recent discoveries, which include some found using the Einstein@Home global volunteer computing system. We also discuss expected future PALFA results.

  12. 2-O-[2-(Methylthio)ethyl]-Modified Oligonucleotide: An Analog of 2-O-[2-(Methoxy)ethyl]-Modified Oligonucleotide with Improved Protein Binding Properties and High Binding Affinity to Target RNA

    Energy Technology Data Exchange (ETDEWEB)

    Prakash, T.P.; Manoharan, M.; Fraser, A.S.; Kawasaki, A.M.; Lesnik, E.; Sioufi, N.; Leeds, J.M.; Teplova, M.; Egli, M.

    2010-03-08

    A novel 2'-modification, 2'-O-[2-(methylthio)ethyl] or 2'-O-MTE, has been incorporated into oligonucleotides and evaluated for properties relevant to antisense activity. The results were compared with the previously characterized 2'-O-[2-(methoxy)ethyl] 2'-O-MOE modification. As expected, the 2'-O-MTE modified oligonucleotides exhibited improved binding to human serum albumin compared to the 2'-O-MOE modified oligonucleotides. The 2'-O-MTE oligonucleotides maintained high binding affinity to target RNA. Nuclease digestion of 2'-O-MTE oligonucleotides showed that they have limited resistance to exonuclease degradation. We analyzed the crystal structure of a decamer DNA duplex containing the 2'-O-MTE modifcation. Analysis of the crystal structure provides insight into the improved RNA binding affinity, protein binding affinity and limited resistance of 2'-O-MTE modified oligonucleotides to exonuclease degradation.

  13. DNA sequence analysis by hybridization with oligonucleotide microchips : MALDI mass spectrometry identification of 5mers contiguously stacked to microchip oligonucleotides.

    Energy Technology Data Exchange (ETDEWEB)

    Stomakhin, A. A.; Vasiliskov, V. A.; Timofeev, E.; Schulga, D.; Cotter, R. J.; Mirzabekov, A. D.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology; Moscow Inst. of Physics and Technology; Middle Atlantic Mass Spectrometry Lab.; Johns Hopkins Univ. School of Medicine

    2000-01-01

    Matrix-assisted laser desorption ionization mass spectrometry (MALDI MS) has been applied to increase the informational output from DNA sequence analysis. It has been used to analyze DNA by hybridization with microarrays of gel-immobilized oligonucleotides extended with stacked 5mers. In model experiments, a 28 nt long DNA fragment was hybridized with 10 immobilized, overlapping 8mers. Then, in a second round of hybridization DNA-8mer duplexes were hybridized with a mixture of 10 5mers. The stability of the 5mer complex with DNA was increased to raise the melting temperature of the duplex by 10-15{sup o}C as a result of stacking interaction with 8mers. Contiguous 13 bp duplexes containing an internal break were formed. MALDI MS identified one or, in some cases, two 5mers contiguously stacked to each DNA-8mer duplex formed on the microchip. Incorporating a mass label into 5mers optimized MALDI MS monitoring. This procedure enabled us to reconstitute the sequence of a model DNA fragment and identify polymorphic nucleotides. The application of MALDI MS identification of contiguously stacked 5mers to increase the length of DNA for sequence analysis is discussed.

  14. Self-Assembly of G-Rich Oligonucleotides Incorporating a 3'-3' Inversion of Polarity Site: A New Route Towards G-Wire DNA Nanostructures.

    Science.gov (United States)

    Oliviero, Giorgia; D'Errico, Stefano; Pinto, Brunella; Nici, Fabrizia; Dardano, Principia; Rea, Ilaria; De Stefano, Luca; Mayol, Luciano; Piccialli, Gennaro; Borbone, Nicola

    2017-08-01

    Obtaining DNA nanostructures with potential applications in drug discovery, diagnostics, and electronics in a simple and affordable way represents one of the hottest topics in nanotechnological and medical sciences. Herein, we report a novel strategy to obtain structurally homogeneous DNA G-wire nanostructures of known length, starting from the short unmodified G-rich oligonucleotide d(5'-CGGT-3'-3'-GGC-5') (1) incorporating a 3'-3' inversion of polarity site. The reported approach allowed us to obtain long G-wire assemblies through 5'-5' π-π stacking interactions in between the tetramolecular G-quadruplex building blocks that form when 1 is annealed in the presence of potassium ions. Our results expand the repertoire of synthetic methodologies to obtain new tailored DNA G-wire nanostructures.

  15. Coupling in reflector arrays

    DEFF Research Database (Denmark)

    Appel-Hansen, Jørgen

    1968-01-01

    In order to reduce the space occupied by a reflector array, it is desirable to arrange the array antennas as close to each other as possible; however, in this case coupling between the array antennas will reduce the reflecting properties of the reflector array. The purpose of the present communic...

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

    CERN Document Server

    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.

  17. A simple and rapid method for the preparation of homologous DNA oligonucleotide hybridization probes from heterologous gene sequences and probes.

    Science.gov (United States)

    Maxwell, E S; Sarge, K D

    1988-11-30

    We describe a simple and rapid method for the preparation of homologous DNA oligonucleotide probes for hybridization analysis and/or cDNA/genomic library screening. With this method, a synthetic DNA oligonucleotide derived from a known heterologous DNA/RNA/protein sequence is annealed to an RNA preparation containing the gene transcript of interest. Any unpaired 3'-terminal oligonucleotides of the heterologous DNA primer are then removed using the 3' exonuclease activity of the DNA Polymerase I Klenow fragment before primer extension/dideoxynucleotide sequencing of the annealed RNA species with AMV reverse transcriptase. From the determined RNA sequence, a completely homologous DNA oligonucleotide probe is then prepared. This approach has been used to prepare a homologous DNA oligonucleotide probe for the successful library screening of the yeast hybRNA gene starting with a heterologous mouse hybRNA DNA oligonucleotide probe.

  18. 'Specific' oligonucleotides often recognize more than one gene: the limits of in situ hybridization applied to GABA receptors.

    Science.gov (United States)

    Mladinic, M; Didelon, F; Cherubini, E; Bradbury, A

    2000-05-15

    As exquisite probes for gene sequences, oligonucleotides are one of the most powerful tools of recombinant molecular biology. In studying the GABA receptor subunits in the neonatal hippocampus we have used oligonucleotide probes in in situ hybridization and cloning techniques. The oligonucleotides used and assumed to be specific for the target gene, actually recognized more than one gene, leading to surprising and contradictory results. In particular, we found that a GABA(A)-rho specific oligonucleotide recognized an abundant, previously unknown, transcription factor in both in situ and library screening, while oligos 'specific' for GABA(A) subunits were able to recognize 30 additional unrelated genes in library screening. This suggests that positive results obtained with oligonucleotides should be interpreted with caution unless confirmed by identical results with oligonucleotides from different parts of the same gene, or cDNA library screening excludes the presence of other hybridizing species.

  19. A Novel Family of Small Molecules that Enhance the Intracellular Delivery and Pharmacological Effectiveness of Antisense and Splice Switching Oligonucleotides.

    Science.gov (United States)

    Wang, Ling; Ariyarathna, Yamuna; Ming, Xin; Yang, Bing; James, Lindsey I; Kreda, Silvia M; Porter, Melissa; Janzen, William; Juliano, Rudolph L

    2017-08-18

    The pharmacological effectiveness of oligonucleotides has been hampered by their tendency to remain entrapped in endosomes, thus limiting their access to cytosolic or nuclear targets. We have previously reported a group of small molecules that enhance the effects of oligonucleotides by causing their release from endosomes. Here, we describe a second novel family of oligonucleotide enhancing compounds (OECs) that is chemically distinct from the compounds reported previously. We demonstrate that these molecules substantially augment the actions of splice switching oligonucleotides (SSOs) and antisense oligonucleotides (ASOs) in cell culture. We also find enhancement of SSO effects in a murine model. These new compounds act by increasing endosome permeability and causing partial release of entrapped oligonucleotides. While they also affect the permeability of lysosomes, they are clearly different from typical lysosomotropic agents. Current members of this compound family display a relatively narrow window between effective dose and toxic dose. Thus, further improvements are necessary before these agents can become suitable for therapeutic use.

  20. Profiled support vector machines for antisense oligonucleotide efficacy prediction

    Directory of Open Access Journals (Sweden)

    Martín-Guerrero José D

    2004-09-01

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

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

    Science.gov (United States)

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

    2016-02-01

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

  2. Liposome-coated lipoplex-based carrier for antisense oligonucleotides.

    Science.gov (United States)

    Wyrozumska, Paulina; Meissner, Justyna; Toporkiewicz, Monika; Szarawarska, Marta; Kuliczkowski, Kazimierz; Ugorski, Maciej; Walasek, Marta A; Sikorski, Aleksander F

    2015-01-01

    The chemical nature of genetic drugs (e.g. antisense oligonucleotides, siRNA, vectors) requires a suitable carrier system to protect them from enzymatic degradation without changing their properties and enable efficient delivery into target cells. Lipid vectors for nucleic acid delivery that have been widely investigated for years can be very effective. As the majority of attempts made in the field of cancer gene therapy have focused on solid tumors, while blood cancer cells have attracted less attention, the latter became the subject of our investigation. The lipid carrier proposed here is based on liposomes constructed by others but the lipid composition is original. A liposome-coated lipoplex (L-cL) consists of a core arising from complexation of positively charged lipid and negatively charged oligodeoxynucleotide (ODN) or plasmid DNA coated by a neutral or anionic lipid bilayer. Moreover, our lipid vector demonstrates size stability and is able to retain a high content of enclosed plasmid DNA or antisense oligodeoxynucleotides (asODNs). Observed transfection efficacies of the tested preparation using a plasmid coding for fluorescent protein were up to 60-85% of examined leukemia cells (Jurkat T and HL-60 lines) in the absence or the presence of serum. When BCL‑2 asODN was encapsulated in the L-cL, specific silencing of this gene product at both the mRNA and protein level and also a markedly decreased cell survival rate were observed in vitro. Moreover, biodistribution analysis in mice indicates prolonged circulation characteristic for PEG-modified liposomal carriers. Experiments on tumor-engrafted animals indicate substantial inhibition of tumor growth.

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

    Science.gov (United States)

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

    2015-03-19

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

  4. Application of heteronuclear couplings to conformational analysis of oligonucleotides

    Energy Technology Data Exchange (ETDEWEB)

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

    1994-12-01

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

  5. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides

    OpenAIRE

    Pawel Bialk; Brett Sansbury; Natalia Rivera-Torres; Kevin Bloh; Dula Man; Kmiec, Eric B.

    2016-01-01

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRIS...

  6. Analyses of point mutation repair and allelic heterogeneity generated by CRISPR/Cas9 and single-stranded DNA oligonucleotides

    OpenAIRE

    Pawel Bialk; Brett Sansbury; Natalia Rivera-Torres; Kevin Bloh; Dula Man; Kmiec, Eric B.

    2016-01-01

    The repair of a point mutation can be facilitated by combined activity of a single-stranded oligonucleotide and a CRISPR/Cas9 system. While the mechanism of action of combinatorial gene editing remains to be elucidated, the regulatory circuitry of nucleotide exchange executed by oligonucleotides alone has been largely defined. The presence of the appropriate CRISPR/Cas9 system leads to an enhancement in the frequency of gene editing directed by single-stranded DNA oligonucleotides. While CRIS...

  7. arrayCGHbase: an analysis platform for comparative genomic hybridization microarrays

    Directory of Open Access Journals (Sweden)

    Moreau Yves

    2005-05-01

    Full Text Available Abstract Background The availability of the human genome sequence as well as the large number of physically accessible oligonucleotides, cDNA, and BAC clones across the entire genome has triggered and accelerated the use of several platforms for analysis of DNA copy number changes, amongst others microarray comparative genomic hybridization (arrayCGH. One of the challenges inherent to this new technology is the management and analysis of large numbers of data points generated in each individual experiment. Results We have developed arrayCGHbase, a comprehensive analysis platform for arrayCGH experiments consisting of a MIAME (Minimal Information About a Microarray Experiment supportive database using MySQL underlying a data mining web tool, to store, analyze, interpret, compare, and visualize arrayCGH results in a uniform and user-friendly format. Following its flexible design, arrayCGHbase is compatible with all existing and forthcoming arrayCGH platforms. Data can be exported in a multitude of formats, including BED files to map copy number information on the genome using the Ensembl or UCSC genome browser. Conclusion ArrayCGHbase is a web based and platform independent arrayCGH data analysis tool, that allows users to access the analysis suite through the internet or a local intranet after installation on a private server. ArrayCGHbase is available at http://medgen.ugent.be/arrayCGHbase/.

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

    Directory of Open Access Journals (Sweden)

    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

  9. Archaeological Discoveries in Liaoning

    Institute of Scientific and Technical Information of China (English)

    1996-01-01

    LIAONING Province, in northeastern China, has been inhabited by many ethnic groups since ancient times. It is one of the sites of China’s earliest civilization. Since the 1950s many archaeological discoveries from periods beginning with the Paleolithic of 200,000 years ago, and through all the following historic periods, have been made in the province.

  10. Discovery of TUG-770

    DEFF Research Database (Denmark)

    Christiansen, Elisabeth; Hansen, Steffen V F; Urban, Christian;

    2013-01-01

    Free fatty acid receptor 1 (FFA1 or GPR40) enhances glucose-stimulated insulin secretion from pancreatic β-cells and currently attracts high interest as a new target for the treatment of type 2 diabetes. We here report the discovery of a highly potent FFA1 agonist with favorable physicochemical a...

  11. Ayurvedic drug discovery.

    Science.gov (United States)

    Balachandran, Premalatha; Govindarajan, Rajgopal

    2007-12-01

    Ayurveda is a major traditional system of Indian medicine that is still being successfully used in many countries. Recapitulation and adaptation of the older science to modern drug discovery processes can bring renewed interest to the pharmaceutical world and offer unique therapeutic solutions for a wide range of human disorders. Eventhough time-tested evidences vouch immense therapeutic benefits for ayurvedic herbs and formulations, several important issues are required to be resolved for successful implementation of ayurvedic principles to present drug discovery methodologies. Additionally, clinical examination in the extent of efficacy, safety and drug interactions of newly developed ayurvedic drugs and formulations are required to be carefully evaluated. Ayurvedic experts suggest a reverse-pharmacology approach focusing on the potential targets for which ayurvedic herbs and herbal products could bring tremendous leads to ayurvedic drug discovery. Although several novel leads and drug molecules have already been discovered from ayurvedic medicinal herbs, further scientific explorations in this arena along with customization of present technologies to ayurvedic drug manufacturing principles would greatly facilitate a standardized ayurvedic drug discovery.

  12. The Scholarship of Discovery.

    Science.gov (United States)

    Dobos, Jean

    2000-01-01

    Contributes to a special issue on how the reconsideration of what scholarship is affects the way in which scholarship is assessed. Examines traditional criteria for evaluating faculty research. Identifies activities pertinent to the scholarship of discovery, and the assessment practices in the field of communication as well as in general use. (SR)

  13. Discovery Education: A Definition.

    Science.gov (United States)

    Wilson, Harold C.

    2002-01-01

    Discovery Education is based on the writings of Henry David Thoreau, an early champion of experiential learning. After 2 months of preparation, 10th-grade students spent 4 days in the wilderness reenacting a piece of history, such as the Lewis and Clark Expedition. The interdisciplinary approach always included journal-writing. Students gained…

  14. Discovery through Gossip

    CERN Document Server

    Haeupler, Bernhard; Peleg, David; Rajaraman, Rajmohan; Sun, Zhifeng

    2012-01-01

    We study randomized gossip-based processes in dynamic networks that are motivated by discovery processes in large-scale distributed networks like peer-to-peer or social networks. A well-studied problem in peer-to-peer networks is the resource discovery problem. There, the goal for nodes (hosts with IP addresses) is to discover the IP addresses of all other hosts. In social networks, nodes (people) discover new nodes through exchanging contacts with their neighbors (friends). In both cases the discovery of new nodes changes the underlying network - new edges are added to the network - and the process continues in the changed network. Rigorously analyzing such dynamic (stochastic) processes with a continuously self-changing topology remains a challenging problem with obvious applications. This paper studies and analyzes two natural gossip-based discovery processes. In the push process, each node repeatedly chooses two random neighbors and puts them in contact (i.e., "pushes" their mutual information to each oth...

  15. The Long Wavelength Array

    Science.gov (United States)

    Taylor, G. B.

    2006-08-01

    The Long Wavelength Array (LWA) will be a new, open, user-oriented astronomical instrument operating in the poorly explored window from 20-80 MHz at arcsecond level resolution and mJy level sensitivity. Key science drivers include (1) acceleration, propagation, and turbulence in the ISM, including the space-distribution and spectrum of Galactic cosmic rays, supernova remnants, and pulsars; (2) the high redshift universe, including the most distant radio galaxies and clusters - tools for understanding the earliest black holes and the cosmological evolution of Dark Matter and Dark Energy; (3) planetary, solar, and space science, including space weather prediction and extra-solar planet searches; and (4) the radio transient universe: including the known (e.g., SNe, GRBs) and the unknown. Because the LWA will explore one of the last and least investigated regions of the spectrum, the potential for new discoveries, including new classes of physical phenomena, is high, and there is a strong synergy with exciting new X-ray and Gamma-ray measurements, e.g. for cosmic ray acceleration, transients, and galaxy clusters. Operated by the University of New Mexico on behalf of the South West Consortium (SWC) the LWA will also provide a unique training ground for the next generation of radio astronomers. Students may also put skills learned on the LWA to work in computer science, electrical engineering, and the communications industry, among others. The development of the LWA will follow a phased build, which benefits from lessons learned at each phase. Four university-based Scientific Testing and Evaluation (ST&E) teams with different areas of concentration (1. High resolution imaging and particle acceleration; 2. Wide field imaging and large scale structures; 3. Ionosphere, and 4. RFI suppression and transient detection) will provide the feedback needed to assure that science objectives are met as the build develops. Currently in its first year of construction funding, the LWA

  16. A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide

    Directory of Open Access Journals (Sweden)

    Roh C

    2012-05-01

    Full Text Available Changhyun RohDivision of Biotechnology, Advanced Radiation Technology Institute (ARTI, Korea Atomic Energy Research Institute (KAERI, Jeongeup, Republic of KoreaAbstract: Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS, and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV nucleocapsid (N protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (--catechin gallate and (--gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (--catechin gallate and (--gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 µg mL–1, (--catechin gallate and (--gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.Keywords: SARS, RNA oligonucleotide, quantum dots, inhibitor, screening

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

    Science.gov (United States)

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

    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.

  18. Combined in vitro transcription and reverse transcription to amplify and label complex synthetic oligonucleotide probe libraries.

    Science.gov (United States)

    Murgha, Yusuf; Beliveau, Brian; Semrau, Kassandra; Schwartz, Donald; Wu, Chao-Ting; Gulari, Erdogan; Rouillard, Jean-Marie

    2015-06-01

    Oligonucleotide microarrays allow the production of complex custom oligonucleotide libraries for nucleic acid detection-based applications such as fluorescence in situ hybridization (FISH). We have developed a PCR-free method to make single-stranded DNA (ssDNA) fluorescent probes through an intermediate RNA library. A double-stranded oligonucleotide library is amplified by transcription to create an RNA library. Next, dye- or hapten-conjugate primers are used to reverse transcribe the RNA to produce a dye-labeled cDNA library. Finally the RNA is hydrolyzed under alkaline conditions to obtain the single-stranded fluorescent probes library. Starting from unique oligonucleotide library constructs, we present two methods to produce single-stranded probe libraries. The two methods differ in the type of reverse transcription (RT) primer, the incorporation of fluorescent dye, and the purification of fluorescent probes. The first method employs dye-labeled reverse transcription primers to produce multiple differentially single-labeled probe subsets from one microarray library. The fluorescent probes are purified from excess primers by oligonucleotide-bead capture. The second method uses an RNA:DNA chimeric primer and amino-modified nucleotides to produce amino-allyl probes. The excess primers and RNA are hydrolyzed under alkaline conditions, followed by probe purification and labeling with amino-reactive dyes. The fluorescent probes created by the combination of transcription and reverse transcription can be used for FISH and to detect any RNA and DNA targets via hybridization.

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

    Indian Academy of Sciences (India)

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

    Abel, B.; Aslan, K. [Morgan State University, Department of Chemistry, 1700 East Cold Spring Lane, Baltimore, MD 21251 (United States)

    2012-11-15

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

  1. Novel Efficient Cell-Penetrating, Peptide-Mediated Strategy for Enhancing Telomerase Inhibitor Oligonucleotides.

    Science.gov (United States)

    Muñoz-Alarcón, Andrés; Eriksson, Jonas; Langel, Ülo

    2015-12-01

    At present, there are several therapeutic approaches for targeting telomerase in tumors. One in particular, currently undergoing clinical trials, is based on synthetic lipid-modified oligonucleotide antagonists aimed at inhibiting the ribonucleoprotein subunit of human telomerase. However, while enabling efficient uptake, the lipid modifications reduce the potency of the therapeutic oligonucleotides compared to nonmodified oligonucleotides. Moreover, lipid modification may increase oligonucleotide accumulation in the liver causing undesirable hepatotoxicity. Noncovalent complexation strategies for cell-penetrating peptide (CPP)-mediated delivery present an option to circumvent the need for potency-reducing modifications, while allowing for a highly efficient uptake, and could significantly improve the efficiency of telomerase-targeting cancer therapeutics. Delivery of a nonlipidated locked nucleic acid/2'-O-methyl mixmer significantly inhibits the telomerase activity in treated HeLa cells. The inhibitory effect was further improved through addition of a CPP. Furthermore, calculated IC50-values for the oligonucleotide delivered by CPPs into HeLa cells are more than 20 times lower than telomerase inhibitor Imetelstat, currently undergoing clinical trials. These results emphasize the potential of CPP-mediated delivery of future pharmaceuticals and provide means by which to enhance an already promising therapeutic strategy for cancer treatment.

  2. Aspects of oligonucleotide and peptide sequencing with MALDI and electrospray mass spectrometry.

    Science.gov (United States)

    Owens, D R; Bothner, B; Phung, Q; Harris, K; Siuzdak, G

    1998-09-01

    Biopolymer sequencing with mass spectrometry has become increasingly important and accessible with the development of matrix-assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Here we examine the use of sequential digestion for the rapid identification of proteolytic fragments, in turn highlighting the general utility of enzymatic MALDI ladder sequencing and ESI tandem mass spectrometry. Analyses were performed on oligonucleotides ranging in size from 2 to 50 residues, on peptides ranging in size from 7 to 44 residues and on viral coat proteins. MALDI ladder sequencing using exonuclease digestion generated a uniform distribution of ions and provided complete sequence information on the oligonucleotides 2-30 nucleic acid residues long. Only partial sequence information was obtained on the longer oligonucleotides. C-terminal peptide ladder sequencing typically provided information from 4 to 7 amino acids into the peptide. Sequential digestion, or endoprotease followed by exoprotease exposure, was also successfully applied to a trypsin digest of viral proteins. Analysis of ladder sequenced peptides by LCMS generated less information than in the MALDI-MS analysis and ESI-MS2 normally provided partial sequence information on both the small oligonucleotides and peptides. In general, MALDI ladder sequencing offered information on a broader mass range of biopolymers than ESI-MS2 and was relatively straightforward to interpret, especially for oligonucleotides.

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

    Directory of Open Access Journals (Sweden)

    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.

  4. Oligonucleotide-induced alternative splicing of serotonin 2C receptor reduces food intake.

    Science.gov (United States)

    Zhang, Zhaiyi; Shen, Manli; Gresch, Paul J; Ghamari-Langroudi, Masoud; Rabchevsky, Alexander G; Emeson, Ronald B; Stamm, Stefan

    2016-08-01

    The serotonin 2C receptor regulates food uptake, and its activity is regulated by alternative pre-mRNA splicing. Alternative exon skipping is predicted to generate a truncated receptor protein isoform, whose existence was confirmed with a new antiserum. The truncated receptor sequesters the full-length receptor in intracellular membranes. We developed an oligonucleotide that promotes exon inclusion, which increases the ratio of the full-length to truncated receptor protein. Decreasing the amount of truncated receptor results in the accumulation of full-length, constitutively active receptor at the cell surface. After injection into the third ventricle of mice, the oligonucleotide accumulates in the arcuate nucleus, where it changes alternative splicing of the serotonin 2C receptor and increases pro-opiomelanocortin expression. Oligonucleotide injection reduced food intake in both wild-type and ob/ob mice. Unexpectedly, the oligonucleotide crossed the blood-brain barrier and its systemic delivery reduced food intake in wild-type mice. The physiological effect of the oligonucleotide suggests that a truncated splice variant regulates the activity of the serotonin 2C receptor, indicating that therapies aimed to change pre-mRNA processing could be useful to treat hyperphagia, characteristic for disorders like Prader-Willi syndrome.

  5. Hard Transparent Arrays for Polymer Pen Lithography.

    Science.gov (United States)

    Hedrick, James L; Brown, Keith A; Kluender, Edward J; Cabezas, Maria D; Chen, Peng-Cheng; Mirkin, Chad A

    2016-03-22

    Patterning nanoscale features across macroscopic areas is challenging due to the vast range of length scales that must be addressed. With polymer pen lithography, arrays of thousands of elastomeric pyramidal pens can be used to write features across centimeter-scales, but deformation of the soft pens limits resolution and minimum feature pitch, especially with polymeric inks. Here, we show that by coating polymer pen arrays with a ∼175 nm silica layer, the resulting hard transparent arrays exhibit a force-independent contact area that improves their patterning capability by reducing the minimum feature size (∼40 nm), minimum feature pitch (<200 nm for polymers), and pen to pen variation. With these new arrays, patterns with as many as 5.9 billion features in a 14.5 cm(2) area were written using a four hundred thousand pyramid pen array. Furthermore, a new method is demonstrated for patterning macroscopic feature size gradients that vary in feature diameter by a factor of 4. Ultimately, this form of polymer pen lithography allows for patterning with the resolution of dip-pen nanolithography across centimeter scales using simple and inexpensive pen arrays. The high resolution and density afforded by this technique position it as a broad-based discovery tool for the field of nanocombinatorics.

  6. Factor XI antisense oligonucleotide for prevention of venous thrombosis.

    Science.gov (United States)

    Büller, Harry R; Bethune, Claudette; Bhanot, Sanjay; Gailani, David; Monia, Brett P; Raskob, Gary E; Segers, Annelise; Verhamme, Peter; Weitz, Jeffrey I

    2015-01-15

    Experimental data indicate that reducing factor XI levels attenuates thrombosis without causing bleeding, but the role of factor XI in the prevention of postoperative venous thrombosis in humans is unknown. FXI-ASO (ISIS 416858) is a second-generation antisense oligonucleotide that specifically reduces factor XI levels. We compared the efficacy and safety of FXI-ASO with those of enoxaparin in patients undergoing total knee arthroplasty. In this open-label, parallel-group study, we randomly assigned 300 patients who were undergoing elective primary unilateral total knee arthroplasty to receive one of two doses of FXI-ASO (200 mg or 300 mg) or 40 mg of enoxaparin once daily. The primary efficacy outcome was the incidence of venous thromboembolism (assessed by mandatory bilateral venography or report of symptomatic events). The principal safety outcome was major or clinically relevant nonmajor bleeding. Around the time of surgery, the mean (±SE) factor XI levels were 0.38±0.01 units per milliliter in the 200-mg FXI-ASO group, 0.20±0.01 units per milliliter in the 300-mg FXI-ASO group, and 0.93±0.02 units per milliliter in the enoxaparin group. The primary efficacy outcome occurred in 36 of 134 patients (27%) who received the 200-mg dose of FXI-ASO and in 3 of 71 patients (4%) who received the 300-mg dose of FXI-ASO, as compared with 21 of 69 patients (30%) who received enoxaparin. The 200-mg regimen was noninferior, and the 300-mg regimen was superior, to enoxaparin (P<0.001). Bleeding occurred in 3%, 3%, and 8% of the patients in the three study groups, respectively. This study showed that factor XI contributes to postoperative venous thromboembolism; reducing factor XI levels in patients undergoing elective primary unilateral total knee arthroplasty was an effective method for its prevention and appeared to be safe with respect to the risk of bleeding. (Funded by Isis Pharmaceuticals; FXI-ASO TKA ClinicalTrials.gov number, NCT01713361.).

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

    Science.gov (United States)

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

    2003-04-01

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

  8. Immunomodulatory oligonucleotide IMT504: Effects on mesenchymal stem cells as a first-in-class immunoprotective/immunoregenerative therapy

    Science.gov (United States)

    Zorzopulos, Jorge; Opal, Steven M; Hernando-Insúa, Andrés; Rodriguez, Juan M; Elías, Fernanda; Fló, Juan; López, Ricardo A; Chasseing, Norma A; Lux-Lantos, Victoria A; Coronel, Maria F; Franco, Raul; Montaner, Alejandro D; Horn, David L

    2017-01-01

    The immune responses of humans and animals to insults (i.e., infections, traumas, tumoral transformation and radiation) are based on an intricate network of cells and chemical messengers. Abnormally high inflammation immediately after insult or abnormally prolonged pro-inflammatory stimuli bringing about chronic inflammation can lead to life-threatening or severely debilitating diseases. Mesenchymal stem cell (MSC) transplant has proved to be an effective therapy in preclinical studies which evaluated a vast diversity of inflammatory conditions. MSCs lead to resolution of inflammation, preparation for regeneration and actual regeneration, and then ultimate return to normal baseline or homeostasis. However, in clinical trials of transplanted MSCs, the expectations of great medical benefit have not yet been fulfilled. As a practical alternative to MSC transplant, a synthetic drug with the capacity to boost endogenous MSC expansion and/or activation may also be effective. Regarding this, IMT504, the prototype of a major class of immunomodulatory oligonucleotides, induces in vivo expansion of MSCs, resulting in a marked improvement in preclinical models of neuropathic pain, osteoporosis, diabetes and sepsis. IMT504 is easily manufactured and has an excellent preclinical safety record. In the small number of patients studied thus far, IMT504 has been well-tolerated, even at very high dosage. Further clinical investigation is necessary to demonstrate the utility of IMT504 for resolution of inflammation and regeneration in a broad array of human diseases that would likely benefit from an immunoprotective/immunoregenerative therapy.

  9. Development of a Method for Profiling Protein Interactions with LNA-Modified Antisense Oligonucleotides Using Protein Microarrays.

    Science.gov (United States)

    Kakiuchi-Kiyota, Satoko; Whiteley, Lawrence O; Ryan, Anne M; Mathialagan, Nagappan

    2016-04-01

    Development of locked nucleic acid (LNA) gapmers, antisense oligonucleotides used for efficient inhibition of target RNA expression, is limited by nontarget-mediated hepatotoxicity. Increased binding of hepatocellular proteins to toxic LNA gapmers may be one of the mechanisms contributing to LNA gapmer-induced hepatotoxicity in vivo. In the present study, we investigated the protein binding propensity of nontoxic sequence-1 (NTS-1), toxic sequence-2 (TS-2), and severely highly toxic sequence-3 (HTS-3) LNA gapmers using human protein microarrays. We previously demonstrated by the transcription profiling analysis of liver RNA isolated from mice that TS-2 and HTS-3 gapmers modulate different transcriptional pathways in mice leading to hepatotoxicity. Our protein array profiling demonstrated that a greater number of proteins, including ones associated with hepatotoxicity, hepatic system disorder, and cell functions, were bound by TS-2 and HTS-3 compared with NTS-1. However, the profiles of proteins bound by TS-2 and HTS-3 were similar and did not distinguish proteins contributing to severe in vivo toxicity. These results, together with the previous transcription profiling analysis, indicate that the combination of sequence-dependent transcription modulation and increased protein binding of toxic LNA gapmers contributes to hepatotoxicity.

  10. Creating A Guided- discovery Lesson

    Institute of Scientific and Technical Information of China (English)

    田枫

    2005-01-01

    In a guided - discovery lesson, students sequentially uncover layers of mathematical information one step at a time and learn new mathematics. We have identified eight critical steps necessary in developing a successful guided- discovery lesson.

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

    Science.gov (United States)

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

    2002-08-01

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

  12. Oligonucleotides conjugated with short chemically defined polyethylene glycol chains are efficient antisense agents.

    Science.gov (United States)

    Shokrzadeh, Nasrin; Winkler, Anna-Maria; Dirin, Mehrdad; Winkler, Johannes

    2014-12-15

    Ligand conjugation is an attractive approach to rationally modify the poor pharmacokinetic behavior and cellular uptake properties of antisense oligonucleotides. Polyethylene glycol (PEG) attachment is a method to increase solubility of oligonucleotides and prevent the rapid elimination, thus increasing tissue distribution. On the other hand, the attachment of long PEG chains negatively influences the pharmacodynamic effect by reducing the hybridization efficiency. We examined the use of short PEG ligands on the in vitro effect of antisense agents. Circular dichroism showed that the tethering of PEG12-chains to phosphodiester and phosphorothioate oligonucleotides had no influence on their secondary structure and did not reduce the affinity to the counter strand. In an in vitro tumor model, a luciferase reporter assay indicated unchanged gene silencing activity compared to unmodified compounds, and even slightly superior target down regulation was found after treatment with a phosphorothioate modified conjugate. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  13. Gene silencing by siRNAs and antisense oligonucleotides in the laboratory and the clinic

    Science.gov (United States)

    Watts, Jonathan K.; Corey, David R.

    2014-01-01

    Synthetic nucleic acids are commonly used laboratory tools for modulating gene expression and have the potential to be widely used in the clinic. Progress towards nucleic acid drugs, however, has been slow and many challenges remain to be overcome before their full impact on patient care can be understood. Antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs) are the two most widely used strategies for silencing gene expression. We first describe these two approaches and contrast their relative strengths and weaknesses for laboratory applications. We then review the choices faced during development of clinical candidates and the current state of clinical trials. Attitudes towards clinical development of nucleic acid silencing strategies have repeatedly swung from optimism to depression during the past twenty years. Our goal is to provide the information needed to design robust studies with oligonucleotides, making use of the strengths of each oligonucleotide technology. PMID:22069063

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

    Science.gov (United States)

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

    1996-11-01

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

  15. ProbeMaker: an extensible framework for design of sets of oligonucleotide probes

    Science.gov (United States)

    Stenberg, Johan; Nilsson, Mats; Landegren, Ulf

    2005-01-01

    Background Procedures for genetic analyses based on oligonucleotide probes are powerful tools that can allow highly parallel investigations of genetic material. Such procedures require the design of large sets of probes using application-specific design constraints. Results ProbeMaker is a software framework for computer-assisted design and analysis of sets of oligonucleotide probe sequences. The tool assists in the design of probes for sets of target sequences, incorporating sequence motifs for purposes such as amplification, visualization, or identification. An extension system allows the framework to be equipped with application-specific components for evaluation of probe sequences, and provides the possibility to include support for importing sequence data from a variety of file formats. Conclusion ProbeMaker is a suitable tool for many different oligonucleotide design and analysis tasks, including the design of probe sets for various types of parallel genetic analyses, experimental validation of design parameters, and in silico testing of probe sequence evaluation algorithms. PMID:16171527

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

    Directory of Open Access Journals (Sweden)

    Roberta D'Agata

    2017-01-01

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

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

    Science.gov (United States)

    Sebastiani, F.; Longo, M.; Orecchini, A.; Comez, L.; De Francesco, A.; Muthmann, M.; Teixeira, S. C. M.; Petrillo, C.; Sacchetti, F.; Paciaroni, A.

    2015-07-01

    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.

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2015-07-07

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

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

    DEFF Research Database (Denmark)

    Taskova, Maria; Madsen, Charlotte Stahl; Jensen, Knud Jørgen

    2017-01-01

    , most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides...... by the internal attachment of peptides but not by LNA alone. Independently of the peptide sequence, the conjugates are stable for up to 24 h in 90% human serum and duplexes of POCs with complementary DNA for up to 160 h in 90% human serum. Such excellent stability has not been previously reported for DNA...... and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media....

  20. Kinetic effects on signal normalization in oligonucleotide microchips with labeled immobilized probes.

    Science.gov (United States)

    Pan'kov, S V; Chechetkin, V R; Somova, O G; Antonova, O V; Moiseeva, O V; Prokopenko, D V; Yurasov, R A; Gryadunov, D A; Chudinov, A V

    2009-10-01

    Among various factors affecting operation of oligonucleotide microchips, the variations in concentration and in homogeneous distribution of immobilized probes over the cells are one of the most important. The labeling of immobilized probes ensures the complete current monitoring on the probe distribution and is reliable and convenient. Using hydrogel-based oligonucleotide microchips, the applicability of Cy3-labeled immobilized probes for quality control and signal normalization after hybridization with Cy5-labeled target DNA was investigated. This study showed that proper signal normalization should be different in thermodynamic conditions and in transient regime with hybridization far from saturation. This kinetic effect holds for both hydrogel-based and surface oligonucleotide microchips. Besides proving basic features, the technique was assessed on a sampling batch of 50 microchips developed for identifying mutations responsible for rifampicin and isoniazid resistance of Mycobacterium tuberculosis.

  1. Dynamics of human telomerase RNA structure revealed by antisense oligonucleotide technique.

    Science.gov (United States)

    Vasilkova, Daria V; Azhibek, Dulat M; Zatsepin, Timofei S; Naraikina, Yulia V; Prassolov, Vladimir S; Prokofjeva, Maria M; Zvereva, Maria I; Rubtsova, Maria P

    2013-12-01

    Telomeres are the nucleoprotein complexes that cap the linear chromosome ends. Telomerase is a ribonucleoprotein that maintains telomere length in stem, embryonic and cancer cells. Somatic cells don't contain active telomerase and telomere function as mitotic clock and telomere length determines the number of cell divisions. Telomerase RNA (TER) contains the template for telomere synthesis and serves as a structural scaffold for holoenzyme assembly. We compared different oligonucleotide based methods for telomerase RNA inhibition, such as antisense oligonucleotides, knockdown by transient siRNA transfection and silencing by miRNA derived from short expressed RNA hairpin in HEK293 cells. All of these methods were applied to different TER regions. Our results revealed that CR2/CR3 domain of TER is accessible in vitro and in vivo and could serve as an optimal site for oligonucleotide-based telomerase silencing.

  2. Gigapixel imaging with microlens arrays

    Science.gov (United States)

    Orth, Antony; Schonbrun, Ethan

    2016-03-01

    A crucial part of the drug discovery process involves imaging the response of thousands of cell cultures to candidate drugs. Quantitative parameters from these "high content screens", such as protein expression and cell morphology, are extracted from fluorescence and brightfield micrographs. Due to the sheer number of cells that need to imaged for adequate statistics, the imaging time itself is a major bottleneck. Automated microscopes image small fields-of-view (FOVs) serially, which are then stitched together to form gigapixel-scale mosaics. We have developed a microscopy architecture that reduces mechanical overhead of traditional large field-of-view by parallelizing the image capture process. Instead of a single objective lens imaging FOVs one by one, we employ a microlens array for continuous photon capture, resulting in a 3-fold throughput increase. In this contribution, we present the design and imaging results of this microscopy architecture in three different contrast modes: multichannel fluorescence, hyperspectral fluorescence and brightfield.

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

    Science.gov (United States)

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

    2016-01-01

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

  4. Acute kidney injury during therapy with an antisense oligonucleotide directed against PCSK9.

    Science.gov (United States)

    van Poelgeest, Eveline P; Swart, Reinout M; Betjes, Michiel G H; Moerland, Matthijs; Weening, Jan J; Tessier, Yann; Hodges, Michael R; Levin, Arthur A; Burggraaf, Jacobus

    2013-10-01

    Antisense oligonucleotides have been explored widely in clinical trials and generally are considered to be nontoxic for the kidney, even at high concentrations. We report a case of toxic acute tubular injury in a healthy 56-year-old female volunteer after a pharmacologically active dose of a locked nucleic acid antisense oligonucleotide was administered. The patient received 3 weekly subcutaneous doses of experimental drug SPC5001, an antisense oligonucleotide directed against PCSK9 (proprotein convertase subtilisin/kexin type 9) that is under investigation as an agent to reduce low-density lipoprotein cholesterol levels. Five days after the last dose, the patient's serum creatinine level increased from 0.81 mg/dL at baseline (corresponding to an estimated glomerular filtration rate [eGFR] of 78 mL/min/1.73 m(2)) to 2.67 mg/dL (eGFR, 20 mL/min/1.73 m(2)), and this increase coincided with the presence of white blood cells, granular casts, and minimal hematuria on urine microscopy. The patient's serum creatinine level peaked at 3.81 mg/dL (eGFR, 13 mL/min/1.73 m(2)) 1 week after the last oligonucleotide dose. Kidney biopsy showed multifocal tubular necrosis and signs of oligonucleotide accumulation. Upon conservative treatment, the patient's serum creatinine level gradually decreased and reached her baseline level 44 days after the last oligonucleotide was administered. The patient recovered fully and kidney function was normal at every follow-up visit. Copyright © 2013 National Kidney Foundation, Inc. Published by Elsevier Inc. All rights reserved.

  5. Atlas of Astronomical Discoveries

    CERN Document Server

    Schilling, Govert

    2011-01-01

    Four hundred years ago in Middelburg, in the Netherlands, the telescope was invented. The invention unleashed a revolution in the exploration of the universe. Galileo Galilei discovered mountains on the Moon, spots on the Sun, and moons around Jupiter. Christiaan Huygens saw details on Mars and rings around Saturn. William Herschel discovered a new planet and mapped binary stars and nebulae. Other astronomers determined the distances to stars, unraveled the structure of the Milky Way, and discovered the expansion of the universe. And, as telescopes became bigger and more powerful, astronomers delved deeper into the mysteries of the cosmos. In his Atlas of Astronomical Discoveries, astronomy journalist Govert Schilling tells the story of 400 years of telescopic astronomy. He looks at the 100 most important discoveries since the invention of the telescope. In his direct and accessible style, the author takes his readers on an exciting journey encompassing the highlights of four centuries of astronomy. Spectacul...

  6. Chronicles in drug discovery.

    Science.gov (United States)

    Davies, Shelley L; Moral, Maria Angels; Bozzo, Jordi

    2007-03-01

    Chronicles in Drug Discovery features special interest reports on advances in drug discovery. This month we highlight agents that target and deplete immunosuppressive regulatory T cells, which are produced by tumor cells to hinder innate immunity against, or chemotherapies targeting, tumor-associated antigens. Antiviral treatments for respiratory syncytial virus, a severe and prevalent infection in children, are limited due to their side effect profiles and cost. New strategies currently under clinical development include monoclonal antibodies, siRNAs, vaccines and oral small molecule inhibitors. Recent therapeutic lines for Huntington's disease include gene therapies that target the mutated human huntingtin gene or deliver neuroprotective growth factors and cellular transplantation in apoptotic regions of the brain. Finally, we highlight the antiinflammatory and antinociceptive properties of new compounds targeting the somatostatin receptor subtype sst4, which warrant further study for their potential application as clinical analgesics.

  7. Use of thiolated oligonucleotides as anti-fouling diluents in electrochemical peptide-based sensors.

    Science.gov (United States)

    McQuistan, Adam; Zaitouna, Anita J; Echeverria, Elena; Lai, Rebecca Y

    2014-05-11

    We incorporated short thiolated oligonucleotides as passivating diluents in the fabrication of electrochemical peptide-based (E-PB) sensors, with the goal of creating a negatively charged layer capable of resisting non-specific adsorption of matrix contaminants. The E-PB HIV sensors fabricated using these diluents were found to be more specific and selective, while retaining attributes similar to the sensor fabricated without these diluents. Overall, these results highlight the advantages of using oligonucleotides as anti-fouling diluents in self-assembled monolayer-based sensors.

  8. Oligonucleotide-templated chemical reactions: pushing the boundaries of a nature-inspired process.

    Science.gov (United States)

    Percivalle, Claudia; Bartolo, Jean-François; Ladame, Sylvain

    2013-01-07

    Widespread in nature, oligonucleotide-templated reactions of phosphodiester bond formation have inspired chemists who are now applying this elegant strategy to the catalysis of a broad range of otherwise inefficient reactions. This review highlights the increasing diversity of chemical reactions that can be efficiently catalysed by an oligonucleotide template, using Watson-Crick base-pairing to bring both reagents in close enough proximity to react, thus increasing significantly their effective molarity. The applications of this elegant concept for nucleic acid sensing and controlled organic synthesis will also be discussed.

  9. Transcriptional inhibition of the bacteriophage T7 early promoter region by oligonucleotide triple helix formation.

    Science.gov (United States)

    Ross, C; Samuel, M; Broitman, S L

    1992-12-30

    We have identified a purine-rich triplex binding sequence overlapping a -35 transcriptional early promoter region of the bacteriophage T7. Triplex-forming oligonucleotide designed to bind this target was annealed to T7 templates and introduced into in vitro transcription systems under conditions favoring specific initiation from this promoter. These templates demonstrated significant transcriptional inhibition relative to naked genomic templates and templates mixed with non-triplex-forming oligonucleotide. It is suggested that triplex formation along this target interferes with transcriptional initiation, and this mechanism may hold potential to disrupt bacteriophage T7 early transcription in vivo.

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

    Science.gov (United States)

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

    1994-09-01

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

  11. Customized oligonucleotide microchips that convert multiple genetic information to simple patterns, are portable and reusable

    Science.gov (United States)

    Mirzabekov, Andrei; Guschin, Dmitry Y.; Chik, Valentine; Drobyshev, Aleksei; Fotin, Alexander; Yershov, Gennadiy; Lysov, Yuri

    2002-01-01

    This invention relates to using customized oligonucleotide microchips as biosensors for the detection and identification of nucleic acids specific for different genes, organisms and/or individuals in the environment, in food and in biological samples. The microchips are designed to convert multiple bits of genetic information into simpler patterns of signals that are interpreted as a unit. Because of an improved method of hybridizing oligonucleotides from samples to microchips, microchips are reusable and transportable. For field study, portable laser or bar code scanners are suitable.

  12. Sequence selective naked-eye detection of DNA harnessing extension of oligonucleotide-modified nucleotides.

    Science.gov (United States)

    Verga, Daniela; Welter, Moritz; Marx, Andreas

    2016-02-01

    DNA polymerases can efficiently and sequence selectively incorporate oligonucleotide (ODN)-modified nucleotides and the incorporated oligonucleotide strand can be employed as primer in rolling circle amplification (RCA). The effective amplification of the DNA primer by Φ29 DNA polymerase allows the sequence-selective hybridisation of the amplified strand with a G-quadruplex DNA sequence that has horse radish peroxidase-like activity. Based on these findings we develop a system that allows DNA detection with single-base resolution by naked eye.

  13. The discovery of quarks.

    Science.gov (United States)

    Riordan, M

    1992-05-29

    Quarks are widely recognized today as being among the elementary particles of which matter is composed. The key evidence for their existence came from a series of inelastic electron-nucleon scattering experiments conducted between 1967 and 1973 at the Stanford Linear Accelerator Center. Other theoretical and experimental advances of the 1970s confirmed this discovery, leading to the present standard model of elementary particle physics.

  14. Discovery as a process

    Energy Technology Data Exchange (ETDEWEB)

    Loehle, C.

    1994-05-01

    The three great myths, which form a sort of triumvirate of misunderstanding, are the Eureka! myth, the hypothesis myth, and the measurement myth. These myths are prevalent among scientists as well as among observers of science. The Eureka! myth asserts that discovery occurs as a flash of insight, and as such is not subject to investigation. This leads to the perception that discovery or deriving a hypothesis is a moment or event rather than a process. Events are singular and not subject to description. The hypothesis myth asserts that proper science is motivated by testing hypotheses, and that if something is not experimentally testable then it is not scientific. This myth leads to absurd posturing by some workers conducting empirical descriptive studies, who dress up their study with a ``hypothesis`` to obtain funding or get it published. Methods papers are often rejected because they do not address a specific scientific problem. The fact is that many of the great breakthroughs in silence involve methods and not hypotheses or arise from largely descriptive studies. Those captured by this myth also try to block funding for those developing methods. The third myth is the measurement myth, which holds that determining what to measure is straightforward, so one doesn`t need a lot of introspection to do science. As one ecologist put it to me ``Don`t give me any of that philosophy junk, just let me out in the field. I know what to measure.`` These myths lead to difficulties for scientists who must face peer review to obtain funding and to get published. These myths also inhibit the study of science as a process. Finally, these myths inhibit creativity and suppress innovation. In this paper I first explore these myths in more detail and then propose a new model of discovery that opens the supposedly miraculous process of discovery to doser scrutiny.

  15. Discovery Systems for Manufacturing

    Science.gov (United States)

    1994-01-01

    the backbone of most of the materials discovery activities we pursue. We call the internal structure of a qualitative model an envisionment . A simple... envisionment (discussed in detail in the next chapter) from the polymer curing domain that is illustrated in figure 1-7. The initial conditions are...TSC "fires" IF-THEN rules which the initial conditions enable, building an envisonment. A stylized envisionment for the immunology knowledge base looks

  16. Synthesis, Dynamic Combinatorial Chemistry, and PCR Amplification of 3'-5' and 3'-6' Disulfide-linked Oligonucleotides

    DEFF Research Database (Denmark)

    Hansen, Dennis Jul; Manuguerra, Ilenia; Kjelstrup, Michael Brøndum;

    2014-01-01

    Disulfide dithymidines linked 3'-5' or 3'-6' were synthesized and incorporated into oligonucleotides through a combined phosphotriester and phosphoramidite solid-phase oligonucleotide synthesis approach. The disulfide links are cleaved and formed reversibly in the presence of thiols and oligonucl...

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

    Institute of Scientific and Technical Information of China (English)

    TianXiaobing; ZhangLihe; MinJimei

    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.

  18. Oligonucleotides Containing Aminated 2'-Amino-LNA Nucleotides: Synthesis and Strong Binding to Complementary DNA and RNA.

    Science.gov (United States)

    Lou, Chenguang; Samuelsen, Simone V; Christensen, Niels Johan; Vester, Birte; Wengel, Jesper

    2017-04-05

    Mono- and diaminated 2'-amino-LNA monomers were synthesized and introduced into oligonucleotides. Each modification imparts significant stabilization of nucleic acid duplexes and triplexes, excellent sequence selectivity, and significant nuclease resistance. Molecular modeling suggested that structural stabilization occurs via intrastrand electrostatic attraction between the protonated amino groups of the aminated 2'-amino-LNA monomers and the host oligonucleotide backbone.

  19. Clocked combustor can array

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Won-Wook; McMahan, Kevin Weston; Srinivasan, Shiva Kumar

    2017-01-17

    The present application provides a clocked combustor can array for coherence reduction in a gas turbine engine. The clocked combustor can array may include a number of combustor cans positioned in a circumferential array. A first set of the combustor cans may have a first orientation and a second set of the combustor cans may have a second orientation.

  20. Axiom turkey genotyping array

    Science.gov (United States)

    The Axiom®Turkey Genotyping Array interrogates 643,845 probesets on the array, covering 643,845 SNPs. The array development was led by Dr. Julie Long of the USDA-ARS Beltsville Agricultural Research Center under a public-private partnership with Hendrix Genetics, Aviagen, and Affymetrix. The Turk...

  1. Clocked combustor can array

    Science.gov (United States)

    Kim, Won-Wook; McMahan, Kevin Weston; Srinivasan, Shiva Kumar

    2017-01-17

    The present application provides a clocked combustor can array for coherence reduction in a gas turbine engine. The clocked combustor can array may include a number of combustor cans positioned in a circumferential array. A first set of the combustor cans may have a first orientation and a second set of the combustor cans may have a second orientation.

  2. Towards the Long Wavelength Array

    Science.gov (United States)

    Kassim, N. E.; Erickson, W. C.

    2008-08-01

    Nearly three decades ago, the Very Large Array (VLA) opened the cm-wavelength radio sky to high-dynamic range imaging. By developing and exploiting new techniques to mitigate ionospheric phase fluctuations, the VLA 74 MHz system is providing the first sub-arcminute resolution view of the meter-wavelength radio universe. This technical innovation has inspired an emerging suite of much more powerful low-frequency instruments, including the Long Wavelength Array (LWA). The LWA, with its great collecting area (approaching one square kilometer at 20 MHz) and long baselines (up to 400 km), will surpass, by up to 2--3 orders of magnitude, the imaging power of any previous low-frequency interferometer. LWA science goals include Cosmic Evolution, the Acceleration of Relativistic Particles, Plasma Astrophysics, and Ionospheric & Space Weather Science. Because it will explore one of the last and most poorly investigated regions of the spectrum, the potential for unexpected new discoveries is high. For more on the LWA, see http://lwa.unm.edu. The LWA project is led by the University of New Mexico, and includes the Naval Research Laboratory, Applied Research Laboratories of U. Texas, Los Alamos National Laboratory, Virginia Tech, and U. Iowa, with cooperation from the National Radio Astronomy Observatory.

  3. HNA and ANA high-affinity arrays for detections of DNA and RNA single-base mismatches.

    Science.gov (United States)

    Abramov, Mikhail; Schepers, Guy; Van Aerschot, Arthur; Van Hummelen, Paul; Herdewijn, Piet

    2008-06-15

    DNA microarrays and sensors have become essential tools in the functional analysis of sequence information. Recently we reported that chimeric hexitol (HNA) and altritol (ANA) nucleotide monomers with an anhydrohexitol sugar moiety are easily available and proved their chemistry to be compatible with DNA and RNA synthesis. In this communication we describe a novel analytical platform based on HNA and ANA units to be used as synthetic oligonucleotide arrays on a glass solid support for match/mismatch detection of DNA and RNA targets. Arrays were fabricated by immobilization of diene-modified oligonucleotides on maleimido-activated glass slides. To demonstrate the selectivity and sensitivity of the HNA/ANA arrays and to compare their properties with regular DNA arrays, sequences in the reverse transcriptase gene (codon 74) and the protease gene of HIV-1 (codon 10) were selected. Both, the relative intensity of the signal and match/mismatch discrimination increased up to fivefold for DNA targets and up to 3-3.5-fold for RNA targets applying HNA or ANA arrays (ANA>HNA>DNA). Certainly in the new field of miRNA detection, ANA arrays could prove very beneficial and their properties should be investigated in more detail.

  4. Negative electrospray ionization mass spectrometry of synthetic and chemically modified oligonucleotides

    NARCIS (Netherlands)

    Potier, N.; Van Dorsselaer, A.; Cordier, Y.; Roch, O.; Bischoff, Rainer

    1994-01-01

    We report here on the analysis of synthetic oligonucleotides by electrospray ionization mass spectrometry (ESI-MS). After intensive removal of salt ions (especially sodium cations), negative ion mass spectra, allowing mass measurement with an accuracy of 0.01%, were obtained on several oligonucleoti

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    operons in E. coli using this method, which we call Microarray-Oligonucleotide (MO)-MAGE. The resulting mutant library was characterized by high-throughput sequencing to show that all attempted insertions were estimated to have occurred at an average frequency of 0.02 % per loci with 0.4 average...

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

    Institute of Scientific and Technical Information of China (English)

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

    2008-01-01

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

  7. An oligonucleotide-tagged microarray for routine diagnostics of colon cancer by genotyping KRAS mutations

    DEFF Research Database (Denmark)

    Liu, Yuliang; Guðnason, Haukur; Li, Yiping

    2014-01-01

    or spiked fecal samples. The immobilized tag-probes were stable under multiple thermal cycling treatments, allowing re-use of the tag-microarray and further optimization to solid PCR. Our results demonstrated that a novel oligonucleotide-tagged microarray system has been developed which would be suitable...

  8. Application of decoy oligonucleotides as novel therapeutic strategy: a contemporary overview.

    Science.gov (United States)

    Ahmad, Mohammad Zaki; Akhter, Sohail; Mallik, Neha; Anwar, Mohammad; Tabassum, Wajda; Ahmad, Farhan Jalees

    2013-03-01

    Molecular therapy is emerging as a potential strategy for the treatment of many diseases. Correct regulation of gene expression is essential for both, to normal development and proper functioning of the all the organisms. Even after four decades of intensive research, it is still a major problem from regulatory and technical point of view, to replace defective genes. The technology of decoy oligonucleotides has received considerable attention to treat and cure a variety of diseases and abnormal physiological conditions, because they provide a rational way to design and selective regulation of a specific gene expression. Decoy oligonucleotides are widely used as inhibitors of specific gene expression because they can offer exciting possibility of expression and blocking of a particular gene without any changes in the functions of other genes. Advances in the decoy oligonucleotides are rapidly paving the way to new insights into the origin and treatment of inflammatory, cancer and/or other immune disorders. The review covers the progress achieved towards the development of decoy oligonucleotides as a potential strategy in a new class of molecular therapy.

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

    Science.gov (United States)

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

    2016-05-11

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

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

    Directory of Open Access Journals (Sweden)

    Yusuf E Murgha

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

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

    Science.gov (United States)

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

    2014-01-01

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

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

    Directory of Open Access Journals (Sweden)

    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.

  13. Synthesis and antisense properties of 2'-O-(2S-methoxypropyl)-RNA-modified gapmer antisense oligonucleotides.

    Science.gov (United States)

    Yu, Jinghua; Pandey, Sanjay K; Khatri, Hetal; Prakash, Thazha P; Swayze, Eric E; Seth, Punit P

    2014-09-01

    To ascertain whether increasing hydrophobicity can enhance the activity of second-generation antisense oligonucleotides (ASOs) in muscle, we investigated the antisense properties of 2'-O-(2S-methoxypropyl)-RNA (2S-MOP)-modified ASOs. Synthesis of the 2S-MOP 5-methyl uridine phosphoramidite was accomplished on a multi-gram scale by Lewis-acid-catalyzed ring opening of 5'-O-tert-butyldiphenylsilyl ether-protected 2,2'-anhydro-5-methyl uridine with 2S-methoxy-1-propanol. Synthesis of the 2S-MOP 5-methyl cytidine nucleoside from the corresponding 5-methyl uridine nucleoside was accomplished by formation and displacement of a 4-triazolide intermediate with aqueous ammonia. 2S-MOP-modified oligonucleotides were prepared on an automated DNA synthesizer and showed similar enhancements in duplex thermal stability as 2'-O-methoxyethyl RNA (MOE)-modified oligonucleotides. 2S-MOP-containing antisense oligonucleotides were evaluated in Balb-c mice and showed good activity for decreasing the expression levels of scavenger receptor B1 (Srb1) and phosphatase and tensin homologue (PTEN) mRNA in liver and muscle tissue. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

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

    Science.gov (United States)

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

    2012-02-01

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

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

    NARCIS (Netherlands)

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

    2003-01-01

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

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

    NARCIS (Netherlands)

    Heemskerk, Johannes Antonius

    2011-01-01

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

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

    NARCIS (Netherlands)

    Heemskerk, Johannes Antonius

    2011-01-01

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

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

    DEFF Research Database (Denmark)

    Lindholm, Marie W; Elmén, Joacim; Fisker, Niels

    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...

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

    DEFF Research Database (Denmark)

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

    2016-01-01

    In continuation of our investigation of characteristics and thermodynamic properties of the i-motif 5′-d[(CCCTAA)3CCCT)] upon insertion of intercalating nucleotides into the cytosine-rich oligonucleotide, this article evaluates the stabilities of i-motif oligonucleotides upon insertion of naphtha......In continuation of our investigation of characteristics and thermodynamic properties of the i-motif 5′-d[(CCCTAA)3CCCT)] upon insertion of intercalating nucleotides into the cytosine-rich oligonucleotide, this article evaluates the stabilities of i-motif oligonucleotides upon insertion...... of naphthalimide (1H-benzo[de]isoquinoline-1,3(2H)-dione) as the intercalating nucleic acid. The stabilities of i-motif structures with inserted naphthalimide intercalating nucleotides were studied using UV melting temperatures (Tm) and circular dichroism spectra at different pH values and conditions (crowding...... and non-crowding). This study indicated a positive effect of the naphthalimide intercalating nucleotides on the stabilities of the i-motif structures compared to the wild-type structure which is in contrast to a previous observation for a pyrene-intercalating nucleotide showing a decrease in Tm values....

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

    Science.gov (United States)

    2013-01-01

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

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

    NARCIS (Netherlands)

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

    2013-01-01

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

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

    Science.gov (United States)

    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.

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

    DEFF Research Database (Denmark)

    Kumar, Rajesh; Ries, Annika; Wengel, Jesper

    2017-01-01

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

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

    NARCIS (Netherlands)

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

    2013-01-01

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

  5. Precise construction of oligonucleotide-Fab fragment conjugate for homogeneous immunoassay using HaloTag technology.

    Science.gov (United States)

    Päkkilä, Henna; Peltomaa, Riikka; Lamminmäki, Urpo; Soukka, Tero

    2015-03-01

    The use of oligonucleotide-protein conjugates enables the development of novel types of bioanalytical assays. However, convenient methods for producing covalent and stoichiometric oligonucleotide-protein conjugates are still rare. Here we demonstrate, for the first time, covalent conjugation of DNA oligonucleotide to Fab fragments with a 1:1 ratio using HaloTag self-labeling technology. The oligonucleotide coupling was carried out while the Fab was attached to protein G matrix, thereby enabling straightforward production of covalent conjugates. Furthermore, it allowed convenient purification of the product because the unreacted components were easily removed before the elution of the high-purity conjugate. The prepared conjugate was employed in a homogeneous immunoassay where prostate-specific antigen was used as a model analyte. Switchable lanthanide luminescence was used for detection, and the obtained limit of detection was 0.27 ng/ml. In the future, the developed method for covalent conjugation and successive purification in protein G column could also be applied for introducing other kinds of modifications to Fab fragments in a simple and site-specific manner.

  6. Rapid identification of bacteria in blood cultures by using fluorescently labeled oligonucleotide probes

    NARCIS (Netherlands)

    Jansen, GJ; Mooibroek, M; Idema, J; Harmsen, HJM; Welling, GW; Degener, JE

    2000-01-01

    The applicability of whole-cell hybridization for the identification of pathogenic bacteria in blood from septic patients was examined. Oligonucleotide probes, fluorescently labeled with fluorescein isothiocyanate, directed against the variable regions of the 16S rRNAs of the following bacterial spe

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

    Directory of Open Access Journals (Sweden)

    Eric Fouquet

    2013-11-01

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

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

    DEFF Research Database (Denmark)

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

    1995-01-01

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

  9. A facile inhibitor screening of SARS coronavirus N protein using nanoparticle-based RNA oligonucleotide.

    Science.gov (United States)

    Roh, Changhyun

    2012-01-01

    Hundreds of million people worldwide have been infected with severe acute respiratory syndrome (SARS), and the rate of global death from SARS has remarkably increased. Hence, the development of efficient drug treatments for the biological effects of SARS is highly needed. We have previously shown that quantum dots (QDs)-conjugated RNA oligonucleotide is sensitive to the specific recognition of the SARS-associated coronavirus (SARS-CoV) nucleocapsid (N) protein. In this study, we found that a designed biochip could analyze inhibitors of the SARS-CoV N protein using nanoparticle-based RNA oligonucleotide. Among the polyphenolic compounds examined, (-)-catechin gallate and (-)-gallocatechin gallate demonstrated a remarkable inhibition activity on SARS-CoV N protein. (-)-catechin gallate and (-)-gallocatechin gallate attenuated the binding affinity in a concentrated manner as evidenced by QDs-conjugated RNA oligonucleotide on a designed biochip. At a concentration of 0.05 μg mL(-1), (-)-catechin gallate and (-)-gallocatechin gallate showed more than 40% inhibition activity on a nanoparticle-based RNA oligonucleotide biochip system.

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

    Science.gov (United States)

    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.

  11. In situ entry of oligonucleotides into brain cells can occur through a nucleic acid channel

    NARCIS (Netherlands)

    Shi, Fuxin; Gounko, Natasha V.; Wang, Xiaoqin; Ronken, Eric; Hoekstra, Dick

    2007-01-01

    Brain tissue has become a challenging therapeutic target, in part because of failure of conventional treatments of brain tumors and a gradually increasing number of neurodegenerative diseases. Because antisense oligonucleotides are readily internalized by neuronal cells in culture, these compounds c

  12. Effect of iontophoresis on the in vitro trans-scleral transport of three single stranded oligonucleotides.

    Science.gov (United States)

    Pescina, Silvia; Antopolsky, Maxim; Santi, Patrizia; Nicoli, Sara; Murtomäki, Lasse

    2013-05-13

    Oligonucleotides represent a subject of clinical interest due to their potential ability to treat several diseases, including those affecting the posterior segment of the eye. Unfortunately, therapeutic oligonucleotides are currently administered by means of highly invasive approaches, such as intravitreal injections. The aim of the present work was to study in vitro, across isolated bovine sclera, the effect of iontophoresis on the transport of three single stranded oligonucleotides (ssDNA), 12-, 24- and 36-mer, selected as reference compounds in view of a non-invasive drug delivery to the back of the eye. All the three sequences were able to cross bovine sclera in vitro without iontophoresis. When anodal iontophoresis was applied, no change in flux was observed, while in the presence of cathodal iontophoresis the permeability coefficients increased four-fold compared to passive conditions. This behavior can be ascribed to the electrorepulsive mechanism, due to the negative charge of the nucleic acid backbone. It was also observed that the molecular weights of the three sequences did not affect trans-scleral transport, neither in passive, nor in current assisted permeation. Furthermore, increasing the current intensity from 1.75 mA to 3 mA, no effect on the trans-scleral transport of the 24-mer was noticed. Although preliminary, the results demonstrate that cathodal iontophoresis enhances trans-scleral transport of single stranded oligonucleotides and suggest its use as a novel non-invasive approach for the treatment of diseases affecting the posterior segment of the eye.

  13. Stable gene targeting in human cells using single-strand oligonucleotides with modified bases.

    Directory of Open Access Journals (Sweden)

    Xavier Rios

    Full Text Available Recent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of ~0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells. Further optimization of this method should allow rapid and scalable genome engineering in human cells.

  14. Optimization of single-base-pair mismatch discrimination in oligonucleotide microarrays

    NARCIS (Netherlands)

    Urakawa, H.; Fantroussi, El S.; Smidt, H.; Smoot, J.C.; Tribou, E.H.; Kelly, J.J.; Noble, P.A.; Stahl, D.A.

    2003-01-01

    The discrimination between perfect-match and single-base-pair-mismatched nucleic acid duplexes was investigated by using oligonucleotide DNA microarrays and nonequilibrium dissociation rates (melting profiles). DNA and RNA versions of two synthetic targets corresponding to the 16S rRNA sequences of

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

    Science.gov (United States)

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

    2011-08-01

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

  16. Tetrahedron-structured DNA and functional oligonucleotide for construction of an electrochemical DNA-based biosensor.

    Science.gov (United States)

    Bu, Nan-Nan; Tang, Chun-Xia; He, Xi-Wen; Yin, Xue-Bo

    2011-07-21

    Tetrahedron-structured DNA (ts-DNA) in combination with a functionalized oligonucleotide was used to develop a "turn-on" biosensor for Hg(2+) ions. The ts-DNA provided an improved sensitivity and was used to block the active sites.

  17. The Discovery Channel Telescope

    Science.gov (United States)

    Millis, R. L.; Dunham, E. W.; Sebring, T. A.; Smith, B. W.; de Kock, M.; Wiecha, O.

    2004-11-01

    The Discovery Channel Telescope (DCT) is a 4.2-m telescope to be built at a new site near Happy Jack, Arizona. The DCT features a large prime focus mosaic CCD camera with a 2-degree-diameter field of view especially designed for surveys of KBOs, Centaurs, NEAs and other moving or time-variable targets. The telescope can be switched quickly to a Ritchey-Chretien configuration for optical/IR spectroscopy or near-IR imaging. This flexibility allows timely follow-up physical studies of high priority objects discovered in survey mode. The ULE (ultra-low-expansion) meniscus primary and secondary mirror blanks for the telescope are currently in fabrication by Corning Glass. Goodrich Aerospace, Vertex RSI, M3 Engineering and Technology Corp., and e2v Technologies have recently completed in-depth conceptual design studies of the optics, mount, enclosure, and mosaic focal plane, respectively. The results of these studies were subjected to a formal design review in July, 2004. Site testing at the 7760-ft altitude Happy Jack site began in 2001. Differential image motion observations from 117 nights since January 1, 2003 gave median seeing of 0.84 arcsec FWHM, and the average of the first quartile was 0.62 arcsec. The National Environmental Policy Act (NEPA) process for securing long-term access to this site on the Coconino National Forest is nearing completion and ground breaking is expected in the spring of 2005. The Discovery Channel Telescope is a project of the Lowell Observatory with major financial support from Discovery Communications, Inc. (DCI). DCI plans ongoing television programming featuring the construction of the telescope and the research ultimately undertaken with the DCT. An additional partner can be accommodated in the project. Interested parties should contact the lead author.

  18. Low-cost, flexible polymer arrays for long-term neuronal culture.

    Science.gov (United States)

    Hogan, N Catherine; Talei-Franzesi, Giovanni; Abudayyeh, Omar; Taberner, Andrew; Hunter, Ian

    2012-01-01

    Conducting polymers are promising materials for fabrication of microelectrode arrays for both neural stimulation and recording. Our ability to engineer the morphology and composition of polypyrrole together with its suitability as an electrically addressable tissue/cell substrate have been used to develop an inexpensive, disposable three-dimensional polymeric array for use in neuronal culture and drug discovery. These arrays could be interfaced with a fixed, parallel stimulation and optical imaging system, amenable to automated handling and data analysis.

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

    Directory of Open Access Journals (Sweden)

    Paula Fernandez

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

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

    Science.gov (United States)

    Taskova, Maria; Madsen, Charlotte S; Jensen, Knud J; Hansen, Lykke Haastrup; Vester, Birte; Astakhova, Kira

    2017-03-15

    Specific target binding and stability in diverse biological media is of crucial importance for applications of synthetic oligonucleotides as diagnostic and therapeutic tools. So far, these issues have been addressed by chemical modification of oligonucleotides and by conjugation with a peptide, most often at the terminal position of the oligonucleotide. Herein, we for the first time systematically investigate the influence of internally attached short peptides on the properties of antisense oligonucleotides. We report the synthesis and internal double labeling of 21-mer oligonucleotides that target the BRAF V600E oncogene, with a library of rationally designed peptides employing CuAAC "click" chemistry. The peptide sequence has an influence on the specificity and affinity of target DNA/RNA binding. We also investigated the impact of locked nucleic acids (LNAs) on the latter. Lysine residues improve binding of POCs to target DNA and RNA, whereas the distance to lysine correlates exclusively with a decrease in binding of mismatched RNA targets. Glycine and tyrosine residues affect target binding as well. Importantly, the resistance of POCs to enzymatic degradation is dramatically improved by the internal attachment of peptides but not by LNA alone. Independently of the peptide sequence, the conjugates are stable for up to 24 h in 90% human serum and duplexes of POCs with complementary DNA for up to 160 h in 90% human serum. Such excellent stability has not been previously reported for DNA and makes internally labeled POCs an exciting object of study, i.e., showing high target specificity and simultaneous stability in biological media.

  1. Osmolality of antisense oligonucleotide parenteral formulations: Implications on counterion dissociation and recommended osmometry techniques.

    Science.gov (United States)

    Lim, Marc; Dibble, Andrew

    2016-12-30

    The intrinsic osmolality of aqueous solutions of sodium salt antisense oligonucleotides (ASOs) has been studied to inform formulation practices, understand the molecular basis underlying the difference between theoretical and empirical results, and determine suitable measurement methods. It was found that regardless of nucleotide sequence, ASO concentration of ∼140mg/mL has isotonic osmolality of ∼290mOsm/kg water (SI unit: mmol osmotically-active particles/kg water), such that lower concentration formulations require excipients for tonicity adjustment. The range of osmolality values at a given active ingredient concentration can be ascribed to drug substance lot-to-lot purity differences impacting total oligonucleotide content (i.e., including oligonucleotide-related impurities). Empirical osmolality measurements were found to be ∼70% of theoretical values, which corresponds to an osmotic coefficient value of ∼0.7, thus inferring incomplete counterion dissociation. When comparing theoretical (ideal) osmolality of multiple sequences with various nucleotide compositions and chemistries at the same w/v concentration, the "average osmolar mass" (molar mass of the oligonucleotide, including the sodium counterions, divided by the ideal Van't Hoff factor, i(id)) appears to be the strongest factor governing theoretical osmolality values. Other factors examined were the sequence length, backbone chemistry, 2' sugar chemistry, and nucleotide composition. A head-to-head comparison between two osmolality techniques showed that vapor pressure osmometry is generally more suitable than freezing point osmometry for oligonucleotide solutions greater than ∼150mg/mL due to viscosity effects, but the two techniques are comparable otherwise. Copyright © 2016 Elsevier B.V. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Muyrers Joep PP

    2003-01-01

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

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

    Directory of Open Access Journals (Sweden)

    Parekh-Olmedo Hetal

    2006-10-01

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

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

    Directory of Open Access Journals (Sweden)

    Elela Sherif

    2006-01-01

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

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

    Science.gov (United States)

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

    2012-01-01

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

  6. Application of lean manufacturing concepts to drug discovery: rapid analogue library synthesis.

    Science.gov (United States)

    Weller, Harold N; Nirschl, David S; Petrillo, Edward W; Poss, Michael A; Andres, Charles J; Cavallaro, Cullen L; Echols, Martin M; Grant-Young, Katherine A; Houston, John G; Miller, Arthur V; Swann, R Thomas

    2006-01-01

    The application of parallel synthesis to lead optimization programs in drug discovery has been an ongoing challenge since the first reports of library synthesis. A number of approaches to the application of parallel array synthesis to lead optimization have been attempted over the years, ranging from widespread deployment by (and support of) individual medicinal chemists to centralization as a service by an expert core team. This manuscript describes our experience with the latter approach, which was undertaken as part of a larger initiative to optimize drug discovery. In particular, we highlight how concepts taken from the manufacturing sector can be applied to drug discovery and parallel synthesis to improve the timeliness and thus the impact of arrays on drug discovery.

  7. A light emitting diode based photoelectrochemical screener for distributed combinatorial materials discovery.

    Science.gov (United States)

    Winkler, Gates R; Winkler, Jay R

    2011-11-01

    Combinatorial approaches for targeted discovery of new materials require rapid screening systems to evaluate large numbers of new material compositions. High-throughput combinatorial materials discovery is a capital-intensive undertaking requiring sophisticated robotic sample preparation and rapid screening assays. A distributed approach to combinatorial materials discovery can achieve similar goals by increasing the breadth of participation and reducing the size of the capital investment. The discovery of new photoactive materials for solar fuels production demands a screening device to probe materials for electrochemical current production upon irradiation with visible light. We have developed a system that uses an array of pulsed light-emitting diodes (LEDs) synchronized with a two-electrode potentiostat that can measure the photoelectrochemical responses of combinatorial sample arrays deposited on conducting glass plates. Compared to raster scanning methods, this LED system trades spatial resolution for a substantial reduction in scan time. © 2011 American Institute of Physics

  8. Causality discovery technology

    Science.gov (United States)

    Chen, M.; Ertl, T.; Jirotka, M.; Trefethen, A.; Schmidt, A.; Coecke, B.; Bañares-Alcántara, R.

    2012-11-01

    Causality is the fabric of our dynamic world. We all make frequent attempts to reason causation relationships of everyday events (e.g., what was the cause of my headache, or what has upset Alice?). We attempt to manage causality all the time through planning and scheduling. The greatest scientific discoveries are usually about causality (e.g., Newton found the cause for an apple to fall, and Darwin discovered natural selection). Meanwhile, we continue to seek a comprehensive understanding about the causes of numerous complex phenomena, such as social divisions, economic crisis, global warming, home-grown terrorism, etc. Humans analyse and reason causality based on observation, experimentation and acquired a priori knowledge. Today's technologies enable us to make observations and carry out experiments in an unprecedented scale that has created data mountains everywhere. Whereas there are exciting opportunities to discover new causation relationships, there are also unparalleled challenges to benefit from such data mountains. In this article, we present a case for developing a new piece of ICT, called Causality Discovery Technology. We reason about the necessity, feasibility and potential impact of such a technology.

  9. Automated Supernova Discovery (Abstract)

    Science.gov (United States)

    Post, R. S.

    2015-12-01

    (Abstract only) We are developing a system of robotic telescopes for automatic recognition of Supernovas as well as other transient events in collaboration with the Puckett Supernova Search Team. At the SAS2014 meeting, the discovery program, SNARE, was first described. Since then, it has been continuously improved to handle searches under a wide variety of atmospheric conditions. Currently, two telescopes are used to build a reference library while searching for PSN with a partial library. Since data is taken every night without clouds, we must deal with varying atmospheric and high background illumination from the moon. Software is configured to identify a PSN, reshoot for verification with options to change the run plan to acquire photometric or spectrographic data. The telescopes are 24-inch CDK24, with Alta U230 cameras, one in CA and one in NM. Images and run plans are sent between sites so the CA telescope can search while photometry is done in NM. Our goal is to find bright PSNs with magnitude 17.5 or less which is the limit of our planned spectroscopy. We present results from our first automated PSN discoveries and plans for PSN data acquisition.

  10. Systematic evaluation and optimization of modification reactions of oligonucleotides with amines and carboxylic acids for the synthesis of DNA-encoded chemical libraries.

    Science.gov (United States)

    Franzini, Raphael M; Samain, Florent; Abd Elrahman, Maaly; Mikutis, Gediminas; Nauer, Angela; Zimmermann, Mauro; Scheuermann, Jörg; Hall, Jonathan; Neri, Dario

    2014-08-20

    DNA-encoded chemical libraries are collections of small molecules, attached to DNA fragments serving as identification barcodes, which can be screened against multiple protein targets, thus facilitating the drug discovery process. The preparation of large DNA-encoded chemical libraries crucially depends on the availability of robust synthetic methods, which enable the efficient conjugation to oligonucleotides of structurally diverse building blocks, sharing a common reactive group. Reactions of DNA derivatives with amines and/or carboxylic acids are particularly attractive for the synthesis of encoded libraries, in view of the very large number of building blocks that are commercially available. However, systematic studies on these reactions in the presence of DNA have not been reported so far. We first investigated conditions for the coupling of primary amines to oligonucleotides, using either a nucleophilic attack on chloroacetamide derivatives or a reductive amination on aldehyde-modified DNA. While both methods could be used for the production of secondary amines, the reductive amination approach was generally associated with higher yields and better purity. In a second endeavor, we optimized conditions for the coupling of a diverse set of 501 carboxylic acids to DNA derivatives, carrying primary and secondary amine functions. The coupling efficiency was generally higher for primary amines, compared to secondary amine substituents, but varied considerably depending on the structure of the acids and on the synthetic methods used. Optimal reaction conditions could be found for certain sets of compounds (with conversions >80%), but multiple reaction schemes are needed when assembling large libraries with highly diverse building blocks. The reactions and experimental conditions presented in this article should facilitate the synthesis of future DNA-encoded chemical libraries, while outlining the synthetic challenges that remain to be overcome.

  11. Electronic Switch Arrays for Managing Microbattery Arrays

    Science.gov (United States)

    Mojarradi, Mohammad; Alahmad, Mahmoud; Sukumar, Vinesh; Zghoul, Fadi; Buck, Kevin; Hess, Herbert; Li, Harry; Cox, David

    2008-01-01

    Integrated circuits have been invented for managing the charging and discharging of such advanced miniature energy-storage devices as planar arrays of microscopic energy-storage elements [typically, microscopic electrochemical cells (microbatteries) or microcapacitors]. The architecture of these circuits enables implementation of the following energy-management options: dynamic configuration of the elements of an array into a series or parallel combination of banks (subarrarys), each array comprising a series of parallel combination of elements; direct addressing of individual banks for charging/or discharging; and, disconnection of defective elements and corresponding reconfiguration of the rest of the array to utilize the remaining functional elements to obtain the desited voltage and current performance. An integrated circuit according to the invention consists partly of a planar array of field-effect transistors that function as switches for routing electric power among the energy-storage elements, the power source, and the load. To connect the energy-storage elements to the power source for charging, a specific subset of switches is closed; to connect the energy-storage elements to the load for discharging, a different specific set of switches is closed. Also included in the integrated circuit is circuitry for monitoring and controlling charging and discharging. The control and monitoring circuitry, the switching transistors, and interconnecting metal lines are laid out on the integrated-circuit chip in a pattern that registers with the array of energy-storage elements. There is a design option to either (1) fabricate the energy-storage elements in the corresponding locations on, and as an integral part of, this integrated circuit; or (2) following a flip-chip approach, fabricate the array of energy-storage elements on a separate integrated-circuit chip and then align and bond the two chips together.

  12. Spider venomics: implications for drug discovery.

    Science.gov (United States)

    Pineda, Sandy S; Undheim, Eivind A B; Rupasinghe, Darshani B; Ikonomopoulou, Maria P; King, Glenn F

    2014-10-01

    Over a period of more than 300 million years, spiders have evolved complex venoms containing an extraordinary array of toxins for prey capture and defense against predators. The major components of most spider venoms are small disulfide-bridged peptides that are highly stable and resistant to proteolytic degradation. Moreover, many of these peptides have high specificity and potency toward molecular targets of therapeutic importance. This unique combination of bioactivity and stability has made spider-venom peptides valuable both as pharmacological tools and as leads for drug development. This review describes recent advances in spider-venom-based drug discovery pipelines. We discuss spider-venom-derived peptides that are currently under investigation for treatment of a diverse range of pathologies including pain, stroke and cancer.

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

    Directory of Open Access Journals (Sweden)

    Ingrid E C Verhaart

    2014-01-01

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

  14. Triple helix-forming oligonucleotides conjugated to indolocarbazole poisons direct topoisomerase I-mediated DNA cleavage to a specific site.

    Science.gov (United States)

    Arimondo, P B; Bailly, C; Boutorine, A S; Moreau, P; Prudhomme, M; Sun, J S; Garestier, T; Hélène, C

    2001-01-01

    Topoisomerase I is an ubiquitous DNA-cleaving enzyme and an important therapeutic target in cancer chemotherapy for camptothecins as well as for indolocarbazole antibiotics such as rebeccamycin. To achieve a sequence-specific cleavage of DNA by topoisomerase I, a triple helix-forming oligonucleotide was covalently linked to indolocarbazole-type topoisomerase I poisons. The three indolocarbazole-oligonucleotide conjugates investigated were able to direct topoisomerase I cleavage at a specific site based upon sequence recognition by triplex formation. The efficacy of topoisomerase I-mediated DNA cleavage depends markedly on the intrinsic potency of the drug. We show that DNA cleavage depends also upon the length of the linker arm between the triplex-forming oligonucleotide and the drug. Based on a known structure of the DNA-topoisomerase I complex, a molecular model of the oligonucleotide conjugates bound to the DNA-topoisomerase I complex was elaborated to facilitate the design of a potent topoisomerase I inhibitor-oligonucleotide conjugate with an optimized linker between the two moieties. The resulting oligonucleotide-indolocarbazole conjugate at 10 nM induced cleavage at the triple helix site 2-fold more efficiently than 5 microM of free indolocarbazole, while the other drug-sensitive sites were not cleaved. The rational design of drug-oligonucleotide conjugates carrying a DNA topoisomerase poison may be exploited to improve the efficacy and selectivity of chemotherapeutic cancer treatments by targeting specific genes and reducing drug toxicity.

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

    Directory of Open Access Journals (Sweden)

    Ajay Kumar

    2010-01-01

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

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

    Institute of Scientific and Technical Information of China (English)

    李中国; 张虹

    2004-01-01

    The effects of suppression of CD44 by CD44-specific antisense oligonucleotide on attachment of human trabecular meshwork cells to hyaluronic acid (HA) were observed and the possible relationship between CD44 and primary open-angle glaucoma (POAG) investigated. CD44-specific antisense oligonucleotide was delivered with cationic lipid to cultured human trabecular meshwork cells. The expression of CD44 suppressed by CD44-specific antisense oligonucleotide was detected by RT-PCR and Western blotting. The effect of CD44 suppression by specific antisense oligonucleotide on attachment of trabecular meshwork cells to HA was measured by MTT assay. Results showed that expression of CD44 was suppressed by CD4, specific antisense oligonucleotide. Antisense oligonucleotide also suppressed the adhesion of human trabecular meshwork cells to HA in a concentration dependent manner. It was concluded that attachment of human trabecular meshwork cells to HA was decreased when CD44 was suppressed by specific antisense oligonucleotide. CD44might play a role in pathogenesis of POAG by affecting the adhesion of trabecular meshwork cells to HA.

  17. Representation Discovery using Harmonic Analysis

    CERN Document Server

    Mahadevan, Sridhar

    2008-01-01

    Representations are at the heart of artificial intelligence (AI). This book is devoted to the problem of representation discovery: how can an intelligent system construct representations from its experience? Representation discovery re-parameterizes the state space - prior to the application of information retrieval, machine learning, or optimization techniques - facilitating later inference processes by constructing new task-specific bases adapted to the state space geometry. This book presents a general approach to representation discovery using the framework of harmonic analysis, in particu

  18. Array Antenna Limitations

    CERN Document Server

    Jonsson, B L G; Hussain, N

    2013-01-01

    This letter defines a physical bound based array figure of merit that provides a tool to compare the performance of both single and multi-band array antennas with respect to return-loss, thickness of the array over the ground-plane, and scan-range. The result is based on a sum-rule result of Rozanov-type for linear polarization. For single-band antennas it extends an existing limit for a given fixed scan-angle to include the whole scan-range of the array, as well as the unit-cell structure in the bound. The letter ends with an investigation of the array figure of merit for some wideband and/or wide-scan antennas with linear polarization. We find arrays with a figure of merit >0.6 that empirically defines high-performance antennas with respect to this measure.

  19. Carbon nanotube nanoelectrode arrays

    Science.gov (United States)

    Ren, Zhifeng; Lin, Yuehe; Yantasee, Wassana; Liu, Guodong; Lu, Fang; Tu, Yi

    2008-11-18

    The present invention relates to microelectode arrays (MEAs), and more particularly to carbon nanotube nanoelectrode arrays (CNT-NEAs) for chemical and biological sensing, and methods of use. A nanoelectrode array includes a carbon nanotube material comprising an array of substantially linear carbon nanotubes each having a proximal end and a distal end, the proximal end of the carbon nanotubes are attached to a catalyst substrate material so as to form the array with a pre-determined site density, wherein the carbon nanotubes are aligned with respect to one another within the array; an electrically insulating layer on the surface of the carbon nanotube material, whereby the distal end of the carbon nanotubes extend beyond the electrically insulating layer; a second adhesive electrically insulating layer on the surface of the electrically insulating layer, whereby the distal end of the carbon nanotubes extend beyond the second adhesive electrically insulating layer; and a metal wire attached to the catalyst substrate material.

  20. Synthesis and evaluation of a fluorine-18 labeled antisense oligonucleotide as a potential PET tracer for iNOS mRNA expression

    Energy Technology Data Exchange (ETDEWEB)

    Vries, Erik F.J. de E-mail: e.f.j.de.vries@pet.azg.nl; Vroegh, Joke; Dijkstra, Gerard; Moshage, Han; Elsinga, Philip H.; Jansen, Peter L.M.; Vaalburg, Willem

    2004-07-01

    Inducible NO synthase (iNOS) is overexpressed in inflammatory bowel diseases. An antisense oligonucleotide with good hybridization properties for iNOS mRNA was selected using RT-PCR. The oligonucleotide was reliably labeled with fluorine-18 using N-(4-[{sup 18}F]fluorobenzyl)-2-bromoacetamide. Cellular uptake and efflux of oligonucleotide complexed with FuGENE-6 were rapid, unlike naked oligonucleotide, which hardly accumulated. However, neither uptake nor efflux showed any selectivity for iNOS expressing cells. The oligonucleotide showed a high level of non-specific binding, which may have obscured its specific hybridization to iNOS mRNA.

  1. Pacific Array (Transportable Broadband Ocean Floor Array)

    Science.gov (United States)

    Kawakatsu, Hitoshi; Ekstrom, Goran; Evans, Rob; Forsyth, Don; Gaherty, Jim; Kennett, Brian; Montagner, Jean-Paul; Utada, Hisashi

    2016-04-01

    Based on recent developments on broadband ocean bottom seismometry, we propose a next generation large-scale array experiment in the ocean. Recent advances in ocean bottom broadband seismometry1, together with advances in the seismic analysis methodology, have enabled us to resolve the regional 1-D structure of the entire lithosphere/asthenosphere system, including seismic anisotropy (azimuthal, and hopefully radial), with deployments of ~15 broadband ocean bottom seismometers (BBOBSs). Having ~15 BBOBSs as an array unit for a 2-year deployment, and repeating such deployments in a leap-frog way or concurrently (an array of arrays) for a decade or so would enable us to cover a large portion of the Pacific basin. Such efforts, not only by giving regional constraints on the 1-D structure beneath Pacific ocean, but also by sharing waveform data for global scale waveform tomography, would drastically increase our knowledge of how plate tectonics works on this planet, as well as how it worked for the past 150 million years. International collaborations is essential: if three countries/institutions participate this endeavor together, Pacific Array may be accomplished within five-or-so years.

  2. Denton Vacuum Discovery-550 Sputterer

    Data.gov (United States)

    Federal Laboratory Consortium — Description: CORAL Name: Sputter 2 Similar to the existing 4-Gun Denton Discovery 22 Sputter system, with the following enhancements: Specifications / Capabilities:...

  3. Optogenetics enlightens neuroscience drug discovery.

    Science.gov (United States)

    Song, Chenchen; Knöpfel, Thomas

    2016-02-01

    Optogenetics - the use of light and genetics to manipulate and monitor the activities of defined cell populations - has already had a transformative impact on basic neuroscience research. Now, the conceptual and methodological advances associated with optogenetic approaches are providing fresh momentum to neuroscience drug discovery, particularly in areas that are stalled on the concept of 'fixing the brain chemistry'. Optogenetics is beginning to translate and transit into drug discovery in several key domains, including target discovery, high-throughput screening and novel therapeutic approaches to disease states. Here, we discuss the exciting potential of optogenetic technologies to transform neuroscience drug discovery.

  4. Dynamically Reconfigurable Microphone Arrays

    Science.gov (United States)

    2011-05-01

    Static + 2 Wireless Using only a standard computer sound card, a robot is limited to binaural inputs. Even when using wireless microphones, the audio...Abstract—Robotic sound localization has traditionally been restricted to either on-robot microphone arrays or embedded microphones in aware...a microphone array has a significant impact on the mathematics of sound source localization. Arrays, for instance, are commonly designed to

  5. Optimization and scale-up of oligonucleotide synthesis in packed bed reactors using computational fluid dynamics modeling.

    Science.gov (United States)

    Wolfrum, Christian; Josten, Andre; Götz, Peter

    2014-01-01

    A computational fluid dynamics (CFD) model for the analysis of oligonucleotide synthesis in packed bed reactors was developed and used to optimize the scale up of the process. The model includes reaction kinetics data obtained under well defined conditions comparable to the situation in the packed bed. The model was validated in terms of flow conditions and reaction kinetics by comparison with experimental data. Experimental validation and the following model parameter studies by simulation were performed on the basis of a column with 0.3 g oligonucleotide capacity. The scale-up studies based on CFD modelling were calculated on a 440 g scale (oligonucleotide capacity).

  6. Characterization of the nanostructure of complexes formed by single- or double-stranded oligonucleotides with a cationic surfactant.

    Science.gov (United States)

    Liu, Xiaoyang; Abbott, Nicholas L

    2010-12-02

    We report the use of dynamic light scattering (DLS), small-angle neutron scattering (SANS), and small-angle X-ray scattering (SAXS) to characterize the nanostructure of complexes formed by either single- or double-stranded oligonucleotides with a cationic surfactant (cetyltrimethylammonium bromide, CTAB) in aqueous solution (1 mM Li(2)SO(4)). For single-stranded oligonucleotides 5'-A(20)-3' and 5'-CCCCATTCTAGCAGCCCGGG-3', both the appearance of two Bragg peaks (at 0.14 and 0.28 Å(-1)) in SAXS spectra with a spacing of 1:2 and form factor fits to SANS spectra are consistent with the presence of multilamellar vesicles (with, on average, 6-9 layers with a periodicity of 45-48 Å). Some samples showed evidence of an additional Bragg peak (at 0.20 Å(-1)) associated with periodic packing (with a periodicity of 31 Å) of the oligonucleotides within the lamellae of the nanostructure. The nucleotide composition of the single-stranded oligonucleotides was also found to impact the number and size of the complexes formed with CTAB. In contrast to 5'-A(20)-3' and 5'-CCCCATTCTAGCAGCCCGGG-3', 5'-T(20)-3' did not change the state of aggregation of CTAB (globular micelles) over a wide range of oligonucleotide:CTAB charge ratios. These results support the proposition that hydrophobic interactions, as well as electrostatics, play a central role in the formation of complexes between cationic amphiphiles and single-stranded oligonucleotides and thus give rise to nanostructures that depend on nucleotide composition. In contrast to the single-stranded oligonucleotides, for double-stranded oligonucleotides mixed with CTAB, three Bragg peaks (0.13, 0.23, and 0.25 Å(-1)) in SAXS spectra with a spacing ratio of 1:√3:√4 and characteristic changes in SANS spectra indicate formation of a hexagonal nanostructure. Also, the composition of the double-stranded oligonucleotides did not measurably impact the nanostructure of complexes formed with CTAB, suggesting that electrostatic

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

    DEFF Research Database (Denmark)

    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....

  8. OligoPrep PVA support for oligonucleotide synthesis in columns on a scale up to 10 micromol.

    Science.gov (United States)

    Aitken, Sheena; Anderson, Emma

    2007-01-01

    OligoPrep is a macroporous polyvinylacetate (PVA) biodegradable support that has been designed for cost-effective automated synthesis of oligonucleotides using standard phosphoramidite chemistry. Originally developed for large-scale oligonucleotide synthesis in beds and reactors, we present here its utility for medium-scale work of 1-10 micromol in column syntheses on standard DNA synthesizers. We show how an increase in scale, and, therefore, yield, can be achieved without significant increase in reagent quantity. Additional deblock and oxidation cycles can provide high coupling yields, and the use of concentrated ammonia in aqueous methylamine (AMA) for oligonucleotide cleavage and deprotection results in excellent recovery.

  9. Integrated avalanche photodiode arrays

    Science.gov (United States)

    Harmon, Eric S.

    2015-07-07

    The present disclosure includes devices for detecting photons, including avalanche photon detectors, arrays of such detectors, and circuits including such arrays. In some aspects, the detectors and arrays include a virtual beveled edge mesa structure surrounded by resistive material damaged by ion implantation and having side wall profiles that taper inwardly towards the top of the mesa structures, or towards the direction from which the ion implantation occurred. Other aspects are directed to masking and multiple implantation and/or annealing steps. Furthermore, methods for fabricating and using such devices, circuits and arrays are disclosed.

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

    Science.gov (United States)

    Straarup, Ellen Marie; Fisker, Niels; Hedtjärn, Maj; Lindholm, Marie W; Rosenbohm, Christoph; Aarup, Vibeke; Hansen, Henrik Frydenlund; Ørum, Henrik; Hansen, Jens B Rode; Koch, Troels

    2010-11-01

    The potency and specificity of locked nucleic acid (LNA) antisense oligonucleotides was investigated as a function of length and affinity. The oligonucleotides were designed to target apolipoprotein B (apoB) and were investigated both in vitro and in vivo. The high affinity of LNA enabled the design of short antisense oligonucleotides (12- to 13-mers) that possessed high affinity and increased potency both in vitro and in vivo compared to longer oligonucleotides. The short LNA oligonucleotides were more target specific, and they exhibited the same biodistribution and tissue half-life as longer oligonucleotides. Pharmacology studies in both mice and non-human primates were conducted with a 13-mer LNA oligonucleotide against apoB, and the data showed that repeated dosing of the 13-mer at 1-2 mg/kg/week was sufficient to provide a significant and long lasting lowering of non-high-density lipoprotein (non-HDL) cholesterol without increasing serum liver toxicity markers. The data presented here show that oligonucleotide length as a parameter needs to be considered in the design of antisense oligonucleotide and that potent short oligonucleotides with sufficient target affinity can be generated using the LNA chemistry. Conclusively, we present a 13-mer LNA oligonucleotide with therapeutic potential that produce beneficial cholesterol lowering effect in non-human primates.

  11. Interpretation of a discovery

    Directory of Open Access Journals (Sweden)

    Vučković Vladan

    2006-01-01

    Full Text Available The paper presents the development of the theory of asynchronous motors since Tesla’s discovery until the present day. The theory of steady state, as we know it today, was completed already during the first dozen of years. That was followed by a period of stagnation during a number of decades, when the theory of asynchronous motors was developed only in the framework of the general theory of electric machines, which was stimulated by the problems of the development of synchronous generators and big electric networks. It is only in our time that this simple motor, which was used for a long time just to perform crude tasks, became again the inspiration for the researchers and engineers who enabled it, with the help of power electronics and semi-conductor technology, to be used in the finest drives.

  12. Discovery with RSS retracted

    Science.gov (United States)

    1997-01-01

    The Rotating Service Structure has been retracted at Pad 39A. Discovery, the Space Shuttle for STS-82 Mission is ready for the launch of the second Hubble Space Telescope service mission. The payload consists of the Near Infrared Camera and Multi-Object Spectrometer (NICMOS) Which will be installed, the Fine Guidance Sensor #1 (FGS-1) and the Space Telescope Imaging Spectrograph (STIS) which will be installed. STS-82 will launch with a crew of seven at 3:54 a.m. February 11, 1997. The launch window is 65 minutes. The Mission Commander for STS-82 is Ken Bowersox. The purpose of the mission is to upgrade the scientific capabilities, service or replace aging components on the Telescope and provide a reboost to the optimum altitude.

  13. Focal plane array with modular pixel array components for scalability

    Energy Technology Data Exchange (ETDEWEB)

    Kay, Randolph R; Campbell, David V; Shinde, Subhash L; Rienstra, Jeffrey L; Serkland, Darwin K; Holmes, Michael L

    2014-12-09

    A modular, scalable focal plane array is provided as an array of integrated circuit dice, wherein each die includes a given amount of modular pixel array circuitry. The array of dice effectively multiplies the amount of modular pixel array circuitry to produce a larger pixel array without increasing die size. Desired pixel pitch across the enlarged pixel array is preserved by forming die stacks with each pixel array circuitry die stacked on a separate die that contains the corresponding signal processing circuitry. Techniques for die stack interconnections and die stack placement are implemented to ensure that the desired pixel pitch is preserved across the enlarged pixel array.

  14. Improving selective androgen receptor modulator discovery and preclinical evaluation.

    Science.gov (United States)

    Jones, Jeremy Orion

    2009-09-01

    Selective androgen receptor modulators (SARMs) represent a new class of pharmaceuticals that may find wide clinical use. However, selectivity is not understood at the molecular level, which has made the discovery and preclinical evaluation of SARMs difficult. We review the current state of SARM discovery and preclinical evaluation, as well as our current understanding of the molecular mechanisms controlling AR selectivity. We then discuss how increasing our molecular knowledge of AR selectivity will help create better discovery and evaluation methods and lead to a wider array of safer SARMs. The SARM field has advanced rapidly, but without a solid foundation of molecular knowledge to inform discovery and preclinical evaluation methods. The field has also taken a narrow view of selectivity, disregarding many androgen-responsive tissues, which could lead to unforeseen and detrimental side effects with chronic administration of SARMs. An investment in basic research could accelerate the discovery of a new generation of more selective and safer SARMs that could be used to treat an expanded range of clinical conditions.

  15. Analysis of VCSEL Array Module Using a Simple Microlens Array

    Institute of Scientific and Technical Information of China (English)

    Hen-Wai; Tsao; Shyh-Lin; Tsao

    2003-01-01

    A simple microlens array is designed between VCSEL array and fiber array for integration of array module. We increase the optical coupling efficiency from -32.057 dBm to -0.9054 dBm by using our designed microlens array.

  16. Analysis of VCSEL Array Module Using a Simple Microlens Array

    Institute of Scientific and Technical Information of China (English)

    Wen-Ming Cheng; Hen-Wai Tsao; Shyh-Lin Tsao

    2003-01-01

    A simple microlens array is designed between VCSEL array and fiber array for integration of array module. We increase the optical coupling efficiency from-32.057 dBm to-0.9054 dBm by using our designed microlens array.

  17. Discoveries of isotopes by fission

    Indian Academy of Sciences (India)

    M Thoennessen

    2015-09-01

    Of the about 3000 isotopes presently known, about 20% have been discovered in fission. The history of fission as it relates to the discovery of isotopes as well as the various reaction mechanisms leading to isotope discoveries involving fission are presented.

  18. Service discovery using Bloom filters

    NARCIS (Netherlands)

    Goering, P.T.H.; Heijenk, Gerhard J.; Lelieveldt, B.P.F.; Haverkort, Boudewijn R.H.M.; de Laat, C.T.A.M.; Heijnsdijk, J.W.J.

    A protocol to perform service discovery in adhoc networks is introduced in this paper. Attenuated Bloom filters are used to distribute services to nodes in the neighborhood and thus enable local service discovery. The protocol has been implemented in a discrete event simulator to investigate the

  19. Discovery Learning Strategies in English

    Science.gov (United States)

    Singaravelu, G.

    2012-01-01

    The study substantiates that the effectiveness of Discovery Learning method in learning English Grammar for the learners at standard V. Discovery Learning is particularly beneficial for any student learning a second language. It promotes peer interaction and development of the language and the learning of concepts with content. Reichert and…

  20. Solar array deployment mechanism

    Science.gov (United States)

    Calassa, Mark C.; Kackley, Russell

    1995-05-01

    This paper describes a Solar Array Deployment Mechanism (SADM) used to deploy a rigid solar array panel on a commercial spacecraft. The application required a deployment mechanism design that was not only lightweight, but also could be produced and installed at the lowest possible cost. This paper covers design, test, and analysis of a mechanism that meets these requirements.

  1. Array for detecting microbes

    Energy Technology Data Exchange (ETDEWEB)

    Andersen, Gary L.; DeSantis, Todd D.

    2014-07-08

    The present embodiments relate to an array system for detecting and identifying biomolecules and organisms. More specifically, the present embodiments relate to an array system comprising a microarray configured to simultaneously detect a plurality of organisms in a sample at a high confidence level.

  2. 29 CFR 2700.56 - Discovery; general.

    Science.gov (United States)

    2010-07-01

    ...(c) or 111 of the Act has been filed. 30 U.S.C. 815(c) and 821. (e) Completion of discovery... 29 Labor 9 2010-07-01 2010-07-01 false Discovery; general. 2700.56 Section 2700.56 Labor... Hearings § 2700.56 Discovery; general. (a) Discovery methods. Parties may obtain discovery by one or...

  3. Photovoltaic array loss mechanisms

    Science.gov (United States)

    Gonzalez, Charles

    1986-10-01

    Loss mechanisms which come into play when solar cell modules are mounted in arrays are identified. Losses can occur either from a reduction in the array electrical performance or with nonoptimal extraction of power from the array. Electrical performance degradation is caused by electrical mismatch, transmission losses from cell surface soiling and steep angle of reflectance, and electrical losses from field wiring resistance and the voltage drop across blocking diodes. The second type of loss, concerned with the operating points of the array, can involve nonoptimal load impedance and limiting the operating envelope of the array to specific ranges of voltage and current. Each of the loss mechanisms are discussed and average energy losses expected from soiling, steep reflectance angles and circuit losses are calculated.

  4. Microfabricated ion trap array

    Science.gov (United States)

    Blain, Matthew G.; Fleming, James G.

    2006-12-26

    A microfabricated ion trap array, comprising a plurality of ion traps having an inner radius of order one micron, can be fabricated using surface micromachining techniques and materials known to the integrated circuits manufacturing and microelectromechanical systems industries. Micromachining methods enable batch fabrication, reduced manufacturing costs, dimensional and positional precision, and monolithic integration of massive arrays of ion traps with microscale ion generation and detection devices. Massive arraying enables the microscale ion traps to retain the resolution, sensitivity, and mass range advantages necessary for high chemical selectivity. The reduced electrode voltage enables integration of the microfabricated ion trap array with on-chip circuit-based rf operation and detection electronics (i.e., cell phone electronics). Therefore, the full performance advantages of the microfabricated ion trap array can be realized in truly field portable, handheld microanalysis systems.

  5. Antisense Oligonucleotide Inhibition of Apolipoprotein C-III Reduces Plasma Triglycerides in Rodents, Nonhuman Primates, and Humans

    National Research Council Canada - National Science Library

    Graham, Mark J; Lee, Richard G; Bell, III, Thomas A; Fu, Wuxia; Mullick, Adam E; Alexander, Veronica J; Singleton, Walter; Viney, Nick; Geary, Richard; Su, John; Baker, Brenda F; Burkey, Jennifer; Crooke, Stanley T; Crooke, Rosanne M

    2013-01-01

    .... METHODS AND RESULTS:Rodent- and human-specific second-generation antisense oligonucleotides were identified and evaluated in preclinical models, including rats, mice, human apoC-III transgenic mice, and nonhuman...

  6. Gene expression visualisation with antisense oligonucleotides; Visualisation de l'expression d'un gene: la strategie antisens

    Energy Technology Data Exchange (ETDEWEB)

    Brard, P.Y.; Gauchez, A.S.; Vuillez, J.P. [Universite Joseph-Fourier, Grenoble I, INSERM E 03-40, Radiopharmaceutiques Biocliniques, Faculte de Medecine, 38 (France); Defrancq, E. [Universite Joseph-Fourier, Grenoble I, UMR CNRS 5616 - LEDSS, Faculte de Medecine, 38 (France)

    2004-08-01

    Using radiolabelled antisense oligonucleotides to target mRNAs is a very promising method to study gene expression in vivo. This molecular imaging technique has the aim to identify cellular modifications in a very early stage of disease. During the last ten years, the number of published studies concerning in vivo tumor specific imaging is small. This fact depends on numerous biological challenges. In fact, gene specific oligonucleotides must be chemically modified to increase nuclease resistance and permit labelling with radionuclide. To be used as imaging radiopharmaceutical agent, a good antisense oligonucleotide need to valid a lot of steps: in vivo stability, cell membrane passage and durable hybridization to mRNA to obtain a kinetic which depends directly on gene expression level. We can get over these difficulties, we will illustrate with our experience on chemo-resistance imaging with antisense oligonucleotides which target h-mdr 1, in vitro and in vivo. (author)

  7. Crystallization of a member of the recFOR DNA repair pathway, RecO, with and without bound oligonucleotide

    Energy Technology Data Exchange (ETDEWEB)

    Aono, Shelly; Hartsch, Thomas; Schulze-Gahmen, Ursula

    2003-01-22

    RecFOR proteins are important for DNA repair by homologous recombination in bacteria. The RecO protein from Thermus thermophilus was cloned, purified and characterized for its binding to oligonucleotides. The protein was crystallized alone and in complex with a 14-mer oligonucleotide. Both crystal forms grow under different crystallization conditions in the same space group, P3121 or P3221, with almost identical unit cell parameters. Complete data sets were collected to 2.8 Angstrom and 2.5 Angstrom for RecO alone and the RecO-oligonucleotide complex, respectively. Visual comparison of the diffraction patterns between the two crystal forms and calculation of an Rmerge of 33.9 percent on F indicate that one of the crystal forms is indeed a complex of RecO with bound oligonucleotide.

  8. Synthesis of high-quality libraries of long (150mer) oligonucleotides by a novel depurination controlled process

    National Research Council Canada - National Science Library

    LeProust, Emily M; Peck, Bill J; Spirin, Konstantin; McCuen, Heather Brummel; Moore, Bridget; Namsaraev, Eugeni; Caruthers, Marvin H

    2010-01-01

    ...) in fmol amounts using parallel synthesis of DNA on microarrays. The sequence accuracy of the oligonucleotides in such large-scale syntheses has been limited by the yields and side reactions of the DNA synthesis process used...

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

    Science.gov (United States)

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

    2014-07-01

    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 enhance antisense activity. The TCP1-β subunit co-localizes with PS-ASOs in distinct nuclear structures, termed phosphorothioate bodies or PS-bodies. Upon Ras-related nuclear protein (RAN) depletion, cytoplasmic PS-body-like structures were observed and nuclear concentrations of PS-ASOs were reduced, suggesting that TCP1-β can interact with PS-ASOs in the cytoplasm and that the nuclear import of PS-ASOs is at least partially through the RAN-mediated pathway. Upon free uptake, PS-ASOs co-localize with TCP1 proteins in cytoplasmic foci related to endosomes/lysosomes. Together, our results indicate that the TCP1 complex binds oligonucleotides with TCP1-β subunit being a nuclear PS-body component and suggest that the TCP1 complex may facilitate PS-ASO uptake and/or release from the endocytosis pathway. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  10. Co-Administration of an Excipient Oligonucleotide Helps Delineate Pathways of Productive and Nonproductive Uptake of Phosphorothioate Antisense Oligonucleotides in the Liver.

    Science.gov (United States)

    Donner, Aaron J; Wancewicz, Edward V; Murray, Heather M; Greenlee, Sarah; Post, Noah; Bell, Melanie; Lima, Walt F; Swayze, Eric E; Seth, Punit P

    2017-08-01

    Phosphorothioate (PS) modified antisense oligonucleotides (ASOs) have progressed rapidly in the clinic for treating a variety of disease indications. We previously demonstrated that the activity of PS ASOs in the liver can be enhanced by co-infusion of an excipient oligonucleotide (EON). It was posited that the EON saturates a nonproductive uptake pathway(s) thereby permitting accumulation of the PS ASO in a productive tissue compartment. In this report, we measured PS ASO activity following administration by bolus, infusion or co-fusion with EON within hepatocytes and nonparenchymal cells (NPCs), of the liver. This revealed that while ASOs accumulate preferentially in NPCs, they are intrinsically more active in hepatocytes. Furthermore, we show that the EON enhances ASO potency when infused up to 72 h before or after administration of the active ASO suggesting that the EON can saturate and displace the ASO from nonproductive to productive compartments. Physical presence of the EON in tissues was required for optimal potentiation suggesting that there is a dynamic distribution of the ASO and EON between the compartments. Lastly, using a candidate approach, we confirmed Stabilin-2 as a molecular pathway for ASO uptake in sinusoidal endothelial cells and the ASGR as a pathway for ASO uptake into hepatocytes in the liver.

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

    Science.gov (United States)

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

    2006-03-01

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

  12. Efficient Synthesis and Biological Evaluation of 5'-GalNAc Conjugated Antisense Oligonucleotides.

    Science.gov (United States)

    Østergaard, Michael E; Yu, Jinghua; Kinberger, Garth A; Wan, W Brad; Migawa, Michael T; Vasquez, Guillermo; Schmidt, Karsten; Gaus, Hans J; Murray, Heather M; Low, Audrey; Swayze, Eric E; Prakash, Thazha P; Seth, Punit P

    2015-08-19

    Conjugation of triantennary N-acetyl galactosamine (GalNAc) to oligonucleotide therapeutics results in marked improvement in potency for reducing gene targets expressed in hepatocytes. In this report we describe a robust and efficient solution-phase conjugation strategy to attach triantennary GalNAc clusters (mol. wt. ∼2000) activated as PFP (pentafluorophenyl) esters onto 5'-hexylamino modified antisense oligonucleotides (5'-HA ASOs, mol. wt. ∼8000 Da). The conjugation reaction is efficient and was used to prepare GalNAc conjugated ASOs from milligram to multigram scale. The solution phase method avoids loading of GalNAc clusters onto solid-support for automated synthesis and will facilitate evaluation of GalNAc clusters for structure activity relationship (SAR) studies. Furthermore, we show that transfer of the GalNAc cluster from the 3'-end of an ASO to the 5'-end results in improved potency in cells and animals.

  13. Photoswitchable oligonucleotide-modified gold nanoparticles: controlling hybridization stringency with photon dose.

    Science.gov (United States)

    Yan, Yunqi; Chen, Jennifer I L; Ginger, David S

    2012-05-09

    We describe a new class of stimulus-responsive DNA-functionalized gold nanoparticles that incorporate azobenzene-modified oligonucleotides. Beyond the classic directed assembly and sensing behaviors associated with oligonucleotide-modified nanoparticles, these particles also exhibit reversible photoswitching of their assembly behavior. Exposure to UV light induces a trans-cis isomerization of the azobenzene which destabilizes the DNA duplex, resulting in dissociation of the nanoparticle assemblies. The isomerization is reversible upon exposure to blue light, resulting in rehybridization and reassembly of the DNA-linked nanoparticle clusters. We show that perfectly complementary and partially mismatched strands exhibit clearly distinguishable photoinduced melting properties, and we demonstrate that photon dose can thus be used in place of temperature or ionic strength to control hybridization stringency with the ability to discriminate single-base mismatches.

  14. The role of antisense oligonucleotide therapy in patients with familial hypercholesterolemia: risks, benefits, and management recommendations.

    Science.gov (United States)

    Agarwala, Anandita; Jones, Peter; Nambi, Vijay

    2015-01-01

    Antisense oligonucleotide therapy is a promising approach for the treatment of a broad variety of medical conditions. It functions at the cellular level by interfering with RNA function, often leading to degradation of specifically targeted abnormal gene products implicated in the disease process. Mipomersen is a novel antisense oligonucleotide directed at apolipoprotein (apoB)-100, the primary apolipoprotein associated with low-density lipoprotein cholesterol (LDL-C), which has recently been approved for the treatment of familial hypercholesterolemia. A number of clinical studies have demonstrated its efficacy in lowering LDL-C and apoB levels in patients with elevated LDL-C despite maximal medical therapy using conventional lipid-lowering agents. This review outlines the risks and benefits of therapy and provides recommendations on the use of mipomersen.

  15. Computer selection of oligonucleotide probes from amino acid sequences for use in gene library screening.

    Science.gov (United States)

    Yang, J H; Ye, J H; Wallace, D C

    1984-01-11

    We present a computer program, FINPROBE, which utilizes known amino acid sequence data to deduce minimum redundancy oligonucleotide probes for use in screening cDNA or genomic libraries or in primer extension. The user enters the amino acid sequence of interest, the desired probe length, the number of probes sought, and the constraints on oligonucleotide synthesis. The computer generates a table of possible probes listed in increasing order of redundancy and provides the location of each probe in the protein and mRNA coding sequence. Activation of a next function provides the amino acid and mRNA sequences of each probe of interest as well as the complementary sequence and the minimum dissociation temperature of the probe. A final routine prints out the amino acid sequence of the protein in parallel with the mRNA sequence listing all possible codons for each amino acid.

  16. Antibacterial Activity of DNA-Stabilized Silver Nanoclusters Tuned by Oligonucleotide Sequence.

    Science.gov (United States)

    Javani, Siamak; Lorca, Romina; Latorre, Alfonso; Flors, Cristina; Cortajarena, Aitziber L; Somoza, Álvaro

    2016-04-27

    Silver nanoclusters (AgNCs) stabilized by DNA are promising materials with tunable fluorescent properties, which have been employed in a plethora of sensing systems. In this report, we explore their antimicrobial properties in Gram-positive and Gram-negative bacteria. After testing 9 oligonucleotides with different sequence and length, we found that the antibacterial activity depends on the sequence of the oligonucleotide employed. The sequences tested yielded fluorescent AgNCs, which can be grouped in blue, yellow, and red emitters. Interestingly, blue emitters yielded poor antibacterial activity, whereas yellow and red emitters afforded an activity similar to silver nitrate. Furthermore, structural studies using circular dichroism indicate the formation of complexes with different stability and structure, which might be one of the factors that modulate their activity. Finally, we prepared a trimeric structure containing the sequence that afforded the best antimicrobial activity, which inhibited the growth of Gram-positive and negative bacteria in the submicromolar range.

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

    Science.gov (United States)

    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.

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

    CERN Document Server

    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...

  19. The Role of Fluorinated Alcohols as Mobile Phase Modifiers for LC-MS Analysis of Oligonucleotides

    Science.gov (United States)

    Basiri, Babak; van Hattum, Hilde; van Dongen, William D.; Murph, Mandi M.; Bartlett, Michael G.

    2016-09-01

    Hexafluoroisopropanol (HFIP) has been widely used as an acidic modifier for mobile phases for liquid chromatography-mass spectrometry (LC-MS) analysis of oligonucleotides ever since the first report of its use for this purpose. This is not surprising, considering the exceptional performance of HFIP compared with carboxylic acids, which cause significant MS signal suppression in electrospray ionization. However, we have found that other fluorinated alcohols can also be utilized for mobile phase preparation and the choice of optimal fluorinated alcohol is determined by the ion-pairing (IP) agent. Although HFIP is a very good choice to be used alongside less hydrophobic IP agents, other fluorinated alcohols such as 1,1,1,3,3,3-hexafluoro-2-methyl-2-propanol (HFMIP) can significantly outperform HFIP when used with more hydrophobic IP agents. We also found that more acidic fluorinated alcohols assist with the transfer of oligonucleotides with secondary structure (e.g., folded strands and hairpins) into the gas phase.

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

    DEFF Research Database (Denmark)

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

    2015-01-01

    results by electronic structure calculations. Functionalized oligonucleotides were prepared in good yields by protein-mediated CuAAC click reactions for the first time with a human copper-binding chaperon. The carbohydrate, peptide, and fluorescent derivatives display high binding affinity and selectivity...... 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...