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Sample records for affymetrix oligonucleotide array

  1. Qualitative assessment of gene expression in affymetrix genechip arrays

    NASA Astrophysics Data System (ADS)

    Nagarajan, Radhakrishnan; Upreti, Meenakshi

    2007-01-01

    Affymetrix Genechip microarrays are used widely to determine the simultaneous expression of genes in a given biological paradigm. Probes on the Genechip array are atomic entities which by definition are randomly distributed across the array and in turn govern the gene expression. In the present study, we make several interesting observations. We show that there is considerable correlation between the probe intensities across the array which defy the independence assumption. While the mechanism behind such correlations is unclear, we show that scaling behavior and the profiles of perfect match (PM) as well as mismatch (MM) probes are similar and immune-to-background subtraction. We believe that the observed correlations are possibly an outcome of inherent non-stationarities or patchiness in the array devoid of biological significance. This is demonstrated by inspecting their scaling behavior and profiles of the PM and MM probe intensities obtained from publicly available Genechip arrays from three eukaryotic genomes, namely: Drosophila melanogaster (fruit fly), Homo sapiens (humans) and Mus musculus (house mouse) across distinct biological paradigms and across laboratories, with and without background subtraction. The fluctuation functions were estimated using detrended fluctuation analysis (DFA) with fourth-order polynomial detrending. The results presented in this study provide new insights into correlation signatures of PM and MM probe intensities and suggests the choice of DFA as a tool for qualitative assessment of Affymetrix Genechip microarrays prior to their analysis. A more detailed investigation is necessary in order to understand the source of these correlations.

  2. High correspondence between Affymetrix exon and standard expression arrays.

    PubMed

    Okoniewski, Michał J; Hey, Yvonne; Pepper, Stuart D; Miller, Crispin J

    2007-02-01

    Exon arrays aim to provide comprehensive gene expression data at the level of individual exons, similar to that provided on a per-gene basis by existing expression arrays. This report describes the performance of Affymetrix GeneChip Human Exon 1.0 ST array by using replicated RNA samples from two human cell lines, MCF7 and MCF10A, hybridized both to Exon 1.0 ST and to HG-U133 Plus2 arrays. Cross-comparison between array types requires an appropriate mapping to be found between individual probe sets. Three possible mappings were considered, reflecting different strategies for dealing with probe sets that target different parts of the same transcript. Irrespective of the mapping used, Exon 1.0 ST and HG-U133 Plus2 arrays show a high degree of correspondence. More than 80% of HG-U133 Plus2 probe sets may be mapped to the Exon chip, and fold changes are found well preserved for over 96% of those probe sets detected present. Since HG-U133 Plus2 arrays have already been extensively validated, these results lend a significant degree of confidence to exon arrays.

  3. Rawcopy: Improved copy number analysis with Affymetrix arrays

    PubMed Central

    Mayrhofer, Markus; Viklund, Björn; Isaksson, Anders

    2016-01-01

    Microarray data is subject to noise and systematic variation that negatively affects the resolution of copy number analysis. We describe Rawcopy, an R package for processing of Affymetrix CytoScan HD, CytoScan 750k and SNP 6.0 microarray raw intensities (CEL files). Noise characteristics of a large number of reference samples are used to estimate log ratio and B-allele frequency for total and allele-specific copy number analysis. Rawcopy achieves better signal-to-noise ratio and higher proportion of validated alterations than commonly used free and proprietary alternatives. In addition, Rawcopy visualizes each microarray sample for assessment of technical quality, patient identity and genome-wide absolute copy number states. Software and instructions are available at http://rawcopy.org. PMID:27796336

  4. Understanding the physics of oligonucleotide microarrays: the Affymetrix spike-in data reanalysed

    NASA Astrophysics Data System (ADS)

    Burden, Conrad J.

    2008-03-01

    The Affymetrix U95 and U133 Latin-Square spike-in datasets are reanalysed, together with a dataset from a version of the U95 spike-in experiment without a complex non-specific background. The approach uses a physico-chemical model which includes the effects of the specific and non-specific hybridization and probe folding at the microarray surface, target folding and hybridization in the bulk RNA target solution and duplex dissociation during the post-hybridization washing phase. The model predicts a three-parameter hyperbolic response function that fits well with fluorescence intensity data from all the three datasets. The importance of the various hybridization and washing effects in determining each of the three parameters is examined, and some guidance is given as to how a practical algorithm for determining specific target concentrations might be developed.

  5. An annotation infrastructure for the analysis and interpretation of Affymetrix exon array data.

    PubMed

    Okoniewski, Michał J; Yates, Tim; Dibben, Siân; Miller, Crispin J

    2007-01-01

    Affymetrix exon arrays contain probesets intended to target every known and predicted exon in the entire genome, posing significant challenges for high-throughput genome-wide data analysis. X:MAP http://xmap.picr.man.ac.uk, an annotation database, and exonmap http://www.bioconductor.org/packages/2.0/bioc/html/exonmap.html, a BioConductor/R package, are designed to support fine-grained analysis of exon array data. The system supports the application of standard statistical techniques, prior to the use of genome scale annotation to provide gene-, transcript- and exon-level summaries and visualization tools.

  6. An annotation infrastructure for the analysis and interpretation of Affymetrix exon array data

    PubMed Central

    Okoniewski, Michał J; Yates, Tim; Dibben, Siân; Miller, Crispin J

    2007-01-01

    Affymetrix exon arrays contain probesets intended to target every known and predicted exon in the entire genome, posing significant challenges for high-throughput genome-wide data analysis. X:MAP , an annotation database, and exonmap , a BioConductor/R package, are designed to support fine-grained analysis of exon array data. The system supports the application of standard statistical techniques, prior to the use of genome scale annotation to provide gene-, transcript- and exon-level summaries and visualization tools. PMID:17498294

  7. Affymetrix Whole-Transcript Human Gene 1.0 ST array is highly concordant with standard 3' expression arrays.

    PubMed

    Pradervand, Sylvain; Paillusson, Alexandra; Thomas, Jérôme; Weber, Johann; Wirapati, Pratyaksha; Hagenbüchle, Otto; Harshman, Keith

    2008-05-01

    The recently released Affymetrix Human Gene 1.0 ST array has two major differences compared with standard 3' based arrays: (i) it interrogates the entire mRNA transcript, and (ii) it uses DNA targets. To assess the impact of these differences on array performance, we performed a series of comparative hybridizations between the Human Gene 1.0 ST and the Affymetrix HG-U133 Plus 2.0 and the Illumina HumanRef-8 BeadChip arrays. Additionally, both RNA and DNA targets were hybridized on HG-U133 Plus 2.0 arrays. The results show that the overall reproducibility of the Gene 1.0 ST array is best. When looking only at the high intensity probes, the reproducibility of the Gene 1.0 ST array and the Illumina BeadChip array is equally good. Concordance of array results was assessed using different inter-platform mappings. Agreements are best between the two labeling protocols using HG-U133 Plus 2.0 array. The Gene 1.0 ST array is most concordant with the HG-U133 array hybridized with cDNA targets. This may reflect the impact of the target type. Overall, the high degree of correspondence provides strong evidence for the reliability of the Gene 1.0 ST array.

  8. Reproducible and inexpensive probe preparation for oligonucleotide arrays.

    PubMed

    Zhang, Y; Price, B D; Tetradis, S; Chakrabarti, S; Maulik, G; Makrigiorgos, G M

    2001-07-01

    We present a new protocol for the preparation of nucleic acids for microarray hybridization. DNA is fragmented quantitatively and reproducibly by using a hydroxyl radical-based reaction, which is initiated by hydrogen peroxide, iron(II)-EDTA and ascorbic acid. Following fragmentation, the nucleic acid fragments are densely biotinylated using a biotinylated psoralen analog plus UVA light and hybridized on microarrays. This non-enzymatic protocol circumvents several practical difficulties associated with DNA preparation for microarrays: the lack of reproducible fragmentation patterns associated with enzymatic methods; the large amount of labeled nucleic acids required by some array designs, which is often combined with a limited amount of starting material; and the high cost associated with currently used biotinylation methods. The method is applicable to any form of nucleic acid, but is particularly useful when applying double-stranded DNA on oligonucleotide arrays. Validation of this protocol is demonstrated by hybridizing PCR products with oligonucleotide-coated microspheres and PCR amplified cDNA with Affymetrix Cancer GeneChip microarrays.

  9. Improvements to previous algorithms to predict gene structure and isoform concentrations using Affymetrix Exon arrays

    PubMed Central

    2010-01-01

    Background Exon arrays provide a way to measure the expression of different isoforms of genes in an organism. Most of the procedures to deal with these arrays are focused on gene expression or on exon expression. Although the only biological analytes that can be properly assigned a concentration are transcripts, there are very few algorithms that focus on them. The reason is that previously developed summarization methods do not work well if applied to transcripts. In addition, gene structure prediction, i.e., the correspondence between probes and novel isoforms, is a field which is still unexplored. Results We have modified and adapted a previous algorithm to take advantage of the special characteristics of the Affymetrix exon arrays. The structure and concentration of transcripts -some of them possibly unknown- in microarray experiments were predicted using this algorithm. Simulations showed that the suggested modifications improved both specificity (SP) and sensitivity (ST) of the predictions. The algorithm was also applied to different real datasets showing its effectiveness and the concordance with PCR validated results. Conclusions The proposed algorithm shows a substantial improvement in the performance over the previous version. This improvement is mainly due to the exploitation of the redundancy of the Affymetrix exon arrays. An R-Package of SPACE with the updated algorithms have been developed and is freely available. PMID:21110835

  10. Exon array data analysis using Affymetrix power tools and R statistical software

    PubMed Central

    2011-01-01

    The use of microarray technology to measure gene expression on a genome-wide scale has been well established for more than a decade. Methods to process and analyse the vast quantity of expression data generated by a typical microarray experiment are similarly well-established. The Affymetrix Exon 1.0 ST array is a relatively new type of array, which has the capability to assess expression at the individual exon level. This allows a more comprehensive analysis of the transcriptome, and in particular enables the study of alternative splicing, a gene regulation mechanism important in both normal conditions and in diseases. Some aspects of exon array data analysis are shared with those for standard gene expression data but others present new challenges that have required development of novel tools. Here, I will introduce the exon array and present a detailed example tutorial for analysis of data generated using this platform. PMID:21498550

  11. Exon array data analysis using Affymetrix power tools and R statistical software.

    PubMed

    Lockstone, Helen E

    2011-11-01

    The use of microarray technology to measure gene expression on a genome-wide scale has been well established for more than a decade. Methods to process and analyse the vast quantity of expression data generated by a typical microarray experiment are similarly well-established. The Affymetrix Exon 1.0 ST array is a relatively new type of array, which has the capability to assess expression at the individual exon level. This allows a more comprehensive analysis of the transcriptome, and in particular enables the study of alternative splicing, a gene regulation mechanism important in both normal conditions and in diseases. Some aspects of exon array data analysis are shared with those for standard gene expression data but others present new challenges that have required development of novel tools. Here, I will introduce the exon array and present a detailed example tutorial for analysis of data generated using this platform.

  12. X:Map: annotation and visualization of genome structure for Affymetrix exon array analysis

    PubMed Central

    Yates, Tim; Okoniewski, Michał J.; Miller, Crispin J.

    2008-01-01

    Affymetrix exon arrays aim to target every known and predicted exon in the human, mouse or rat genomes, and have reporters that extend beyond protein coding regions to other areas of the transcribed genome. This combination of increased coverage and precision is important because a substantial proportion of protein coding genes are predicted to be alternatively spliced, and because many non-coding genes are known also to be of biological significance. In order to fully exploit these arrays, it is necessary to associate each reporter on the array with the features of the genome it is targeting, and to relate these to gene and genome structure. X:Map is a genome annotation database that provides this information. Data can be browsed using a novel Google-maps based interface, and analysed and further visualized through an associated BioConductor package. The database can be found at http://xmap.picr.man.ac.uk. PMID:17932061

  13. EzArray: A web-based highly automated Affymetrix expression array data management and analysis system

    PubMed Central

    Zhu, Yuerong; Zhu, Yuelin; Xu, Wei

    2008-01-01

    Background Though microarray experiments are very popular in life science research, managing and analyzing microarray data are still challenging tasks for many biologists. Most microarray programs require users to have sophisticated knowledge of mathematics, statistics and computer skills for usage. With accumulating microarray data deposited in public databases, easy-to-use programs to re-analyze previously published microarray data are in high demand. Results EzArray is a web-based Affymetrix expression array data management and analysis system for researchers who need to organize microarray data efficiently and get data analyzed instantly. EzArray organizes microarray data into projects that can be analyzed online with predefined or custom procedures. EzArray performs data preprocessing and detection of differentially expressed genes with statistical methods. All analysis procedures are optimized and highly automated so that even novice users with limited pre-knowledge of microarray data analysis can complete initial analysis quickly. Since all input files, analysis parameters, and executed scripts can be downloaded, EzArray provides maximum reproducibility for each analysis. In addition, EzArray integrates with Gene Expression Omnibus (GEO) and allows instantaneous re-analysis of published array data. Conclusion EzArray is a novel Affymetrix expression array data analysis and sharing system. EzArray provides easy-to-use tools for re-analyzing published microarray data and will help both novice and experienced users perform initial analysis of their microarray data from the location of data storage. We believe EzArray will be a useful system for facilities with microarray services and laboratories with multiple members involved in microarray data analysis. EzArray is freely available from . PMID:18218103

  14. Identifying the impact of G-quadruplexes on Affymetrix 3' arrays using cloud computing.

    PubMed

    Memon, Farhat N; Owen, Anne M; Sanchez-Graillet, Olivia; Upton, Graham J G; Harrison, Andrew P

    2010-01-15

    A tetramer quadruplex structure is formed by four parallel strands of DNA/ RNA containing runs of guanine. These quadruplexes are able to form because guanine can Hoogsteen hydrogen bond to other guanines, and a tetrad of guanines can form a stable arrangement. Recently we have discovered that probes on Affymetrix GeneChips that contain runs of guanine do not measure gene expression reliably. We associate this finding with the likelihood that quadruplexes are forming on the surface of GeneChips. In order to cope with the rapidly expanding size of GeneChip array datasets in the public domain, we are exploring the use of cloud computing to replicate our experiments on 3' arrays to look at the effect of the location of G-spots (runs of guanines). Cloud computing is a recently introduced high-performance solution that takes advantage of the computational infrastructure of large organisations such as Amazon and Google. We expect that cloud computing will become widely adopted because it enables bioinformaticians to avoid capital expenditure on expensive computing resources and to only pay a cloud computing provider for what is used. Moreover, as well as financial efficiency, cloud computing is an ecologically-friendly technology, it enables efficient data-sharing and we expect it to be faster for development purposes. Here we propose the advantageous use of cloud computing to perform a large data-mining analysis of public domain 3' arrays.

  15. SFP Genotyping from Affymetrix Arrays is Robust but Largely Detects Cis-acting Expression Regulators

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The recent development of Affymetrix chips designed from assembled EST sequences has spawned considerable interest in identifying single-feature polymorphisms (SFPs) from transcriptome data. SFPs are valuable genetic markers that potentially offer a physical link to the structural genes themselves....

  16. Discovery and mapping of single feature polymorphisms in wheat using affymetrix arrays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Single feature polymorphisms (SFPs) can be a rich source of markers for gene mapping and function studies. To explore the feasibility of using the Affymetrix GeneChip to discover and map SFPs in the large hexaploid wheat genome, six wheat varieties of diverse origins were analyzed for significant pr...

  17. Oligonucleotide-arrayed TFT photosensor applicable for DNA chip technology.

    PubMed

    Tanaka, Tsuyoshi; Hatakeyama, Keiichi; Sawaguchi, Masahiro; Iwadate, Akihito; Mizutani, Yasushi; Sasaki, Kazuhiro; Tateishi, Naofumi; Takeyama, Haruko; Matsunaga, Tadashi

    2006-09-05

    A thin film transistor (TFT) photosensor fabricated by semiconductor integrated circuit (IC) technology was applied to DNA chip technology. The surface of the TFT photosensor was coated with TiO2 using a vapor deposition technique for the fabrication of optical filters. The immobilization of thiolated oligonucleotide probes onto a TiO2-coated TFT photosensor using gamma-aminopropyltriethoxysilane (APTES) and N-(gamma-maleimidobutyloxy) sulfosuccinimide ester (GMBS) was optimized. The coverage value of immobilized oligonucleotides reached a plateau at 33.7 pmol/cm2, which was similar to a previous analysis using radioisotope-labeled oligonucleotides. The lowest detection limits were 0.05 pmol/cm2 for quantum dot and 2.1 pmol/cm2 for Alexa Fluor 350. Furthermore, single nucleotide polymorphism (SNP) detection was examined using the oligonucleotide-arrayed TFT photosensor. A SNP present in the aldehyde dehydrogenase 2 (ALDH2) gene was used as a target. The SNPs in ALDH2*1 and ALDH2*2 target DNA were detected successfully using the TFT photosensor. DNA hybridization in the presence of both ALDH2*1 and ALDH2*2 target DNA was observed using both ALDH2*1 and ALDH2*2 detection oligonucleotides-arrayed TFT photosensor. Use of the TFT photosensor will allow the development of a disposable photodetecting device for DNA chip systems.

  18. Evaluating the performance of Affymetrix SNP Array 6.0 platform with 400 Japanese individuals

    PubMed Central

    Nishida, Nao; Koike, Asako; Tajima, Atsushi; Ogasawara, Yuko; Ishibashi, Yoshimi; Uehara, Yasuka; Inoue, Ituro; Tokunaga, Katsushi

    2008-01-01

    Background With improvements in genotyping technologies, genome-wide association studies with hundreds of thousands of SNPs allow the identification of candidate genetic loci for multifactorial diseases in different populations. However, genotyping errors caused by genotyping platforms or genotype calling algorithms may lead to inflation of false associations between markers and phenotypes. In addition, the number of SNPs available for genome-wide association studies in the Japanese population has been investigated using only 45 samples in the HapMap project, which could lead to an inaccurate estimation of the number of SNPs with low minor allele frequencies. We genotyped 400 Japanese samples in order to estimate the number of SNPs available for genome-wide association studies in the Japanese population and to examine the performance of the current SNP Array 6.0 platform and the genotype calling algorithm "Birdseed". Results About 20% of the 909,622 SNP markers on the array were revealed to be monomorphic in the Japanese population. Consequently, 661,599 SNPs were available for genome-wide association studies in the Japanese population, after excluding the poorly behaving SNPs. The Birdseed algorithm accurately determined the genotype calls of each sample with a high overall call rate of over 99.5% and a high concordance rate of over 99.8% using more than 48 samples after removing low-quality samples by adjusting QC criteria. Conclusion Our results confirmed that the SNP Array 6.0 platform reached the level reported by the manufacturer, and thus genome-wide association studies using the SNP Array 6.0 platform have considerable potential to identify candidate susceptibility or resistance genetic factors for multifactorial diseases in the Japanese population, as well as in other populations. PMID:18803882

  19. Light-generated oligonucleotide arrays for rapid DNA sequence analysis.

    PubMed Central

    Pease, A C; Solas, D; Sullivan, E J; Cronin, M T; Holmes, C P; Fodor, S P

    1994-01-01

    In many areas of molecular biology there is a need to rapidly extract and analyze genetic information; however, current technologies for DNA sequence analysis are slow and labor intensive. We report here how modern photolithographic techniques can be used to facilitate sequence analysis by generating miniaturized arrays of densely packed oligonucleotide probes. These probe arrays, or DNA chips, can then be applied to parallel DNA hybridization analysis, directly yielding sequence information. In a preliminary experiment, a 1.28 x 1.28 cm array of 256 different octanucleotides was produced in 16 chemical reaction cycles, requiring 4 hr to complete. The hybridization pattern of fluorescently labeled oligonucleotide targets was then detected by epifluorescence microscopy. The fluorescence signals from complementary probes were 5-35 times stronger than those with single or double base-pair hybridization mismatches, demonstrating specificity in the identification of complementary sequences. This method should prove to be a powerful tool for rapid investigations in human genetics and diagnostics, pathogen detection, and DNA molecular recognition. Images PMID:8197176

  20. A Single-Array-Based Method for Detecting Copy Number Variants Using Affymetrix High Density SNP Arrays and its Application to Breast Cancer

    PubMed Central

    Li, Ming; Wen, Yalu; Fu, Wenjiang

    2014-01-01

    Cumulative evidence has shown that structural variations, due to insertions, deletions, and inversions of DNA, may contribute considerably to the development of complex human diseases, such as breast cancer. High-throughput genotyping technologies, such as Affymetrix high density single-nucleotide polymorphism (SNP) arrays, have produced large amounts of genetic data for genome-wide SNP genotype calling and copy number estimation. Meanwhile, there is a great need for accurate and efficient statistical methods to detect copy number variants. In this article, we introduce a hidden-Markov-model (HMM)-based method, referred to as the PICR-CNV, for copy number inference. The proposed method first estimates copy number abundance for each single SNP on a single array based on the raw fluorescence values, and then standardizes the estimated copy number abundance to achieve equal footing among multiple arrays. This method requires no between-array normalization, and thus, maintains data integrity and independence of samples among individual subjects. In addition to our efforts to apply new statistical technology to raw fluorescence values, the HMM has been applied to the standardized copy number abundance in order to reduce experimental noise. Through simulations, we show our refined method is able to infer copy number variants accurately. Application of the proposed method to a breast cancer dataset helps to identify genomic regions significantly associated with the disease. PMID:26279618

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

    SciTech Connect

    2000-06-15

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

  2. BEAT: Bioinformatics Exon Array Tool to store, analyze and visualize Affymetrix GeneChip Human Exon Array data from disease experiments

    PubMed Central

    2012-01-01

    Background It is known from recent studies that more than 90% of human multi-exon genes are subject to Alternative Splicing (AS), a key molecular mechanism in which multiple transcripts may be generated from a single gene. It is widely recognized that a breakdown in AS mechanisms plays an important role in cellular differentiation and pathologies. Polymerase Chain Reactions, microarrays and sequencing technologies have been applied to the study of transcript diversity arising from alternative expression. Last generation Affymetrix GeneChip Human Exon 1.0 ST Arrays offer a more detailed view of the gene expression profile providing information on the AS patterns. The exon array technology, with more than five million data points, can detect approximately one million exons, and it allows performing analyses at both gene and exon level. In this paper we describe BEAT, an integrated user-friendly bioinformatics framework to store, analyze and visualize exon arrays datasets. It combines a data warehouse approach with some rigorous statistical methods for assessing the AS of genes involved in diseases. Meta statistics are proposed as a novel approach to explore the analysis results. BEAT is available at http://beat.ba.itb.cnr.it. Results BEAT is a web tool which allows uploading and analyzing exon array datasets using standard statistical methods and an easy-to-use graphical web front-end. BEAT has been tested on a dataset with 173 samples and tuned using new datasets of exon array experiments from 28 colorectal cancer and 26 renal cell cancer samples produced at the Medical Genetics Unit of IRCCS Casa Sollievo della Sofferenza. To highlight all possible AS events, alternative names, accession Ids, Gene Ontology terms and biochemical pathways annotations are integrated with exon and gene level expression plots. The user can customize the results choosing custom thresholds for the statistical parameters and exploiting the available clinical data of the samples for a

  3. Gene Expression Analysis of Cultured Rat-Endothelial Cells after Nd:YAG Laser Irradiation by Affymetrix GeneChip Array

    PubMed Central

    MASUDA, YOSHIKO; YOKOSE, SATOSHI; SAKAGAMI, HIROSHI

    2017-01-01

    Endothelial cells and dental pulp cells enhance osteo-/odontogenic and angiogenic differentiation. In our previous study, rat pulp cells migrated to Nd:YAG laser-irradiated endothelial cells in an insert cell culture system. The purpose of this study was to examine the possible changes in the gene expression of cultured rat aortic endothelial cells after Nd:YAG laser irradiation using affymetrix GeneChip Array. Total RNA was extracted from the cells at 5 h after laser irradiation. Gene expressions were evaluated by DNA array chip. Up-regulated genes were related to cell migration and cell structure (membrane stretch, actin regulation and junctional complexes), neurotransmission and inflammation. Heat-shock 70 kDa protein (Hsp70) was related to the development of tooth germ. This study offers candidate genes for understanding the relationship between the laser-stimulated endothelial cells and dental pulp cells. PMID:28064220

  4. Application of oligonucleotide array technology for the rapid detection of pathogenic bacteria of foodborne infections.

    PubMed

    Hong, Bang-Xing; Jiang, Li-Fang; Hu, Yu-Shan; Fang, Dan-Yun; Guo, Hui-Yu

    2004-09-01

    A rapid and accurate method for detection for common pathogenic bacteria in foodborne infections was established by using oligonucleotide array technology. Nylon membrane was used as the array support. A mutation region of the 23S rRNA gene was selected as the discrimination target from 14 species (genera) of bacteria causing foodborne infections and two unrelated bacterial species. A pair of universal primers was designed for PCR amplification of the 23S rRNA gene. Twenty-one species (genera)-specific oligonucleotide detection probes were synthesized and spotted onto the nylon membranes. The 23S rRNA gene amplification products of 14 species of pathogenic bacteria were hybridized to the oligonucleotide array. Hybridization results were analyzed with digoxigenin-linked enzyme reaction. Results indicated that nine species of pathogenic bacteria (Escherichia coli, Campylobacter jejuni, Shigella dysenteriae, Vibrio cholerae, Vibrio parahaemolyticus, Proteus vulgaris, Bacillus cereus, Listeria monocytogenes and Clostridium botulinum) showed high sensitivity and specificity for the oligonucleotide array. Two other species (Salmonella enterica and Yersinia enterocolitica) gave weak cross-reaction with E. coli, but the reaction did not affect their detection. After redesigning the probes, positive hybridization results were obtained with Staphylococcus aureus, but not with Clostridium perfringens and Streptococcus pyogenes. The oligonucleotide array can also be applied to samples collected in clinical settings of foodborne infections. The superiority of oligonucleotide array over other tests lies on its rapidity, accuracy and efficiency in the diagnosis, treatment and control of foodborne infections.

  5. A sequence-based identification of the genes detected by probesets on the Affymetrix U133 plus 2.0 array.

    PubMed

    Harbig, Jeremy; Sprinkle, Robert; Enkemann, Steven A

    2005-02-18

    One of the biggest problems facing microarray experiments is the difficulty of translating results into other microarray formats or comparing microarray results to other biochemical methods. We believe that this is largely the result of poor gene identification. We re-identified the probesets on the Affymetrix U133 plus 2.0 GeneChip array. This identification was based on the sequence of the probes and the sequence of the human genome. Using the BLAST program, we matched probes with documented and postulated human transcripts. This resulted in the redefinition of approximately 37% of the probes on the U133 plus 2.0 array. This updated identification specifically points out where the identification is complicated by cross-hybridization from splice variants or closely related genes. More than 5000 probesets detect multiple transcripts and therefore the exact protein affected cannot be readily concluded from the performance of one probeset alone. This makes naming difficult and impacts any downstream analysis such as associating gene ontologies, mapping affected pathways or simply validating expression changes. We have now automated the sequence-based identification and can more appropriately annotate any array where the sequence on each spot is known.

  6. Arrays of complementary oligonucleotides for analysing the hybridisation behaviour of nucleic acids.

    PubMed Central

    Southern, E M; Case-Green, S C; Elder, J K; Johnson, M; Mir, K U; Wang, L; Williams, J C

    1994-01-01

    Arrays of oligonucleotides corresponding to a full set of complements of a known sequence can be made in a single series of base couplings in which each base in the complement is added in turn. Coupling is carried out on the surface of a solid support such as a glass plate, using a device which applies reagents in a defined area. The device is displaced by a fixed movement after each coupling reaction so that consecutive couplings overlap only a portion of previous ones. The shape and size of the device and the amount by which it is displaced at each step determines the length of the oligonucleotides. Certain shapes create arrays of oligonucleotides from mononucleotides up to a given length in a single series of couplings. The array is used in a hybridisation reaction to a labelled target sequence, and shows the hybridisation behaviour of every oligonucleotide in the target sequence with its complement in the array. Applications include sequence comparison to test for mutation, analysis of secondary structure, and optimisation of PCR primer and antisense oligonucleotide design. Images PMID:7514785

  7. Effects of stray light on the fidelity of photodirected oligonucleotide array synthesis.

    PubMed

    Garland, Peter B; Serafinowski, Pawel J

    2002-10-01

    Fabrication of high density oligonucleotide arrays using metal on glass photolithographic masks is inflexible and expensive. Maskless methods using computer-controlled projection have been proposed and implemented, but associated stray light effects on photodirected oligonucleotide synthesis and their analysis have not been reported. We have developed a theoretical approach: it predicts that the stray light content of the output of digital micromirror devices and other spatial light modulators of similar performance (contrast ratio approximately 400) will cause extensive random base insertions. For example, use of a digital micromirror device for synthesis of a 20mer array will result in the majority of oligonucleotide chains being 21mers or 22mers. This chain lengthening effect of stray light would not be preventable when synthesis involves a directly photosensitive 5'-blocking group. If the 5'-blocking group is acid labile and released with photogenerated acid, the presence of low concentrations of weak base will prevent the effect of stray light. We have demonstrated experimentally the anticipated chain lengthening effect of stray light on photoacid-dependent synthesis of oligonucleotides and prevention of the effect by low concentrations of n-octylamine. The application of these findings should facilitate the development of maskless fabrication and availability of high density and high fidelity user-designed arrays for research applications.

  8. Comparative transcriptomic profiling of Vitis vinifera under high light using a custom-made array and the Affymetrix GeneChip.

    PubMed

    Carvalho, Luísa C; Vilela, Belmiro J; Mullineaux, Phil M; Amâncio, Sara

    2011-11-01

    Understanding abiotic stress responses is one of the most important issues in plant research nowadays. Abiotic stress, including excess light, can promote the onset of oxidative stress through the accumulation of reactive oxygen species. Oxidative stress also arises when in vitro propagated plants are exposed to high light upon transfer to ex vitro. To determine whether the underlying pathways activated at the transfer of in vitro grapevine to ex vitro conditions reflect the processes occurring upon light stress, we used Vitis vinifera Affymetrix GeneChip (VvGA) and a custom array of genes responsive to light stress (LSCA) detected by real-time reverse transcriptase PCR (qRT-PCR). When gene-expression profiles were compared, 'protein metabolism and modification', 'signaling', and 'anti-oxidative' genes were more represented in LSCA, while, in VvGA, 'cell wall metabolism' and 'secondary metabolism' were the categories in which gene expression varied more significantly. The above functional categories confirm previous studies involving other types of abiotic stresses, enhancing the common attributes of abiotic stress defense pathways. The LSCA analysis of our experimental system detected strong response of heat shock genes, particularly the protein rescuing mechanism involving the cooperation of two ATP-dependent chaperone systems, Hsp100 and Hsp70, which showed an unusually late response during the recovery period, of extreme relevance to remove non-functional, potentially harmful polypeptides arising from misfolding, denaturation, or aggregation brought about by stress. The success of LSCA also proves the feasibility of a custom-made qRT-PCR approach, particularly for species for which no GeneChip is available and for researchers dealing with a specific and focused problem.

  9. A high density COX1 barcode oligonucleotide array for identification and detection of species of Penicillium subgenus Penicillium.

    PubMed

    Chen, W; Seifert, K A; Lévesque, C A

    2009-05-01

    We developed a COX1 barcode oligonucleotide array based on 358 sequences, including 58 known and two new species of Penicillium subgenus Penicillium, and 12 allied species. The array was robotically spotted at near microarray density on membranes. Species and clade-specific oligonucleotides were selected using the computer programs SigOli and Array Designer. Robotic spotting allowed 768 spots with duplicate sets of perfect match and the corresponding mismatch and positive control oligonucleotides, to be printed on 2 × 6 cm(2) nylon membranes. The array was validated with hybridizations between the array and digoxigenin (DIG)-labelled COX1 polymerase chain reaction amplicons from 70 pure DNA samples, and directly from environmental samples (cheese and plants) without culturing. DNA hybridization conditions were optimized, but undesired cross-reactions were detected frequently, reflecting the relatively high sequence similarity of the COX1 gene among Penicillium species. Approximately 60% of the perfect match oligonucleotides were rejected because of low specificity and 76 delivered useful group-specific or species-specific reactions and could be used for detecting certain species of Penicillium in environmental samples. In practice, the presence of weak signals on arrays exposed to amplicons from environmental samples, which could have represented weak detections or weak cross reactions, made interpretation difficult for over half of the oligonucleotides. DNA regions with very few single nucleotide polymorphisms or lacking insertions/deletions among closely related species are not ideal for oligonucleotide-based diagnostics, and supplementing the COX1-based array with oligonucleotides derived from additional genes would result in a more robust hierarchical identification system.

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

    PubMed

    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.

  11. Oligonucleotide Arrays vs. Metaphase-Comparative Genomic Hybridisation and BAC Arrays for Single-Cell Analysis: First Applications to Preimplantation Genetic Diagnosis for Robertsonian Translocation Carriers

    PubMed Central

    Ramos, Laia; del Rey, Javier; Daina, Gemma; García-Aragonés, Manel; Armengol, Lluís; Fernandez-Encinas, Alba; Parriego, Mònica; Boada, Montserrat; Martinez-Passarell, Olga; Martorell, Maria Rosa; Casagran, Oriol; Benet, Jordi; Navarro, Joaquima

    2014-01-01

    Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers. PMID:25415307

  12. Oligonucleotide arrays vs. metaphase-comparative genomic hybridisation and BAC arrays for single-cell analysis: first applications to preimplantation genetic diagnosis for Robertsonian translocation carriers.

    PubMed

    Ramos, Laia; del Rey, Javier; Daina, Gemma; García-Aragonés, Manel; Armengol, Lluís; Fernandez-Encinas, Alba; Parriego, Mònica; Boada, Montserrat; Martinez-Passarell, Olga; Martorell, Maria Rosa; Casagran, Oriol; Benet, Jordi; Navarro, Joaquima

    2014-01-01

    Comprehensive chromosome analysis techniques such as metaphase-Comparative Genomic Hybridisation (CGH) and array-CGH are available for single-cell analysis. However, while metaphase-CGH and BAC array-CGH have been widely used for Preimplantation Genetic Diagnosis, oligonucleotide array-CGH has not been used in an extensive way. A comparison between oligonucleotide array-CGH and metaphase-CGH has been performed analysing 15 single fibroblasts from aneuploid cell-lines and 18 single blastomeres from human cleavage-stage embryos. Afterwards, oligonucleotide array-CGH and BAC array-CGH were also compared analysing 16 single blastomeres from human cleavage-stage embryos. All three comprehensive analysis techniques provided broadly similar cytogenetic profiles; however, non-identical profiles appeared when extensive aneuploidies were present in a cell. Both array techniques provided an optimised analysis procedure and a higher resolution than metaphase-CGH. Moreover, oligonucleotide array-CGH was able to define extra segmental imbalances in 14.7% of the blastomeres and it better determined the specific unbalanced chromosome regions due to a higher resolution of the technique (≈ 20 kb). Applicability of oligonucleotide array-CGH for Preimplantation Genetic Diagnosis has been demonstrated in two cases of Robertsonian translocation carriers 45,XY,der(13;14)(q10;q10). Transfer of euploid embryos was performed in both cases and pregnancy was achieved by one of the couples. This is the first time that an oligonucleotide array-CGH approach has been successfully applied to Preimplantation Genetic Diagnosis for balanced chromosome rearrangement carriers.

  13. Identification of a submicroscopic 3.2 Mb chromosomal 16q12.2-13 deletion in a child with short stature, mild developmental delay, and craniofacial anomalies, by high-density oligonucleotide array-a recognizable syndrome.

    PubMed

    Chang, Ching-Fen; Li, Ling-Hui; Wang, Chung-Hsing; Tsai, Fuu-Jen; Chen, Tsai-Chuan; Wu, Jer-Yuarn; Chen, Yuan-Tsong; Tsai, Anne Chun-Hui

    2010-09-01

    Interstitial deletion of 16q has emerged into a recognizable pattern of congenital malformation. We report on a 9-year-old boy with short stature, psychomotor retardation, high forehead, broad flat nasal bridge, hypertelorism, cup-shaped ears, short neck, and a normal karyotype. Using high-density oligonucleotide array chip (Affymetrix 6.0) to perform parental and proband samples concurrently on three chips and interpreted as a trio set, a de novo 3.2 Mb deletion from bands q12.2 to q13 on chromosome 16 (from 52.08 to 55.3 Mb) of paternal origin was identified. The deletion was confirmed by quantitative genomic PCR and the break points were defined by junction PCR. Our study demonstrated the power of high-density oligonucleotide array chip in identifying novel submicroscopic deletions that were not detectable using G-banding cytogenetic technology. Furthermore, our result narrowed down the critical region for craniofacial features in interstitial 16q11.2-q13 deletion syndrome. In patients who have high forehead, broad flat nasal bridge, hypertelorism, cup-shaped ears, short neck and short stature, high-density array should be included in initial work up.

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

    PubMed

    Jung, Ki-Hong; Dardick, Christopher; Bartley, Laura E; Cao, Peijian; Phetsom, Jirapa; Canlas, Patrick; Seo, Young-Su; Shultz, Michael; Ouyang, Shu; Yuan, Qiaoping; Frank, Bryan C; Ly, Eugene; Zheng, Li; Jia, Yi; Hsia, An-Ping; An, Kyungsook; Chou, Hui-Hsien; Rocke, David; Lee, Geun Cheol; Schnable, Patrick S; An, Gynheung; Buell, C Robin; Ronald, Pamela C

    2008-10-06

    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. Malignant canine mammary tumours: Preliminary genomic insights using oligonucleotide array comparative genomic hybridisation analysis.

    PubMed

    Santos, Marta; Dias-Pereira, Patrícia; Williams, Christina; Lopes, Carlos; Breen, Matthew

    2017-03-28

    Neoplastic mammary disease in female dogs represents a major health concern for dog owners and veterinarians, but the genomic basis of the disease is poorly understood. In this study, we performed high resolution oligonucleotide array comparative genomic hybridisation (oaCGH) to assess genome wide DNA copy number changes in 10 malignant canine mammary tumours from seven female dogs, including multiple tumours collected at one time from each of three female dogs. In all but two tumours, genomic imbalances were detected, with losses being more common than gains. Canine chromosomes 9, 22, 26, 27, 34 and X were most frequently affected. Dissimilar oaCGH ratio profiles were observed in multiple tumours from the same dogs, providing preliminary evidence for probable independent pathogenesis. Analysis of adjacent samples of one tumour revealed regional differences in the number of genomic imbalances, suggesting heterogeneity within tumours.

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

    PubMed Central

    Jung, Ki-Hong; Dardick, Christopher; Bartley, Laura E.; Cao, Peijian; Phetsom, Jirapa; Canlas, Patrick; Seo, Young-Su; Shultz, Michael; Ouyang, Shu; Yuan, Qiaoping; Frank, Bryan C.; Ly, Eugene; Zheng, Li; Jia, Yi; Hsia, An-Ping; An, Kyungsook; Chou, Hui-Hsien; Rocke, David; Lee, Geun Cheol; Schnable, Patrick S.; An, Gynheung; Buell, C. Robin; Ronald, Pamela C.

    2008-01-01

    Studies of gene function are often hampered by gene-redundancy, especially in organisms with large genomes such as rice (Oryza sativa). We present an approach for using transcriptomics data to focus functional studies and address redundancy. To this end, we have constructed and validated an inexpensive and publicly available rice oligonucleotide near-whole genome array, called the rice NSF45K array. We generated expression profiles for light- vs. dark-grown rice leaf tissue and validated the 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. PMID:18836531

  17. Kinetics of Oligonucleotide Hybridization to DNA Probe Arrays on High-Capacity Porous Silica Substrates

    PubMed Central

    Glazer, Marc I.; Fidanza, Jacqueline A.; McGall, Glenn H.; Trulson, Mark O.; Forman, Jonathan E.; Frank, Curtis W.

    2007-01-01

    We have investigated the kinetics of DNA hybridization to oligonucleotide arrays on high-capacity porous silica films that were deposited by two techniques. Films created by spin coating pure colloidal silica suspensions onto a substrate had pores of ∼23 nm, relatively low porosity (35%), and a surface area of 17 times flat glass (for a 0.3-μm film). In the second method, latex particles were codeposited with the silica by spin coating and then pyrolyzed, which resulted in larger pores (36 nm), higher porosity (65%), and higher surface area (26 times flat glass for a 0.3-μm film). As a result of these favorable properties, the templated silica hybridized more quickly and reached a higher adsorbed target density (11 vs. 8 times flat glass at 22°C) than the pure silica. Adsorption of DNA onto the high-capacity films is controlled by traditional adsorption and desorption coefficients, as well as by morphology factors and transient binding interactions between the target and the probes. To describe these effects, we have developed a model based on the analogy to diffusion of a reactant in a porous catalyst. Adsorption values (ka, kd, and K) measured on planar arrays for the same probe/target system provide the parameters for the model and also provide an internally consistent comparison for the stability of the transient complexes. The interpretation of the model takes into account factors not previously considered for hybridization in three-dimensional films, including the potential effects of heterogeneous probe populations, partial probe/target complexes during diffusion, and non-1:1 binding structures. The transient complexes are much less stable than full duplexes (binding constants for full duplexes higher by three orders of magnitude or more), which may be a result of the unique probe density and distribution that is characteristic of the photolithographically patterned arrays. The behavior at 22°C is described well by the predictive equations for

  18. VIZARD: analysis of Affymetrix Arabidopsis GeneChip data

    NASA Technical Reports Server (NTRS)

    Moseyko, Nick; Feldman, Lewis J.

    2002-01-01

    SUMMARY: The Affymetrix GeneChip Arabidopsis genome array has proved to be a very powerful tool for the analysis of gene expression in Arabidopsis thaliana, the most commonly studied plant model organism. VIZARD is a Java program created at the University of California, Berkeley, to facilitate analysis of Arabidopsis GeneChip data. It includes several integrated tools for filtering, sorting, clustering and visualization of gene expression data as well as tools for the discovery of regulatory motifs in upstream sequences. VIZARD also includes annotation and upstream sequence databases for the majority of genes represented on the Affymetrix Arabidopsis GeneChip array. AVAILABILITY: VIZARD is available free of charge for educational, research, and not-for-profit purposes, and can be downloaded at http://www.anm.f2s.com/research/vizard/ CONTACT: moseyko@uclink4.berkeley.edu.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Many Phytophthora species are destructive plant pathogens imposing severe threats to both natural and agricultural vegetation. Effective monitoring and accurate early detection are important means of preventing potential epidemics and outbreaks of such diseases. DNA array hybridization technique is ...

  20. Real-time detection of DNA hybridization and melting on oligonucleotide arrays by using optical wave guides.

    PubMed Central

    Stimpson, D I; Hoijer, J V; Hsieh, W T; Jou, C; Gordon, J; Theriault, T; Gamble, R; Baldeschwieler, J D

    1995-01-01

    The challenge of the Human Genome Project is to increase the rate of DNA sequence acquisition by two orders of magnitude to complete sequencing of the human genome by the year 2000. The present work describes a rapid detection method using a two-dimensional optical wave guide that allows measurement of real-time binding or melting of a light-scattering label on a DNA array. A particulate label on the target DNA acts as a light-scattering source when illuminated by the evanescent wave of the wave guide and only the label bound to the surface generates a signal. Imaging/visual examination of the scattered light permits interrogation of the entire array simultaneously. Hybridization specificity is equivalent to that obtained with a conventional system using autoradiography. Wave guide melting curves are consistent with those obtained in the liquid phase and single-base discrimination is facile. Dilution experiments showed an apparent lower limit of detection at 0.4 nM oligonucleotide. This performance is comparable to the best currently known fluorescence-based systems. In addition, wave guide detection allows manipulation of hybridization stringency during detection and thereby reduces DNA chip complexity. It is anticipated that this methodology will provide a powerful tool for diagnostic applications that require rapid cost-effective detection of variations from known sequences. Images Fig. 1 Fig. 2 Fig. 3 PMID:7603999

  1. Development and evaluation of 50-mer oligonucleotide arrays for detecting microbial populations in Acid Mine Drainages and bioleaching systems.

    PubMed

    Yin, Huaqun; Cao, Linhui; Qiu, Guanzhou; Wang, Dianzuo; Kellogg, Laurie; Zhou, Jizhong; Dai, Zhimin; Liu, Xueduan

    2007-07-01

    To effectively monitor microbial populations in acidic environments and bioleaching systems, a comprehensive 50-mer-based oligonucleotide microarray was developed based on most of the known genes associated with the acidophiles. This array contained 1,072 probes in which there were 571 related to 16S rRNA and 501 related to functional genes. The functional genes in the microarray were involved in carbon metabolism (158), nitrogen metabolism (72), sulfur metabolism (39), iron metabolism (68), DNA replication and repair (97), metal-resistance (27), membrane-relate gene (16), transposon (13) and IST sequence (11). Based on the results of microarray hybridizations, specificity tests with representative pure cultures indicated that the designed probes on the arrays appeared to be specific to their corresponding target genes. The detection limit was 5 ng of genomic DNA in the absence of background DNA. Strong linear relationships between the signal intensity and the target DNA were observed (r(2) approximately 0.98). Application of this type of the microarray to analyze the acidic environments and bioleaching systems demonstrated that the developed microarray appeared to be useful for profiling differences in microbial community structures of acidic environments and bioleaching systems. Our results indicate that this technology has potential as a specific, sensitive, and quantitative tool in revealing a comprehensive picture of the compositions of genes related with acidophilic microorganism and the microbial community in acidic environments and bioleaching systems, although more work is needed to improve.

  2. Regions of homozygosity identified by oligonucleotide SNP arrays: evaluating the incidence and clinical utility.

    PubMed

    Wang, Jia-Chi; Ross, Leslie; Mahon, Loretta W; Owen, Renius; Hemmat, Morteza; Wang, Boris T; El Naggar, Mohammed; Kopita, Kimberly A; Randolph, Linda M; Chase, John M; Matas Aguilera, Maria J; Siles, Juan López; Church, Joseph A; Hauser, Natalie; Shen, Joseph J; Jones, Marilyn C; Wierenga, Klaas J; Jiang, Zhijie; Haddadin, Mary; Boyar, Fatih Z; Anguiano, Arturo; Strom, Charles M; Sahoo, Trilochan

    2015-05-01

    Copy neutral segments with allelic homozygosity, also known as regions of homozygosity (ROHs), are frequently identified in cases interrogated by oligonucleotide single-nucleotide polymorphism (oligo-SNP) microarrays. Presence of ROHs may be because of parental relatedness, chromosomal recombination or rearrangements and provides important clues regarding ancestral homozygosity, consanguinity or uniparental disomy. In this study of 14 574 consecutive cases, 832 (6%) were found to harbor one or more ROHs over 10 Mb, of which 651 cases (78%) had multiple ROHs, likely because of identity by descent (IBD), and 181 cases (22%) with ROHs involving a single chromosome. Parental relatedness was predicted to be first degree or closer in 5%, second in 9% and third in 19%. Of the 181 cases, 19 had ROHs for a whole chromosome revealing uniparental isodisomy (isoUPD). In all, 25 cases had significant ROHs involving a single chromosome; 5 cases were molecularly confirmed to have a mixed iso- and heteroUPD15 and 1 case each with segmental UPD9pat and segmental UPD22mat; 17 cases were suspected to have a mixed iso- and heteroUPD including 2 cases with small supernumerary marker and 2 cases with mosaic trisomy. For chromosome 15, 12 (92%) of 13 molecularly studied cases had either Prader-Willi or Angelman syndrome. Autosomal recessive disorders were confirmed in seven of nine cases from eight families because of the finding of suspected gene within a ROH. This study demonstrates that ROHs are much more frequent than previously recognized and often reflect parental relatedness, ascertain autosomal recessive diseases or unravel UPD in many cases.

  3. Comparing three methods for variance estimation with duplicated high density oligonucleotide arrays.

    PubMed

    Huang, Xiaohong; Pan, Wei

    2002-08-01

    Microarray experiments are being increasingly used in molecular biology. A common task is to detect genes with differential expression across two experimental conditions, such as two different tissues or the same tissue at two time points of biological development. To take proper account of statistical variability, some statistical approaches based on the t-statistic have been proposed. In constructing the t-statistic, one needs to estimate the variance of gene expression levels. With a small number of replicated array experiments, the variance estimation can be challenging. For instance, although the sample variance is unbiased, it may have large variability, leading to a large mean squared error. For duplicated array experiments, a new approach based on simple averaging has recently been proposed in the literature. Here we consider two more general approaches based on nonparametric smoothing. Our goal is to assess the performance of each method empirically. The three methods are applied to a colon cancer data set containing 2,000 genes. Using two arrays, we compare the variance estimates obtained from the three methods. We also consider their impact on the t-statistics. Our results indicate that the three methods give variance estimates close to each other. Due to its simplicity and generality, we recommend the use of the smoothed sample variance for data with a small number of replicates.

  4. Unique Combination of 22q11 and 14qter Microdeletion Syndromes Detected Using Oligonucleotide Array-CGH

    PubMed Central

    Zrnová, E.; Vranová, V.; Šoukalová, J.; Slámová, I.; Vilémová, M.; Gaillyová, R.; Kuglík, P.

    2012-01-01

    We report an infant with a unique combination of 22q11 deletion syndrome and 14q terminal deletion syndrome. The proband had clinical symptoms compatible with diagnosis of 22q11 deletion syndrome: microcephaly, micrognathia, high-arched palate, hypertelorism, short palpebral fissures, square nasal root, prominent tubular nose, hypoplastic nasal alae, bulbous nasal tip, dysplastic low-set ears, short philtrum, and heart defect, but no cell-mediated immunodeficiency typical for the syndrome. G-banding and fluorescence in situ hybridization analyses revealed a karyotype 45,XY,der(14)t(14;22)(q32.3;q11.2),-22.ish del(14)(q32.33)(D14S1420-),del(22)(q11.2q11.2)(N25-). Subsequent analyses disclosed a translocation between chromosomes 14 and 22 in the proband's mother with a deleted 14q telomere. Using comparative genome hybridization on oligonucleotide-based microarray (array-CGH), the deletion at 22q11.21 in the size of ∼4.25 Mb was revealed in the proband as well as the deletion of the telomeric area at 14q32.33qter (∼3.24 Mb) in the proband and his mother. However, both the proband and his mother showed mild symptoms (microcephaly, thin lips, carp-shaped mouth) typical for patients with the described terminal 14q deletion syndrome. PMID:22511897

  5. Genome profiling of chondrosarcoma using oligonucleotide array-based comparative genomic hybridization.

    PubMed

    Hameed, Meera; Ulger, Celal; Yasar, Duygu; Limaye, Neha; Kurvathi, Rohini; Streck, Deanna; Benevenia, Joseph; Patterson, Francis; Dermody, James J; Toruner, Gokce A

    2009-07-15

    Chondrosarcomas of the bone are malignant hyaline cartilage-forming tumors with an annual incidence rate of 3.6% of all primary bone malignancies in the United States. Specimens of 25 chondrosarcomas (10 grade I, 9 grade II, 1 grade III, and 5 dedifferentiated) from 23 patients were collected from the Department of Pathology at the University Hospital at UMDNJ-New Jersey Medical School from 1996 to 2007. Array-based comparative genomic hybridization (array-CGH) studies were performed on frozen tumor specimens. Recurrent deletions observed in at least in six tumors were 5q13.2, 5q14.2 approximately q21.3, 6q12 approximately q13, 6q16 approximately q25.3, 9p24.2 approximately q12, and 9p21.3. There was a statistically significant association between high-grade tumor (grade III and dedifferentiated) and the recurrent genetic deletions at 5q14.2 approximately q21.3, 6q16 approximately q25.3, 9p24.2 approximately q12, and 9p21.3. There is consistency between increased levels of aneuploidy and the progression of chondrosarcoma from lower to higher grades.

  6. Gene expression levels in small specimens from patients detected using oligonucleotide arrays.

    PubMed

    Hoffmann, Katrin; Firth, Martin J; Freitas, Joseph R; de Klerk, Nicholas H; Kees, Ursula R

    2005-01-01

    Large-scale gene expression profiling using microarray technology is often limited by the amount of tissue or cells available. A number of RNA amplification protocols have been published to overcome this problem. However, additional amplification steps can result in both a 3' bias and poor reproducibility for low abundance transcripts. We performed microarray experiments using HG-U133A GeneChip arrays to ascertain whether less than the recommended amount of RNA can be used, thus avoiding additional amplification steps. In a titration experiment, 2-10 microg of total RNA from a single cryopreserved patient specimen was used to prepare biotinylated cRNA, and the recommended standard amount of 15 microg of each preparation was used for hybridization. Statistical analysis using box plots, correlation coefficients, MvA plots, and concordance percentages revealed almost identical levels of gene expression, independent of the amount of RNA used for target preparation. Most importantly, there was no statistically significant difference when the concordance percentages for low abundance genes were compared, demonstrating that as little as 2 microg of total RNA is sufficient to perform GeneChip analysis.

  7. Development of a Novel Oligonucleotide Array for Genomic Screening of Chemical Warfare Agent Exposure in Guinea Pigs and Swine

    DTIC Science & Technology

    2004-04-01

    respectively. To address this gap in research tools, we have developed an oligonucleotide microarray that contains representative genes from guinea pig...To address this gap in research tools, we have developed an oligonucleotide microarray that contains representative genes from guinea pig...toxicogenomics. Toxicogenomics is focused on identifying changes in gene expression in response to a toxic chemical and then using that information to

  8. ASSESSMENT OF THE SWINE PROTEIN-ANNOTATED OLIGONUCLEOTIDE MICROARRAY AND UTILITY OF THE ARRAYS FOR EQTL AND TRANSCRIPTIONAL PROFILING STUDIES

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have evaluated the new Swine Protein-Annotated Oligonucleotide Microarray (http://www.pigoligoarray.org) by analyzing transcriptional profiles for longissimus dorsi muscle (LD), Bronchial lymph node (BLN) and Lung. Four LD samples were used to assess the stringency of hybridization conditions com...

  9. Micro-Analyzer: automatic preprocessing of Affymetrix microarray data.

    PubMed

    Guzzi, Pietro Hiram; Cannataro, Mario

    2013-08-01

    A current trend in genomics is the investigation of the cell mechanism using different technologies, in order to explain the relationship among genes, molecular processes and diseases. For instance, the combined use of gene-expression arrays and genomic arrays has been demonstrated as an effective instrument in clinical practice. Consequently, in a single experiment different kind of microarrays may be used, resulting in the production of different types of binary data (images and textual raw data). The analysis of microarray data requires an initial preprocessing phase, that makes raw data suitable for use on existing analysis platforms, such as the TIGR M4 (TM4) Suite. An additional challenge to be faced by emerging data analysis platforms is the ability to treat in a combined way those different microarray formats coupled with clinical data. In fact, resulting integrated data may include both numerical and symbolic data (e.g. gene expression and SNPs regarding molecular data), as well as temporal data (e.g. the response to a drug, time to progression and survival rate), regarding clinical data. Raw data preprocessing is a crucial step in analysis but is often performed in a manual and error prone way using different software tools. Thus novel, platform independent, and possibly open source tools enabling the semi-automatic preprocessing and annotation of different microarray data are needed. The paper presents Micro-Analyzer (Microarray Analyzer), a cross-platform tool for the automatic normalization, summarization and annotation of Affymetrix gene expression and SNP binary data. It represents the evolution of the μ-CS tool, extending the preprocessing to SNP arrays that were not allowed in μ-CS. The Micro-Analyzer is provided as a Java standalone tool and enables users to read, preprocess and analyse binary microarray data (gene expression and SNPs) by invoking TM4 platform. It avoids: (i) the manual invocation of external tools (e.g. the Affymetrix Power

  10. Oligonucleotide-directed self-assembly of proteins: semisynthetic DNA--streptavidin hybrid molecules as connectors for the generation of macroscopic arrays and the construction of supramolecular bioconjugates.

    PubMed Central

    Niemeyer, C M; Sano, T; Smith, C L; Cantor, C R

    1994-01-01

    Modified biomolecules were used for the non-covalent assembly of novel bioconjugates. Hybrid molecules were synthesized from short single-stranded DNA and streptavidin by chemical methods using a heterobispecific crosslinker. The covalent attachment of an oligonucleotide moiety to streptavidin provides a specific recognition domain for a complementary nucleic acid sequence, in addition to the four native biotin-binding sites. These bispecific binding capabilities allow the hybrid molecules to serve as versatile connectors in a variety of applications. Bifunctional constructs have been prepared from two complementary hybrid molecules, each previously conjugated to biotinylated immunoglobulin G or alkaline phosphatase. The use of nucleic acid sequences as a template for the formation of an array of proteins is further demonstrated on two size scales. A macroscopic DNA array on a microtiter plate has been transformed into a comparable protein chip. A nano-scale array was made by hybridizing DNA-tagged proteins to specific positions along a RNA or DNA sequence. The generation of supramolecular bioconjugates was shown by quantitative measurements and gel-retardation assays. Images PMID:7530841

  11. Guidelines for Optimisation of a Multiplex Oligonucleotide Ligation-PCR for Characterisation of Microbial Pathogens in a Microsphere Suspension Array

    PubMed Central

    Wuyts, Véronique; Roosens, Nancy H. C.; Marchal, Kathleen; De Keersmaecker, Sigrid C. J.

    2015-01-01

    With multiplex oligonucleotide ligation-PCR (MOL-PCR) different molecular markers can be simultaneously analysed in a single assay and high levels of multiplexing can be achieved in high-throughput format. As such, MOL-PCR is a convenient solution for microbial detection and identification assays where many markers should be analysed, including for routine further characterisation of an identified microbial pathogenic isolate. For an assay aimed at routine use, optimisation in terms of differentiation between positive and negative results and of cost and effort is indispensable. As MOL-PCR includes a multiplex ligation step, followed by a singleplex PCR and analysis with microspheres on a Luminex device, several parameters are accessible for optimisation. Although MOL-PCR performance may be influenced by the markers used in the assay and the targeted bacterial species, evaluation of the method of DNA isolation, the probe concentration, the amount of microspheres, and the concentration of reporter dye is advisable in the development of any MOL-PCR assay. Therefore, we here describe our observations made during the optimisation of a 20-plex MOL-PCR assay for subtyping of Salmonella Typhimurium with the aim to provide a possible workflow as guidance for the development and optimisation of a MOL-PCR assay for the characterisation of other microbial pathogens. PMID:25705689

  12. Celsius: a community resource for Affymetrix microarray data.

    PubMed

    Day, Allen; Carlson, Marc R J; Dong, Jun; O'Connor, Brian D; Nelson, Stanley F

    2007-01-01

    Celsius is a data warehousing system to aggregate Affymetrix CEL files and associated metadata. It provides mechanisms for importing, storing, querying, and exporting large volumes of primary and pre-processed microarray data. Celsius contains ten billion assay measurements and affiliated metadata. It is the largest publicly available source of Affymetrix microarray data, and through sheer volume it allows a sophisticated, broad view of transcription that has not previously been possible.

  13. Pigeons: A Novel GUI Software for Analysing and Parsing High Density Heterologous Oligonucleotide Microarray Probe Level Data

    PubMed Central

    Lai, Hung-Ming; May, Sean T.; Mayes, Sean

    2014-01-01

    Genomic DNA-based probe selection by using high density oligonucleotide arrays has recently been applied to heterologous species (Xspecies). With the advent of this new approach, researchers are able to study the genome and transcriptome of a non-model or an underutilised crop species through current state-of-the-art microarray platforms. However, a software package with a graphical user interface (GUI) to analyse and parse the oligonucleotide probe pair level data is still lacking when an experiment is designed on the basis of this cross species approach. A novel computer program called Pigeons has been developed for customised array data analysis to allow the user to import and analyse Affymetrix GeneChip® probe level data through XSpecies. One can determine empirical boundaries for removing poor probes based on genomic hybridisation of the test species to the Xspecies array, followed by making a species-specific Chip Description File (CDF) file for transcriptomics in the heterologous species, or Pigeons can be used to examine an experimental design to identify potential Single-Feature Polymorphisms (SFPs) at the DNA or RNA level. Pigeons is also focused around visualization and interactive analysis of the datasets. The software with its manual (the current release number version 1.2.1) is freely available at the website of the Nottingham Arabidopsis Stock Centre (NASC). PMID:27605027

  14. Pentopyranosyl Oligonucleotide Systems

    NASA Technical Reports Server (NTRS)

    Wagner, Thomas; Huyuh, Hoan K.; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert

    2002-01-01

    Beta-D-Xylopyranosyl-(4 - 2 )-oligonucleotides containing adenine and thymine as nucleohases were synthesized as a part of a systematic study of the pairing properties of pentopyranosyl oligonucleotides. Contrary to earlier expectations based on qualitative conformational criteria, Beta-D-xylopyranosyl(4 - 2 )- oligonucleotides show Watson-Crick pairing comparable in strength to that shown by pyranosyl-RNA.

  15. Axiom turkey genotyping array

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  16. Comparison of gene coverage of mouse oligonucleotide microarray platforms

    PubMed Central

    Verdugo, Ricardo A; Medrano, Juan F

    2006-01-01

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

  17. 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.

    PubMed

    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.

  18. Template-Directed Ligation of Peptides to Oligonucleotides

    NASA Technical Reports Server (NTRS)

    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.

  19. Identification of differentially regulated transcripts in mouse striatum following methamphetamine treatment--an oligonucleotide microarray approach.

    PubMed

    Thomas, David M; Francescutti-Verbeem, Dina M; Liu, Xiuli; Kuhn, Donald M

    2004-01-01

    Methamphetamine is an addictive drug of abuse that can produce neurotoxic effects in dopamine nerve endings of the striatum. The purpose of this study was to identify new genes that may play a role in the highly complex cascade of events associated with methamphetamine intoxication. Using Affymetrix oligonucleotide arrays, 12 488 genes were simultaneously interrogated and there were 152 whose expression levels were changed following methamphetamine treatment. The genes are grouped into broad functional categories with inflammatory/immune response elements, receptor/signal transduction components and ion channel/transport proteins among the most populated. Many genes within these categories can be linked to ion regulation and apoptosis, both of which have been implicated in methamphetamine toxicity, and numerous factors associated with microglial activation emerged with significant changes in expression. For example, brain-derived neurotrophic factor (BDNF), chemokine (C-C) receptor 6 (CCr6) and numerous chemokine transcripts were increased or decreased in expression more than 2.8-fold. These results point to activated microglia as a potential source of the reactive oxygen/nitrogen species and cytokines that have been previously associated with methamphetamine toxicity and other neurotoxic conditions.

  20. A novel 4p16.3 microduplication distal to WHSC1 and WHSC2 characterized by oligonucleotide array with new phenotypic features.

    PubMed

    Cyr, Andrew B; Nimmakayalu, Manjunath; Longmuir, Susannah Q; Patil, Shivanand R; Keppler-Noreuil, Kim M; Shchelochkov, Oleg A

    2011-09-01

    Larger imbalances on chromosome 4p in the form of deletions associated with Wolf-Hirschhorn syndrome (WHS) and duplications of chromosome 4p have a defined clinical phenotype. The critical region for both these clinical disorders has been narrowed based on the genotype-phenotype correlations. However, cryptic rearrangements in this region have been reported infrequently. We report on a male patient with a microduplication of chromosome 4p, who presents with findings of macrocephaly, irregular iris pigmentation-heterochromia, and preserved linear growth in addition to overlapping features of trisomy 4p such as seizures, delayed psychomotor development, and dysmorphic features including prominent glabella, low-set ears, and short neck. Using a high-density oligonucleotide microarray, we have identified a novel submicroscopic duplication involving dosage sensitive genes TACC3, FGFR3, and LETM1. The microduplication did not involve WHSC1 and WHSC2 which are considered in the critical region for WHS and trisomy 4p. This patient's presentation and genomic findings help further delineate clinical significance of re-arrangements in the 4p16 region without the involvement of WHS critical region.

  1. Mining Affymetrix microarray data for long non-coding RNAs: altered expression in the nucleus accumbens of heroin abusers.

    PubMed

    Michelhaugh, Sharon K; Lipovich, Leonard; Blythe, Jason; Jia, Hui; Kapatos, Gregory; Bannon, Michael J

    2011-02-01

    Although recent data suggest that some long non-coding RNAs (lncRNAs) exert widespread effects on gene expression and organelle formation, lncRNAs as a group constitute a sizable but poorly characterized fraction of the human transcriptome. We investigated whether some human lncRNA sequences were fortuitously represented on commonly used microarrays, then used this annotation to assess lncRNA expression in human brain. A computational and annotation pipeline was developed to identify lncRNA transcripts represented on Affymetrix U133 arrays. A previously published dataset derived from human nucleus accumbens was then examined for potential lncRNA expression. Twenty-three lncRNAs were determined to be represented on U133 arrays. Of these, dataset analysis revealed that five lncRNAs were consistently detected in samples of human nucleus accumbens. Strikingly, the abundance of these lncRNAs was up-regulated in human heroin abusers compared to matched drug-free control subjects, a finding confirmed by quantitative PCR. This study presents a paradigm for examining existing Affymetrix datasets for the detection and potential regulation of lncRNA expression, including changes associated with human disease. The finding that all detected lncRNAs were up-regulated in heroin abusers is consonant with the proposed role of lncRNAs as mediators of widespread changes in gene expression as occur in drug abuse.

  2. CEL_INTERROGATOR: A FREE AND OPEN SOURCE PACKAGE FOR AFFYMETRIX CEL FILE PARSING

    Technology Transfer Automated Retrieval System (TEKTRAN)

    CEL_Interrogator Package is a suite of programs designed to extract the average probe intensity and other information for each probe sequence from an Affymetrix GeneChip CEL file and unite them with their human-readable Affymetrix consensus sequence names. The resulting text file is suitable for di...

  3. Cross reactive arrays of three-way junction sensors for steroid determination

    NASA Technical Reports Server (NTRS)

    Stojanovic, Milan N. (Inventor); Landry, Donald (Inventor); Nikic, Dragan B. (Inventor)

    2008-01-01

    This invention provides analyte sensitive oligonucleotide compositions for detecting and analyzing analytes in solution, including complex solutions using cross reactive arrays of analyte sensitive oligonucleotide compositions.

  4. MA-SNP--A new genotype calling method for oligonucleotide SNP arrays modeling the batch effect with a normal mixture model.

    PubMed

    Wen, Yalu; Li, Ming; Fu, Wenjiang J

    2011-08-30

    Genome-wide association studies hold great promise in identifying disease-susceptibility variants and understanding the genetic etiology of complex diseases. Microarray technology enables the genotyping of millions of single nucleotide polymorphisms. Many factors in microarray studies, such as probe selection, sample quality, and experimental process and batch, have substantial effect on the genotype calling accuracy, which is crucial for downstream analyses. Failure to account for the variability of these sources may lead to inaccurate genotype calls and false positive and false negative findings. In this study, we develop a SNP-specific genotype calling algorithm based on the probe intensity composite representation (PICR) model, while using a normal mixture model to account for the variability of batch effect on the genotype calls. We demonstrate our method with SNP array data in a few studies, including the HapMap project, the coronary heart disease and the UK Blood Service Control studies by the Wellcome Trust Case-Control Consortium, and a methylation profiling study. Our single array based approach outperforms PICR and is comparable to the best multi-array genotype calling methods.

  5. Identification of a deep intronic mutation in the COL6A2 gene by a novel custom oligonucleotide CGH array designed to explore allelic and genetic heterogeneity in collagen VI-related myopathies

    PubMed Central

    2010-01-01

    Background Molecular characterization of collagen-VI related myopathies currently relies on standard sequencing, which yields a detection rate approximating 75-79% in Ullrich congenital muscular dystrophy (UCMD) and 60-65% in Bethlem myopathy (BM) patients as PCR-based techniques tend to miss gross genomic rearrangements as well as copy number variations (CNVs) in both the coding sequence and intronic regions. Methods We have designed a custom oligonucleotide CGH array in order to investigate the presence of CNVs in the coding and non-coding regions of COL6A1, A2, A3, A5 and A6 genes and a group of genes functionally related to collagen VI. A cohort of 12 patients with UCMD/BM negative at sequencing analysis and 2 subjects carrying a single COL6 mutation whose clinical phenotype was not explicable by inheritance were selected and the occurrence of allelic and genetic heterogeneity explored. Results A deletion within intron 1A of the COL6A2 gene, occurring in compound heterozygosity with a small deletion in exon 28, previously detected by routine sequencing, was identified in a BM patient. RNA studies showed monoallelic transcription of the COL6A2 gene, thus elucidating the functional effect of the intronic deletion. No pathogenic mutations were identified in the remaining analyzed patients, either within COL6A genes, or in genes functionally related to collagen VI. Conclusions Our custom CGH array may represent a useful complementary diagnostic tool, especially in recessive forms of the disease, when only one mutant allele is detected by standard sequencing. The intronic deletion we identified represents the first example of a pure intronic mutation in COL6A genes. PMID:20302629

  6. Mutational analysis using oligonucleotide microarrays

    PubMed Central

    Hacia, J.; Collins, F.

    1999-01-01

    The development of inexpensive high throughput methods to identify individual DNA sequence differences is important to the future growth of medical genetics. This has become increasingly apparent as epidemiologists, pathologists, and clinical geneticists focus more attention on the molecular basis of complex multifactorial diseases. Such undertakings will rely upon genetic maps based upon newly discovered, common, single nucleotide polymorphisms. Furthermore, candidate gene approaches used in identifying disease associated genes necessitate screening large sequence blocks for changes tracking with the disease state. Even after such genes are isolated, large scale mutational analyses will often be needed for risk assessment studies to define the likely medical consequences of carrying a mutated gene.
This review concentrates on the use of oligonucleotide arrays for hybridisation based comparative sequence analysis. Technological advances within the past decade have made it possible to apply this technology to many different aspects of medical genetics. These applications range from the detection and scoring of single nucleotide polymorphisms to mutational analysis of large genes. Although we discuss published scientific reports, unpublished work from the private sector12 could also significantly affect the future of this technology.


Keywords: mutational analysis; oligonucleotide microarrays; DNA chips PMID:10528850

  7. Pentopyranosyl Oligonucleotide Systems

    NASA Technical Reports Server (NTRS)

    Reck, Folkert; Kudick, Rene; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert; Wippo, Harald

    2001-01-01

    To determine whether the remarkable chemical properties of the pyranosyl isomer of RNA as an informational Watson-Crick base-pairing system are unique to the pentopyranosyl-(4 + 2)-oligonucleotide isomer derived from the RNA-building block D-ribose, studies on the entire family of diastereoisomeric pyranosyL(4 - Z)-oligonucleotide systems deriving from D-ribose. L-lyxose. D-xylose, and L-arabinose were carried out. The result of these extended studies is unambiguous: not only pyranosyl-RNA, but all members of the pentopyranosyl(4 + 2)-oligonucleotide family are highly efficient Watson-Crick base-pairing systems. Their synthesis and pairing properties will be described in a series of publications in this journal.

  8. The delivery of therapeutic oligonucleotides

    PubMed Central

    Juliano, Rudolph L.

    2016-01-01

    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. PMID:27084936

  9. Statistical evaluation of transcriptomic data generated using the Affymetrix one-cycle, two-cycle and IVT-Express RNA labelling protocols with the Arabidopsis ATH1 microarray

    PubMed Central

    2010-01-01

    Background Microarrays are a powerful tool used for the determination of global RNA expression. There is an increasing requirement to focus on profiling gene expression in tissues where it is difficult to obtain large quantities of material, for example individual tissues within organs such as the root, or individual isolated cells. From such samples, it is difficult to produce the amount of RNA required for labelling and hybridisation in microarray experiments, thus a process of amplification is usually adopted. Despite the increasing use of two-cycle amplification for transcriptomic analyses on the Affymetrix ATH1 array, there has been no report investigating any potential bias in gene representation that may occur as a result. Results Here we compare transcriptomic data generated using Affymetrix one-cycle (standard labelling protocol), two-cycle (small-sample protocol) and IVT-Express protocols with the Affymetrix ATH1 array using Arabidopsis root samples. Results obtained with each protocol are broadly similar. However, we show that there are 35 probe sets (of a total of 22810) that are misrepresented in the two-cycle data sets. Of these, 33 probe sets were classed as mis-amplified when comparisons of two independent publicly available data sets were undertaken. Conclusions Given the unreliable nature of the highlighted probes, we caution against using data associated with the corresponding genes in analyses involving transcriptomic data generated with two-cycle amplification protocols. We have shown that the Affymetrix IVT-E labelling protocol produces data with less associated bias than the two-cycle protocol, and as such, would recommend this kit for new experiments that involve small samples. PMID:20230623

  10. MicroRNA expression analysis using the Affymetrix Platform.

    PubMed

    Dee, Suzanne; Getts, Robert C

    2012-01-01

    Microarrays have been used extensively for messenger RNA expression monitoring. Recently, microarrays have been designed to interrogate expression levels of noncoding RNAs. Here, we describe methods for RNA labeling and the use of a miRNA array to identify and measure microRNA present in RNA samples.

  11. Methods comparison for high-resolution transcriptional analysis of archival material on Affymetrix Plus 2.0 and Exon 1.0 microarrays.

    PubMed

    Linton, Kim; Hey, Yvonne; Dibben, Sian; Miller, Crispin; Freemont, Anthony; Radford, John; Pepper, Stuart

    2009-07-01

    Microarray gene expression profiling of formalin-fixed paraffin-embedded (FFPE) tissues is a new and evolving technique. This report compares transcript detection rates on Affymetrix U133 Plus 2.0 and Human Exon 1.0 ST GeneChips across several RNA extraction and target labeling protocols, using routinely collected archival FFPE samples. All RNA extraction protocols tested (Ambion-Optimum, Ambion-RecoverAll, and Qiagen-RNeasy FFPE) provided extracts suitable for microarray hybridization. Compared with Affymetrix One-Cycle labeled extracts, NuGEN system protocols utilizing oligo(dT) and random hexamer primers, and cDNA target preparations instead of cRNA, achieved percent present rates up to 55% on Plus 2.0 arrays. Based on two paired-sample analyses, at 90% specificity this equalled an average 30 percentage-point increase (from 50% to 80%) in FFPE transcript sensitivity relative to fresh frozen tissues, which we have assumed to have 100% sensitivity and specificity. The high content of Exon arrays, with multiple probe sets per exon, improved FFPE sensitivity to 92% at 96% specificity, corresponding to an absolute increase of ~600 genes over Plus 2.0 arrays. While larger series are needed to confirm high correspondence between fresh-frozen and FFPE expression patterns, these data suggest that both Plus 2.0 and Exon arrays are suitable platforms for FFPE microarray expression analyses.

  12. A note on oligonucleotide expression values not being normally distributed.

    PubMed

    Hardin, Johanna; Wilson, Jason

    2009-07-01

    Novel techniques for analyzing microarray data are constantly being developed. Though many of the methods contribute to biological discoveries, inability to properly evaluate the novel techniques limits their ability to advance science. Because the underlying distribution of microarray data is unknown, novel methods are typically tested against the assumed normal distribution. However, microarray data are not, in fact, normally distributed, and assuming so can have misleading consequences. Using an Affymetrix technical replicate spike-in data set, we show that oligonucleotide expression values are not normally distributed for any of the standard methods for calculating expression values. The resulting data tend to have a large proportion of skew and heavy tailed genes. Additionally, we show that standard methods can give unexpected and misleading results when the data are not well approximated by the normal distribution. Robust methods are therefore recommended when analyzing microarray data. Additionally, new techniques should be evaluated with skewed and/or heavy-tailed data distributions.

  13. Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours

    PubMed Central

    Linton, K M; Hey, Y; Saunders, E; Jeziorska, M; Denton, J; Wilson, C L; Swindell, R; Dibben, S; Miller, C J; Pepper, S D; Radford, J A; Freemont, A J

    2008-01-01

    Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low (∼50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)–PCR was confirmed, especially for abundant transcripts, and RT–PCR validated the regulation pattern for 19 of 24 candidate genes (overall R2=0.4662). RT–PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET – whose combined expression carried greater prognostic value than tumour grade – and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach. PMID:18382428

  14. Acquisition of biologically relevant gene expression data by Affymetrix microarray analysis of archival formalin-fixed paraffin-embedded tumours.

    PubMed

    Linton, K M; Hey, Y; Saunders, E; Jeziorska, M; Denton, J; Wilson, C L; Swindell, R; Dibben, S; Miller, C J; Pepper, S D; Radford, J A; Freemont, A J

    2008-04-22

    Robust protocols for microarray gene expression profiling of archival formalin-fixed paraffin-embedded tissue (FFPET) are needed to facilitate research when availability of fresh-frozen tissue is limited. Recent reports attest to the feasibility of this approach, but the clinical value of these data is poorly understood. We employed state-of-the-art RNA extraction and Affymetrix microarray technology to examine 34 archival FFPET primary extremity soft tissue sarcomas. Nineteen arrays met stringent QC criteria and were used to model prognostic signatures for metastatic recurrence. Arrays from two paired frozen and FFPET samples were compared: although FFPET sensitivity was low ( approximately 50%), high specificity (95%) and positive predictive value (92%) suggest that transcript detection is reliable. Good agreement between arrays and real time (RT)-PCR was confirmed, especially for abundant transcripts, and RT-PCR validated the regulation pattern for 19 of 24 candidate genes (overall R(2)=0.4662). RT-PCR and immunohistochemistry on independent cases validated prognostic significance for several genes including RECQL4, FRRS1, CFH and MET - whose combined expression carried greater prognostic value than tumour grade - and cmet and TRKB proteins. These molecules warrant further evaluation in larger series. Reliable clinically relevant data can be obtained from archival FFPET, but protocol amendments are needed to improve the sensitivity and broad application of this approach.

  15. Electrophoresis for genotyping: temporal thermal gradient gel electrophoresis for profiling of oligonucleotide dissociation.

    PubMed Central

    Day, I N; O'Dell, S D; Cash, I D; Humphries, S E; Weavind, G P

    1995-01-01

    Traditional use of an oligonucleotide probe to determine genotype depends on perfect base pairing to a single-stranded target which is stable to a higher temperature than when imperfect binding occurs due to a mismatch in the target sequence. Bound oligonucleotide is detected at a predetermined single temperature 'snapshot' of the melting profile, allowing the distinction of perfect from imperfect base pairing. In heterozygotes, the presence of the alternative sequence must be verified with a second oligonucleotide complementary to the variant. Here we describe a system of real-time variable temperature electrophoresis during which the oligonucleotide dissociates from its target. In 20% polyacrylamide the target strand has minimal mobility and released oligonucleotide migrates extremely quickly so that the 'freed' rather than the 'bound' is displayed. The full profile of oligonucleotide dissociation during gel electrophoresis is represented along the gel track, and a single oligonucleotide is sufficient to confirm heterozygosity, since the profile displays two separate peaks. Resolution is great, with use of short track lengths enabling analysis of dense arrays of samples. Each gel track can contain a different target or oligonucleotide and the temperature gradient can accommodate oligonucleotides of different melting temperatures. This provides a convenient system to examine the interaction of many different oligonucleotides and target sequences simultaneously and requires no prior knowledge of the mutant sequence(s) nor of oligonucleotide melting temperatures. The application of the technique is described for screening of a hotspot for mutations in the LDL receptor gene in patients with familial hypercholesterolaemia. Images PMID:7630718

  16. Current progress on aptamer-targeted oligonucleotide therapeutics

    PubMed Central

    Dassie, Justin P; Giangrande, Paloma H

    2014-01-01

    Exploiting the power of the RNAi pathway through the use of therapeutic siRNA drugs has remarkable potential for treating a vast array of human disease conditions. However, difficulties in delivery of these and similar nucleic acid-based pharmacological agents to appropriate organs or tissues, remains a major impediment to their broad clinical application. Synthetic nucleic acid ligands (aptamers) have emerged as effective delivery vehicles for therapeutic oligonucleotides, including siRNAs. In this review, we summarize recent attractive developments in creatively employing cell-internalizing aptamers to deliver therapeutic oligonucleotides (e.g., siRNAs, miRNAs, anti-miRs and antisense oligos) to target cells. We also discuss advancements in aptamer-siRNA chimera technology, as well as, aptamer-functionalized nanoparticles for siRNA delivery. In addition, the challenges and future prospects of aptamer-targeted oligonucleotide drugs for clinical translation are further highlighted. PMID:24304250

  17. POSaM: a fast, flexible, open-source, inkjet oligonucleotide synthesizer and microarrayer

    PubMed Central

    Lausted, Christopher; Dahl, Timothy; Warren, Charles; King, Kimberly; Smith, Kimberly; Johnson, Michael; Saleem, Ramsey; Aitchison, John; Hood, Lee; Lasky, Stephen R

    2004-01-01

    DNA arrays are valuable tools in molecular biology laboratories. Their rapid acceptance was aided by the release of plans for a pin-spotting microarrayer by researchers at Stanford. Inkjet microarraying is a flexible, complementary technique that allows the synthesis of arrays of any oligonucleotide sequences de novo. We describe here an open-source inkjet arrayer capable of rapidly producing sets of unique 9,800-feature arrays. PMID:15287980

  18. Oligonucleotide conjugates for therapeutic applications

    PubMed Central

    Winkler, Johannes

    2013-01-01

    Insufficient pharmacokinetic properties and poor cellular uptake are the main hurdles for successful therapeutic development of oligonucleotide agents. The covalent attachment of various ligands designed to influence the biodistribution and cellular uptake or for targeting specific tissues is an attractive possibility to advance therapeutic applications and to expand development options. In contrast to advanced formulations, which often consist of multiple reagents and are sensitive to a variety of preparation conditions, oligonucleotide conjugates are defined molecules, enabling structure-based analytics and quality control techniques. This review gives an overview of current developments of oligonucleotide conjugates for therapeutic applications. Attached ligands comprise peptides, proteins, carbohydrates, aptamers and small molecules, including cholesterol, tocopherol and folic acid. Important linkage types and conjugation methods are summarized. The distinct ligands directly influence biochemical parameters, uptake machanisms and pharmacokinetic properties. PMID:23883124

  19. Oligonucleotide conjugates for therapeutic applications.

    PubMed

    Winkler, Johannes

    2013-07-01

    Insufficient pharmacokinetic properties and poor cellular uptake are the main hurdles for successful therapeutic development of oligonucleotide agents. The covalent attachment of various ligands designed to influence the biodistribution and cellular uptake or for targeting specific tissues is an attractive possibility to advance therapeutic applications and to expand development options. In contrast to advanced formulations, which often consist of multiple reagents and are sensitive to a variety of preparation conditions, oligonucleotide conjugates are defined molecules, enabling structure-based analytics and quality control techniques. This review gives an overview of current developments of oligonucleotide conjugates for therapeutic applications. Attached ligands comprise peptides, proteins, carbohydrates, aptamers and small molecules, including cholesterol, tocopherol and folic acid. Important linkage types and conjugation methods are summarized. The distinct ligands directly influence biochemical parameters, uptake mechanisms and pharmacokinetic properties.

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

    PubMed Central

    Payton, Paxton; Kottapalli, Kameswara Rao; Rowland, Diane; Faircloth, Wilson; Guo, Baozhu; Burow, Mark; Puppala, Naveen; Gallo, Maria

    2009-01-01

    Background Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently has a significant number of ESTs been released into the public domain. Utilization of these ESTs for oligonucleotide microarrays provides a means to investigate large-scale transcript responses to a variety of developmental and environmental signals, ultimately improving our understanding of plant biology. Results We have developed a high-density oligonucleotide microarray for peanut using 49,205 publicly available ESTs and tested the utility of this array for expression profiling in a variety of peanut tissues. To identify putatively tissue-specific genes and demonstrate the utility of this array for expression profiling in a variety of peanut tissues, we compared transcript levels in pod, peg, leaf, stem, and root tissues. Results from this experiment showed 108 putatively pod-specific/abundant genes, as well as transcripts whose expression was low or undetected in pod compared to peg, leaf, stem, or root. The transcripts significantly over-represented in pod include genes responsible for seed storage proteins and desiccation (e.g., late-embryogenesis abundant proteins, aquaporins, legumin B), oil production, and cellular defense. Additionally, almost half of the pod-abundant genes represent unknown genes allowing for the possibility of associating putative function to these previously uncharacterized genes. Conclusion The peanut oligonucleotide array represents the majority of publicly available peanut ESTs and can be used as a tool for expression profiling studies in diverse tissues. PMID:19523230

  1. A Quantitative Tool for Producing DNA-Based Diagnostic Arrays

    SciTech Connect

    Tom J. Whitaker

    2008-07-11

    The purpose of this project was to develop a precise, quantitative method to analyze oligodeoxynucleotides (ODNs) on an array to enable a systematic approach to quality control issues affecting DNA microarrays. Two types of ODN's were tested; ODN's formed by photolithography and ODN's printed onto microarrays. Initial work in Phase I, performed in conjunction with Affymetrix, Inc. who has a patent on a photolithographic in situ technique for creating DNA arrays, was very promising but did seem to indicate that the atomization process was not complete. Soon after Phase II work was under way, Affymetrix had further developed fluorescent methods and indicated they were no longer interested in our resonance ionization technique. This was communicated to the program manager and it was decided that the project would continue and be focused on printed ODNs. The method being tested is called SIRIS, Sputter-Initiated Resonance Ionization Spectroscopy. SIRIS has been shown to be a highly sensitive, selective, and quantitative tool for atomic species. This project was aimed at determining if an ODN could be labeled in such a way that SIRIS could be used to measure the label and thus provide quantitative measurements of the ODN on an array. One of the largest problems in this study has been developing a method that allows us to know the amount of an ODN on a surface independent of the SIRIS measurement. Even though we could accurately determine the amount of ODN deposited on a surface, the amount that actually attached to the surface is very difficult to measure (hence the need for a quantitative tool). A double-labeling procedure was developed in which 33P and Pt were both used to label ODNs. The radioactive 33P could be measured by a proportional counter that maps the counts in one dimension. This gave a good measurement of the amount of ODN remaining on a surface after immobilization and washing. A second label, Pt, was attached to guanine nucleotides in the ODN. Studies

  2. Thermodynamics of Oligonucleotide Duplex Melting

    NASA Astrophysics Data System (ADS)

    Schreiber-Gosche, Sherrie; Edwards, Robert A.

    2009-05-01

    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 rigorous thermodynamic analysis to an important biochemical problem. Because the stacking of base pairs on top of one another is a significant factor in the energetics of oligonucleotide melting, several investigators have applied van't Hoff analysis to melting temperature data using a nearest-neighbor model and have obtained entropies and enthalpies for the stacking of bases. The present article explains how the equilibrium constant for the dissociation of strands from double-stranded oligonucleotides can be expressed in terms of the total strand concentration and thus how the total strand concentration influences the melting temperature. It also presents a simplified analysis based on the entropies and enthalpies of stacking that is manually tractable so that students can work examples to help them understand the thermodynamics of oligonucleotide melting.

  3. Thermodynamics of Oligonucleotide Duplex Melting

    ERIC Educational Resources Information Center

    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…

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

    PubMed Central

    Ramdas, Latha; Cogdell, David E; Jia, Jack Y; Taylor, Ellen E; Dunmire, Valerie R; Hu, Limei; Hamilton, Stanley R; Zhang, Wei

    2004-01-01

    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. PMID:15196312

  5. Oligonucleotide-based antiviral strategies.

    PubMed

    Schubert, S; Kurreck, J

    2006-01-01

    In the age of extensive global traffic systems, the close neighborhood of man and livestock in some regions of the world, as well as inadequate prevention measures and medical care in poorer countries, greatly facilitates the emergence and dissemination of new virus strains. The appearance of avian influenza viruses that can infect humans, the spread of the severe acute respiratory syndrome (SARS) virus, and the unprecedented raging of human immunodeficiency virus (HIV) illustrate the threat of a global virus pandemic. In addition, viruses like hepatitis B and C claim more than one million lives every year for want of efficient therapy. Thus, new approaches to prevent virus propagation are urgently needed. Antisense strategies are considered a very attractive means of inhibiting viral replication, as oligonucleotides can be designed to interact with any viral RNA, provided its sequence is known. The ensuing targeted destruction of viral RNA should interfere with viral replication without entailing negative effects on ongoing cellular processes. In this review, we will give some examples of the employment of antisense oligonucleotides, ribozymes, and RNA interference strategies for antiviral purposes. Currently, in spite of encouraging results in preclinical studies, only a few antisense oligonucleotides and ribozymes have turned out to be efficient antiviral compounds in clinical trials. The advent of RNA interference now seems to be refueling hopes for decisive progress in the field of therapeutic employment of antisense strategies.

  6. MAAMD: a workflow to standardize meta-analyses and comparison of affymetrix microarray data

    PubMed Central

    2014-01-01

    Background Mandatory deposit of raw microarray data files for public access, prior to study publication, provides significant opportunities to conduct new bioinformatics analyses within and across multiple datasets. Analysis of raw microarray data files (e.g. Affymetrix CEL files) can be time consuming, complex, and requires fundamental computational and bioinformatics skills. The development of analytical workflows to automate these tasks simplifies the processing of, improves the efficiency of, and serves to standardize multiple and sequential analyses. Once installed, workflows facilitate the tedious steps required to run rapid intra- and inter-dataset comparisons. Results We developed a workflow to facilitate and standardize Meta-Analysis of Affymetrix Microarray Data analysis (MAAMD) in Kepler. Two freely available stand-alone software tools, R and AltAnalyze were embedded in MAAMD. The inputs of MAAMD are user-editable csv files, which contain sample information and parameters describing the locations of input files and required tools. MAAMD was tested by analyzing 4 different GEO datasets from mice and drosophila. MAAMD automates data downloading, data organization, data quality control assesment, differential gene expression analysis, clustering analysis, pathway visualization, gene-set enrichment analysis, and cross-species orthologous-gene comparisons. MAAMD was utilized to identify gene orthologues responding to hypoxia or hyperoxia in both mice and drosophila. The entire set of analyses for 4 datasets (34 total microarrays) finished in ~ one hour. Conclusions MAAMD saves time, minimizes the required computer skills, and offers a standardized procedure for users to analyze microarray datasets and make new intra- and inter-dataset comparisons. PMID:24621103

  7. In situ oligonucleotide synthesis on carbon materials: stable substrates for microarray fabrication

    PubMed Central

    Phillips, Margaret F.; Lockett, Matthew R.; Rodesch, Matthew J.; Shortreed, Michael R.; Cerrina, Franco; Smith, Lloyd M.

    2008-01-01

    Glass has become the standard substrate for the preparation of DNA arrays. Typically, glass is modified using silane chemistries to provide an appropriate functional group for nucleic acid synthesis or oligonucleotide immobilization. We have found substantial issues with the stability of these surfaces as manifested in the unwanted release of oligomers from the surface when incubated in aqueous buffers at moderate temperatures. To address this issue, we have explored the use of carbon-based substrates. Here, we demonstrate in situ synthesis of oligonucleotide probes on carbon-based substrates using light-directed photolithographic phosphoramidite chemistry and evaluate the stabilities of the resultant DNA arrays compared to those fabricated on silanized glass slides. DNA arrays on carbon-based substrates are substantially more stable than arrays prepared on glass. This superior stability enables the use of high-density DNA arrays for applications involving high temperatures, basic conditions, or where serial hybridization and dehybridization is desired. PMID:18084027

  8. The prebiotic synthesis of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Oro, J.; Stephen-Sherwood, E.

    1974-01-01

    This paper is primarily a review of recent developments in the abiotic synthesis of nucleotides, short chain oligonucleotides, and their mode of replication in solution. It also presents preliminary results from this laboratory on the prebiotic synthesis of thymidine oligodeoxynucleotides. A discussion, based on the physicochemical properties of RNA and DNA oligomers, relevant to the molecular evolution of these compounds leads to the tentative hypothesis that oligodeoxyribonucleotides of about 12 units may have been of sufficient length to initiate a self replicating coding system. Two models are suggested to account for the synthesis of high molecular weight oligomers using short chain templates and primers.

  9. Marek's disease virus-induced immunosuppression: array analysis of chicken immune response gene expression profiling.

    PubMed

    Heidari, Mohammad; Sarson, Aimie J; Huebner, Marianne; Sharif, Shayan; Kireev, Dmitry; Zhou, Huaijun

    2010-06-01

    Marek's disease (MD) is a lymphoproliferative disease of chickens induced by a highly cell-associated oncogenic alpha-herpesvirus, Marek's disease virus (MDV). MDV replicates in chicken lymphocytes and establishes a latency infection within CD4(+) T cells. Host-virus interaction, immune responses to infection, and transcriptional profiling of chicken gene expression in MD are poorly understood. In this study we conducted a global host gene expression analysis in the splenocytes of MDV-infected chickens using oligonucleotide-based Affymetrix GeneChip Chicken Genome Arrays. These arrays contain probes for more than 32,000 chicken transcripts and most of the known MDV genes and open reading frames. Two-week-old MD-susceptible chickens were inoculated with an oncogenic strain of MDV, and spleen samples were collected 5 and 15 days post-infection (dpi) for RNA isolation and microarray analysis. Array results displayed a significant differential pattern of immune response transcriptome between the two phases of MDV infection. The expression levels of more than 22 immune-response and related genes were downregulated, while the expression levels of at least 58 genes were increased at 5 dpi (cytolytic infection), compared to age-matched control birds. In comparison, out of 73 immune-response and related genes, 67 genes were downregulated, with only 6 genes having higher expression levels at 15 dpi (latency infection). Cytokines, chemokines, MHC molecules and related receptors, and adhesion molecules were among the many MDV-induced downregulated genes that are critical for an effective antiviral immune response. In addition, several apoptosis-associated genes were decreased in expression during latent infection, suggesting an MDV-induced blocking of initiation or progression of programmed cell death processes. These chicken arrays are valuable tools in understanding the molecular mechanisms behind viral pathogenesis and chicken gene expression patterns, and associated

  10. SERS beacons for multiplexed oligonucleotide detection

    NASA Astrophysics Data System (ADS)

    Sun, Jian; Cullum, Brian M.

    2007-09-01

    Gold-based surface-enhanced Raman scattering (SERS) beacons have been developed, which represent a simple, biocompatible and rapid means of performing multiplexed DNA sequence detection in a non-arrayed format. These SERS beacons consist of a simple stem-loop oligonucleotide probe in its native form with one end attached to a SERS active dye molecule and the other to a gold nanoparticle, approximately 50 nm in diameter. The probe sequence is designed to achieve a stem-loop structure, with the loop portion complementary to the target sequence, similar to fluorescent molecular beacons. In the absence of the target DNA sequence, the SERS signal of the associated dye molecule is detected, representing the "ON" state of the probe. When the target sequence is hybridized to the probe, which results in an open conformation, its respective reporter dye is separated from the gold nanoparticle, producing diminished SERS signal. In this paper, the fabrication and characterization of these SERS beacons is described. We also demonstrate selective hybridization of a target sequence to one beacon in a mixture, revealing their potential for use in a multiplexed fashion.

  11. Diagnostic Oligonucleotide Microarray Fingerprinting of Bacillus Isolates

    SciTech Connect

    Chandler, Darrell P.; Alferov, Oleg; Chernov, Boris; Daly, Don S.; Golova, Julia; Perov, Alexander N.; Protic, Miroslava; Robison, Richard; Shipma, Matthew; White, Amanda M.; Willse, Alan R.

    2006-01-01

    A diagnostic, genome-independent microbial fingerprinting method using DNA oligonucleotide microarrays was used for high-resolution differentiation between closely related Bacillus strains, including two strains of Bacillus anthracis that are monomorphic (indistinguishable) via amplified fragment length polymorphism fingerprinting techniques. Replicated hybridizations on 391-probe nonamer arrays were used to construct a prototype fingerprint library for quantitative comparisons. Descriptive analysis of the fingerprints, including phylogenetic reconstruction, is consistent with previous taxonomic organization of the genus. Newly developed statistical analysis methods were used to quantitatively compare and objectively confirm apparent differences in microarray fingerprints with the statistical rigor required for microbial forensics and clinical diagnostics. These data suggest that a relatively simple fingerprinting microarray and statistical analysis method can differentiate between species in the Bacillus cereus complex, and between strains of B. anthracis. A synthetic DNA standard was used to understand underlying microarray and process-level variability, leading to specific recommendations for the development of a standard operating procedure and/or continued technology enhancements for microbial forensics and diagnostics.

  12. Oligonucleotide therapeutics for human leukaemia.

    PubMed

    Gewirtz, A M

    1997-01-01

    The concept of antisense oligonucleotide 'therapeutics' has generated a great deal of controversy. Questions abound regarding the mechanism of action of these compounds, their reliability and their ultimate utility. These problems are compounded by the 'hype', which has attended their development, and the inability of workers in this area to meet the expectations raised by its most zealous proponents. Nevertheless, it is worth pointing out that there have been some notable gene disruption successes with this technique that have stood up to rigorous scrutiny. Our own work with c-myb as a target is perhaps a reasonable example. Though much remains to be accomplished before antisense drugs are commonly, and usefully, employed in the clinic, it is important to remember what motivates their development. Gene-targeted drugs have the promise of exquisite specificity and the potential to do much good with little toxicity. Accordingly, antisense oligonucleotides can serve as a paradigm of rational drug development. For all these reasons then, we believe that efforts should be increased to decipher the mechanism of action of antisense oligodeoxynucleotides, and to learn how they may be successfully employed in the clinic.

  13. The Affymetrix DMET Plus Platform Reveals Unique Distribution of ADME-Related Variants in Ethnic Arabs

    PubMed Central

    Wakil, Salma M.; Nguyen, Cao; Muiya, Nzioka P.; Andres, Editha; Lykowska-Tarnowska, Agnieszka; Baz, Batoul; Meyer, Brian F.; Morahan, Grant

    2015-01-01

    Background. The Affymetrix Drug Metabolizing Enzymes and Transporters (DMET) Plus Premier Pack has been designed to genotype 1936 gene variants thought to be essential for screening patients in personalized drug therapy. These variants include the cytochrome P450s (CYP450s), the key metabolizing enzymes, many other enzymes involved in phase I and phase II pharmacokinetic reactions, and signaling mediators associated with variability in clinical response to numerous drugs not only among individuals, but also between ethnic populations. Materials and Methods. We genotyped 600 Saudi individuals for 1936 variants on the DMET platform to evaluate their clinical potential in personalized medicine in ethnic Arabs. Results. Approximately 49% each of the 437 CYP450 variants, 56% of the 581 transporters, 56% of 419 transferases, 48% of the 104 dehydrogenases, and 58% of the remaining 390 variants were detected. Several variants, such as rs3740071, rs6193, rs258751, rs6199, rs11568421, and rs8187797, exhibited significantly either higher or lower minor allele frequencies (MAFs) than those in other ethnic groups. Discussion. The present study revealed some unique distribution trends for several variants in Arabs, which displayed partly inverse allelic prevalence compared to other ethnic populations. The results point therefore to the need to verify and ascertain the prevalence of a variant as a prerequisite for engaging it in clinical routine screening in personalized medicine in any given population. PMID:25802476

  14. Cellular Uptake and Intracellular Trafficking of Oligonucleotides: Implications for Oligonucleotide Pharmacology

    PubMed Central

    Ming, Xin; Carver, Kyle; Laing, Brian

    2014-01-01

    One of the major constraints on the therapeutic use of oligonucleotides is inefficient delivery to their sites of action in the cytosol or nucleus. Recently it has become evident that the pathways of cellular uptake and intracellular trafficking of oligonucleotides can strongly influence their pharmacological actions. Here we provide background information on the basic processes of endocytosis and trafficking and then review recent literature on targeted delivery and subcellular trafficking of oligonucleotides in that context. A variety of approaches including molecular scale ligand-oligonucleotide conjugates, ligand-targeted nanocarriers, and the use of small molecules to enhance oligonucleotide effects are discussed. PMID:24383421

  15. Bromodeoxyuridine-labeled oligonucleotides as tools for oligonucleotide uptake studies.

    PubMed

    Maszewska, Maria; Kobylańska, Anna; Gendaszewska-Darmach, Edyta; Koziołkiewicz, Maria

    2002-12-01

    The mechanisms by which various oligonucleotides (ODNs) and their analogs enter cells are not fully understood. A common technique used in studies on cellular uptake of ODNs is their conjugation with fluorochromes. However, fluorescently labeled ODNs may vary from the parent compounds in charge and hydrophilicity, and they may interact differently with some components of cellular membranes. In this report, we present an alternative method based on the immunofluorescent detection of ODNs with incorporated 5-bromo-2'-deoxyuridine (BrdUrd). Localization of BrdUrd-modified ODNs has been achieved using FITC-labeled anti-BrdUrd antibodies. This technique allowed determination of the differences in cellular uptake of phosphodiester (PO) and phosphorothioate (PS) ODNs and their derivatives conjugated with cholesterol and menthol. The immunocytochemical method also has shown that the cellular uptake of some ODNs may be influenced by specific sequences that are responsible for the formation of higher-order structures.

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

    PubMed

    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.

  17. Adaptive resolution simulation of oligonucleotides

    NASA Astrophysics Data System (ADS)

    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.

  18. Adaptive resolution simulation of oligonucleotides.

    PubMed

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

    2016-12-21

    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.

  19. Methods of DNA sequencing by hybridization based on optimizing concentration of matrix-bound oligonucleotide and device for carrying out same

    DOEpatents

    Khrapko, Konstantin R.; Khorlin, Alexandr A.; Ivanov, Igor B.; Ershov, Gennady M.; Lysov, Jury P.; Florentiev, Vladimir L.; Mirzabekov, Andrei D.

    1996-09-03

    A method for sequencing DNA by hybridization that includes the following steps: forming an array of oligonucleotides at such concentrations that either ensure the same dissociation temperature for all fully complementary duplexes or allows hybridization and washing of such duplexes to be conducted at the same temperature; hybridizing said oligonucleotide array with labeled test DNA; washing in duplex dissociation conditions; identifying single-base substitutions in the test DNA by analyzing the distribution of the dissociation temperatures and reconstructing the DNA nucleotide sequence based on the above analysis. A device for carrying out the method comprises a solid substrate and a matrix rigidly bound to the substrate. The matrix contains the oligonucleotide array and consists of a multiplicity of gel portions. Each gel portion contains one oligonucleotide of desired length. The gel portions are separated from one another by interstices and have a thickness not exceeding 30 .mu.m.

  20. Complex DNA nanostructures from oligonucleotide ensembles.

    PubMed

    Mathur, Divita; Henderson, Eric R

    2013-04-19

    The first synthetic DNA nanostructures were created by self-assembly of a small number of oligonucleotides. Introduction of the DNA origami method provided a new paradigm for designing and creating two- and three-dimensional DNA nanostructures by folding a large single-stranded DNA and 'stapling' it together with a library of oligonucleotides. Despite its power and wide-ranging implementation, the DNA origami technique suffers from some limitations. Foremost among these is the limited number of useful single-stranded scaffolds of biological origin. This report describes a new approach to creating large DNA nanostructures exclusively from synthetic oligonucleotides. The essence of this approach is to replace the single-stranded scaffold in DNA origami with a library of oligonucleotides termed "scaples" (scaffold staples). Scaples eliminate the need for scaffolds of biological origin and create new opportunities for producing larger and more diverse DNA nanostructures as well as simultaneous assembly of distinct structures in a "single-pot" reaction.

  1. Antisense oligonucleotides as therapeutics for malignant diseases.

    PubMed

    Ho, P T; Parkinson, D R

    1997-04-01

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

  2. Multipathogen oligonucleotide microarray for environmental and biodefense applications.

    PubMed

    Sergeev, Nikolay; Distler, Margaret; Courtney, Shannon; Al-Khaldi, Sufian F; Volokhov, Dmitriy; Chizhikov, Vladimir; Rasooly, Avraham

    2004-11-01

    Food-borne pathogens are a major health problem. The large and diverse number of microbial pathogens and their virulence factors has fueled interest in technologies capable of detecting multiple pathogens and multiple virulence factors simultaneously. Some of these pathogens and their toxins have potential use as bioweapons. DNA microarray technology allows the simultaneous analysis of thousands of sequences of DNA in a relatively short time, making it appropriate for biodefense and for public health uses. This paper describes methods for using DNA microarrays to detect and analyze microbial pathogens. The FDA-1 microarray was developed for the simultaneous detection of several food-borne pathogens and their virulence factors including Listeria spp., Campylobacter spp., Staphylococcus aureus enterotoxin genes and Clostridium perfringens toxin genes. Three elements were incorporated to increase confidence in the microarray detection system: redundancy of genes, redundancy of oligonucleotide probes (oligoprobes) for a specific gene, and quality control oligoprobes to monitor array spotting and target DNA hybridization. These elements enhance the reliability of detection and reduce the chance of erroneous results due to the genetic variability of microbes or technical problems with the microarray. The results presented demonstrate the potential of oligonucleotide microarrays for detection of environmental and biodefense relevant microbial pathogens.

  3. Portable system for microbial sample preparation and oligonucleotide microarray analysis.

    SciTech Connect

    Bavykin, S. G.; Akowski, J. P.; Zakhariev, V. M.; Barsky, V. E.; Mirzabekov, A. D.; Perov, A. N.; Biochip Technology Center; Engelhardt Inst. of Molecular Biology

    2001-02-01

    We have developed a three-component system for microbial identification that consists of (i) a universal syringe-operated silica minicolumn for successive DNA and RNA isolation, fractionation, fragmentation, fluorescent labeling, and removal of excess free label and short oligonucleotides; (ii) microarrays of immobilized oligonucleotide probes for 16S rRNA identification; and (iii) a portable battery-powered device for imaging the hybridization of fluorescently labeled RNA fragments with the arrays. The minicolumn combines a guanidine thiocyanate method of nucleic acid isolation with a newly developed hydroxyl radical-based technique for DNA and RNA labeling and fragmentation. DNA and RNA can also be fractionated through differential binding of double- and single-stranded forms of nucleic acids to the silica. The procedure involves sequential washing of the column with different solutions. No vacuum filtration steps, phenol extraction, or centrifugation is required. After hybridization, the overall fluorescence pattern is captured as a digital image or as a Polaroid photo. This three-component system was used to discriminate Escherichia coli, Bacillus subtilis, Bacillus thuringiensis, and human HL60 cells. The procedure is rapid: beginning with whole cells, it takes approximately 25 min to obtain labeled DNA and RNA samples and an additional 25 min to hybridize and acquire the microarray image using a stationary image analysis system or the portable imager.

  4. Microfabrication of encoded microparticle array for multiplexed DNA hybridization detection.

    PubMed

    Zhi, Zheng-Liang; Morita, Yasutaka; Yamamura, Shouhei; Tamiya, Eiichi

    2005-05-21

    A strategy for the high-sensitivity, high-selectivity, and multiplexed detection of oligonucleotide hybridizations has been developed with an encoded Ni microparticle random array that was manufactured by a "top-down" approach using micromachining and microfabrication techniques.

  5. An oligonucleotide hybridization approach to DNA sequencing.

    PubMed

    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.

  6. Rapid large-scale oligonucleotide selection for microarrays.

    PubMed

    Rahmann, Sven

    2002-01-01

    We present the first algorithm that selects oligonucleotide probes (e.g. 25-mers) for microarray experiments on a large scale. For example, oligos for human genes can be found within 50 hours. This becomes possible by using the longest common substring as a specificity measure for candidate oligos. We present an algorithm based on a suffix array with additional information that is efficient both in terms of memory usage and running time to rank all candidate oligos according to their specificity. We also introduce the concept of master sequences to describe the sequences from which oligos are to be selected. Constraints such as oligo length, melting temperature, and self-complementarity are incorporated in the master sequence at a preprocessing stage and thus kept separate from the main selection problem. As a result, custom oligos can now be designed for any sequenced genome, just as the technology for on-site chip synthesis is becoming increasingly mature.

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

    PubMed

    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.

  8. Oligonucleotide-based therapy for neurodegenerative diseases.

    PubMed

    Magen, Iddo; Hornstein, Eran

    2014-10-10

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

  9. Oligonucleotide recombination in gram negative bacteria

    Technology Transfer Automated Retrieval System (TEKTRAN)

    This report describes several key aspects of a novel form of RecA-independent homologous recombination. We found that synthetic single stranded DNA oligonucleotides (oligos) introduced into bacteria by transformation can site-specifically recombine with bacterial chromosomes in the absence of any a...

  10. Liver as a target for oligonucleotide therapeutics.

    PubMed

    Sehgal, Alfica; Vaishnaw, Akshay; Fitzgerald, Kevin

    2013-12-01

    Oligonucleotide-based therapeutics are an emerging class of drugs that hold the promise for silencing "un-druggable" targets,thus creating unique opportunities for innovative medicines. As opposed to gene therapy, oligonucleotides are considered to be more akin to small molecule therapeutics because they are small,completely synthetic in origin, do not integrate into the host genome,and have a defined duration of therapeutic activity after which effects recover to baseline. They offer a high degree of specificity at the genetic level, thereby reducing off-target effects.At the same time, they provide a strategy for targeting any gene in the genome, including transcripts that produce mutated proteins.Oligonucleotide-based therapeutics include short interfering RNA (siRNA), that degrade target mRNA through RISC mediated RNAi; anti-miRs, that target miRNAs; miRNA mimics, that regulate target mRNA; antisense oligonucleotides, that may be working through RNAseH mediated mRNA decay; mRNA upregulation,by targeting long non-coding RNAs; and oligonucleotides induced alternative splicing [1]. All these approaches require some minimal degree of homology at the nucleic acid sequence level for them to be functional. The different mechanisms of action and their relevant activity are outlined in Fig. 1. Besides homology,RNA secondary structure has also been exploited in the case of ribozymes and aptamers, which act by binding to nucleic acids or proteins, respectively. While there have been many reports of gene knockdown and gene modulation in cell lines and mice with all these methods, very few have advanced to clinical stages.The main obstacle to date has been the safe and effective intracellular delivery of these compounds in higher species, including humans. Indeed, their action requires direct interaction with DNA/RNA within the target cell so even when one solves the issues of tissue and cellular access, intracellular/intranuclear location represents yet another barrier to

  11. Drug targeting: synthesis and endocytosis of oligonucleotide-neoglycoprotein conjugates.

    PubMed Central

    Bonfils, E; Depierreux, C; Midoux, P; Thuong, N T; Monsigny, M; Roche, A C

    1992-01-01

    Inhibition of gene expression by antisense oligonucleotides is limited by their low ability to enter cells. Knowing that sugar binding receptors, also called membrane lectins, efficiently internalize neoglycoproteins bearing the relevant sugar, 6-phosphomannose, for instance, oligonucleotides--substituted on their 5'-end with either a fluorescent probe or a radioactive label on the one hand, and bearing a thiol function on their 3'-end, on the other hand,--were coupled onto 6-phosphomannosylated proteins via a disulfide bridge. The oligonucleotide bound to 6-phosphomannosylated serum albumin is much more efficiently internalized roughly 20 times than the free oligonucleotide. Although most of the oligonucleotides are associated with vesicular compartments, oligonucleotides after releasing from the carrier by reduction of the disulfide bridge may find their way to reach the cytosol and then lead to an increase in the efficiency of the oligonucleotides. Images PMID:1408764

  12. Detection of genetically modified canola using multiplex PCR coupled with oligonucleotide microarray hybridization.

    PubMed

    Schmidt, Anna-Mary; Sahota, Robert; Pope, Derek S; Lawrence, Tracy S; Belton, Mark P; Rott, Michael E

    2008-08-27

    A rapid method was developed for concurrent screening of transgenic elements in GM canola. This method utilizes a single multiplex PCR coupled with an oligonucleotide DNA array capable of simultaneously detecting the 12 approved GM canola lines in Canada. The assay includes construct-specific elements for identification of approved lines, common elements (e.g., CaMV 35S promoter, Agrobacterium tumefaciens nos terminator, or nptII gene) for screening of approved or unapproved lines, a canola-specific endogenous gene, and endogenous genes from heterologous crops to serve as additional controls. Oligonucleotide probes were validated individually for functionality and specificity by amplification of specific transgene sequences from appropriate GM canola lines corresponding to each probe sequence, and hybridization of amplicons to the array. Each target sequence hybridized to its corresponding oligonucleotide probe and no significant cross-hybridization was observed. The limit of detection was examined for the GM lines GT73, T45, and MS8/RF3, and was determined to be 0.1%, 0.1%, and 0.5%, respectively, well within the European food and feed labeling threshold level of 0.9% for approved GM product. Practically, the method was demonstrated to be effective for the detection of GM canola in several types of animal feed, as well as in commercial canola meal.

  13. Antisense oligonucleotides in therapy for neurodegenerative disorders.

    PubMed

    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.

  14. Identification of characteristic oligonucleotides in the bacterial 16S ribosomal RNA sequence dataset

    NASA Technical Reports Server (NTRS)

    Zhang, Zhengdong; Willson, Richard C.; Fox, George E.

    2002-01-01

    MOTIVATION: The phylogenetic structure of the bacterial world has been intensively studied by comparing sequences of 16S ribosomal RNA (16S rRNA). This database of sequences is now widely used to design probes for the detection of specific bacteria or groups of bacteria one at a time. The success of such methods reflects the fact that there are local sequence segments that are highly characteristic of particular organisms or groups of organisms. It is not clear, however, the extent to which such signature sequences exist in the 16S rRNA dataset. A better understanding of the numbers and distribution of highly informative oligonucleotide sequences may facilitate the design of hybridization arrays that can characterize the phylogenetic position of an unknown organism or serve as the basis for the development of novel approaches for use in bacterial identification. RESULTS: A computer-based algorithm that characterizes the extent to which any individual oligonucleotide sequence in 16S rRNA is characteristic of any particular bacterial grouping was developed. A measure of signature quality, Q(s), was formulated and subsequently calculated for every individual oligonucleotide sequence in the size range of 5-11 nucleotides and for 15mers with reference to each cluster and subcluster in a 929 organism representative phylogenetic tree. Subsequently, the perfect signature sequences were compared to the full set of 7322 sequences to see how common false positives were. The work completed here establishes beyond any doubt that highly characteristic oligonucleotides exist in the bacterial 16S rRNA sequence dataset in large numbers. Over 16,000 15mers were identified that might be useful as signatures. Signature oligonucleotides are available for over 80% of the nodes in the representative tree.

  15. The prebiotic synthesis of deoxythymidine oligonucleotides

    NASA Technical Reports Server (NTRS)

    Stephen-Sherwood, E.; Odom, D. G.; Oro, J.

    1974-01-01

    Deoxythymidine 5 prime-triphosphate in the presence of deoxythymidine 5 prime-phosphate, cyanamide and 4-amino-5-imidazole carboxamide polymerizes under drying conditions at moderate temperatures (60 to 90 C) to yield oligonucleotides of up to four units in length. Enzymatic analysis indicated that the majority of these oligomers contained natural 3 prime-5 prime phosphodiester bonds. This reaction offers a possible method for the formation of deoxyoligonucleotides under primitive earth conditions.

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

    PubMed

    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.

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

    PubMed Central

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

    2005-01-01

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

  18. Detection of SPO11-oligonucleotide complexes from mouse testes.

    PubMed

    Pan, Jing; Keeney, Scott

    2009-01-01

    The SPO11 protein generates programmed DNA double-strand breaks (DSBs) that initiate meiotic recombination. Endonucleolytic cleavage 3' to the DSB sites releases SPO11 from DNA, leaving SPO11 covalently associated with an oligonucleotide. This chapter describes detection of the release product, SPO11-oligonucleotide complexes, from mouse testis lysates. The method for determining the size of SPO11-associated oligonucleotides is also provided.

  19. Linear model for fast background subtraction in oligonucleotide microarrays

    PubMed Central

    2009-01-01

    Background One important preprocessing step in the analysis of microarray data is background subtraction. In high-density oligonucleotide arrays this is recognized as a crucial step for the global performance of the data analysis from raw intensities to expression values. Results We propose here an algorithm for background estimation based on a model in which the cost function is quadratic in a set of fitting parameters such that minimization can be performed through linear algebra. The model incorporates two effects: 1) Correlated intensities between neighboring features in the chip and 2) sequence-dependent affinities for non-specific hybridization fitted by an extended nearest-neighbor model. Conclusion The algorithm has been tested on 360 GeneChips from publicly available data of recent expression experiments. The algorithm is fast and accurate. Strong correlations between the fitted values for different experiments as well as between the free-energy parameters and their counterparts in aqueous solution indicate that the model captures a significant part of the underlying physical chemistry. PMID:19917117

  20. Application of oligonucleotide microarrays for bacterial source tracking of environmental Enterococcus sp. isolates.

    PubMed

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

    2005-04-01

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

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

    PubMed Central

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

    2005-01-01

    In an effort towards adapting new and defensible methods for assessing and managing the risk posed by microbial pollution, we evaluated the utility of oligonucleotide microarrays for bacterial source tracking (BST) of environmental Enterococcus sp. isolates derived from various host sources. Current bacterial source tracking approaches rely on various phenotypic and genotypic methods to identify sources of bacterial contamination resulting from point or non-point pollution. For this study 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. PMID:16705816

  2. Template switching between PNA and RNA oligonucleotides

    NASA Technical Reports Server (NTRS)

    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.

  3. BIOCONJUGATION OF OLIGONUCLEOTIDES FOR TREATING LIVER FIBROSIS

    PubMed Central

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

    2009-01-01

    Liver fibrosis results from chronic liver injury due to hepatitis B and C, excessive alcohol ingestion, and metal ion overload. Fibrosis culminates in cirrhosis and results in liver failure. Therefore, a potent antifibrotic therapy is in urgent need to reverse scarring and eliminate progression to cirrhosis. Although activated hepatic stellate cells (HSCs) remains the principle cell type responsible for liver fibrosis, perivascular fibroblasts of portal and central veins as well as periductular fibroblasts are other sources of fibrogenic cells. This review will critically discuss various treatment strategies for liver fibrosis, including prevention of liver injury, reduction of inflammation, inhibition of HSC activation, degradation of scar matrix, and inhibition of aberrant collagen synthesis. Oligonucleotides (ODNs) are short, single-stranded nucleic acids, which disrupt expression of target protein by binding to complementary mRNA or forming triplex with genomic DNA. Triplex forming oligonucleotides (TFOs) provide an attractive strategy for treating liver fibrosis. A series of TFOs have been developed for inhibiting the transcription of α1(I) collagen gene, which opens a new area for antifibrotic drugs. There will be in depth discussion on the use of TFOs and how different bioconjugation strategies can be utilized for their site-specific delivery to HSCs or hepatocytes for enhanced antifibrotic activities. Various insights developed in individual strategy and the need for multipronged approaches will also be discussed. PMID:18154454

  4. Disulfide-linked oligonucleotide phosphorothioates - Novel analogues of nucleic acids

    NASA Technical Reports Server (NTRS)

    Wu, Taifeng; Orgel, Leslie E.

    1991-01-01

    The synthesis of phosphorothioate analogs of oligonucleotides by the oxidation of deoxyadenosine 3',5'-bisphosphorothioate (3) was attempted. Cyclization of 3 is much more efficient than oligomerization under all the conditions investigated. However, a preformed oligonucleotide carrying a 5'-terminal phosphorotioate group undergoes efficient chain-extension when oxidized in the presence of 3.

  5. Oligonucleotide therapies for disorders of the nervous system.

    PubMed

    Khorkova, Olga; Wahlestedt, Claes

    2017-03-01

    Oligonucleotide therapies are currently experiencing a resurgence driven by advances in backbone chemistry and discoveries of novel therapeutic pathways that can be uniquely and efficiently modulated by the oligonucleotide drugs. A quarter of a century has passed since oligonucleotides were first applied in living mammalian brain to modulate gene expression. Despite challenges in delivery to the brain, multiple oligonucleotide-based compounds are now being developed for treatment of human brain disorders by direct delivery inside the blood brain barrier (BBB). Notably, the first new central nervous system (CNS)-targeted oligonucleotide-based drug (nusinersen/Spinraza) was approved by US Food and Drug Administration (FDA) in late 2016 and several other compounds are in advanced clinical trials. Human testing of brain-targeted oligonucleotides has highlighted unusual pharmacokinetic and pharmacodynamic properties of these compounds, including complex active uptake mechanisms, low systemic exposure, extremely long half-lives, accumulation and gradual release from subcellular depots. Further work on oligonucleotide uptake, development of formulations for delivery across the BBB and relevant disease biology studies are required for further optimization of the oligonucleotide drug development process for brain applications.

  6. Voltage-gated calcium channel and antisense oligonucleotides thereto

    NASA Technical Reports Server (NTRS)

    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.

  7. 2'-modified nucleosides for site-specific labeling of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Krider, Elizabeth S.; Miller, Jeremiah E.; Meade, Thomas J.

    2002-01-01

    We report the synthesis of 2'-modified nucleosides designed specifically for incorporating labels into oligonucleotides. Conversion of these nucleosides to phosphoramidite and solid support-bound derivatives proceeds in good yield. Large-scale synthesis of 11-mer oligonucleotides possessing the 2'-modified nucleosides is achieved using these derivatives. Thermal denaturation studies indicate that the presence of 2'-modified nucleosides in 11-mer duplexes has minimal destabilizing effects on the duplex structure when the nucleosides are placed at the duplex termini. The powerful combination of phosphoramidite and support-bound derivatives of 2'-modified nucleosides affords the large-scale preparation of an entirely new class of oligonucleotides. The ability to synthesize oligonucleotides containing label attachment sites at 3', intervening, and 5' locations of a duplex is a significant advance in the development of oligonucleotide conjugates.

  8. The spherulites™: a promising carrier for oligonucleotide delivery

    PubMed Central

    Mignet, Nathalie; Brun, Armelle; Degert, Corinne; Delord, Brigitte; Roux, Didier; Hélène, Claude; Laversanne, René; François, Jean-Christophe

    2000-01-01

    Concentric multilamellar microvesicles, named spherulites™, were evaluated as an oligonucleotide carrier. Up to 80% oligonucleotide was encapsulated in these vesicles. The study was carried out on two different spherulite™ formulations. The spherulite™ size and stability characteristics are presented. Delivery of encapsulated oligonucleotide was performed on a rat hepatocarcinoma and on a lymphoblastoid T cell line, both expressing the luciferase gene. We showed that spherulites™ were able to transfect both adherent and suspension cell lines and deliver the oligonucleotide to the nucleus. Moreover, 48–62% luciferase inhibition was obtained in the rat hepatocarcinoma cell line when the antisense oligonucleotide targeted to the luciferase coding region was encapsulated at 500 nM concentration in spherulites™ of different compositions. PMID:10931929

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

    PubMed

    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.

  10. Survivin Antisense Oligonucleotides Effectively Radiosensitize Colorectal Cancer Cells in Both Tissue Culture and Murine Xenograft Models

    SciTech Connect

    Roedel, Franz; Capalbo, Gianni; Weiss, Christian; Roedel, Claus

    2008-05-01

    Purpose: Survivin shows a radiation resistance factor in colorectal cancer. In the present study, we determined whether survivin messenger RNA levels in patients with rectal cancer predict tumor response after neoadjuvant radiochemotherapy and whether inhibition of survivin by the use of antisense oligonucleotides (ASOs) enhances radiation responses. Methods and Materials: SW480 colorectal carcinoma cells were transfected with survivin ASO (LY2181308) and irradiated with doses ranging from 0-8 Gy. Survivin expression, cell-cycle distribution, {gamma}H2AX fluorescence, and induction of apoptosis were monitored by means of immunoblotting, flow cytometry, and caspase 3/7 activity. Clonogenic survival was determined by using a colony-forming assay. An SW480 xenograft model was used to investigate the effect of survivin attenuation and irradiation on tumor growth. Furthermore, survivin messenger RNA levels were studied in patient biopsy specimens by using Affymetrix microarray analysis. Results: In the translational study of 20 patients with rectal cancer, increased survivin levels were associated with significantly greater risk of local tumor recurrence (p = 0.009). Treatment of SW480 cells with survivin ASOs and irradiation resulted in an increased percentage of apoptotic cells, caspase 3/7 activity, fraction of cells in the G{sub 2}/M phase, and H2AX phosphorylation. Clonogenic survival decreased compared with control-treated cells. Furthermore, treatment of SW480 xenografts with survivin ASOs and irradiation resulted in a significant delay in tumor growth. Conclusion: Survivin appears to be a molecular biomarker in patients with rectal cancer. Furthermore, in vitro and in vivo data suggest a potential role of survivin as a molecular target to improve treatment response to radiotherapy in patients with rectal cancer.

  11. Conjugation of fluorescent proteins with DNA oligonucleotides.

    PubMed

    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.

  12. Abiotic formation of oligonucleotides on basalt surfaces

    NASA Astrophysics Data System (ADS)

    Otroshchenko, V. A.; Vasilyeva, N. V.; Kopilov, A. M.

    1985-06-01

    The complication and further evolution of abiotic syntheses products occurred under environmental influences at the prebiological stage. From this point of view, the influence of some types of irradiation on the organic molecules adsorbed on the surfaces of volcanic rocks, appeared to be of great importance. In this connection, the effect of gamma rays on the AMP molecules adsorbed on mineral surfaces such as cinders and ashes has been studied. It has been shown that they can polymerize with the formation of oligonucleotides. The treatment of oligomers obtained by venom phosphodiesterase has shown that a polymeric product has mainly 3' 5' and 2' 5' bonds between nucleotides. The results obtained have been discussed from the evolutionary aspect.

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

    PubMed

    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.

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

    PubMed

    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.

  15. A facile method for the construction of oligonucleotide microarrays.

    PubMed

    Sethi, Dalip; Kumar, A; Gupta, K C; Kumar, P

    2008-11-19

    In recent years, the oligonucleotide-based microarray technique has emerged as a powerful and promising tool for various molecular biological studies. Here, a facile protocol for the construction of an oligonucleotide microarray is demonstrated that involves immobilization of oligonucleotide-trimethoxysilyl conjugates onto virgin glass microslides. The projected immobilization strategy reflects high immobilization efficiency ( approximately 36-40%) and signal-to-noise ratio ( approximately 98), and hybridization efficiency ( approximately 32-35%). Using the proposed protocol, aminoalkyl, mercaptoalkyl, and phosphorylated oligonucleotides were immobilized onto virgin glass microslides. Briefly, modified oligonucleotides were reacted first with 3-glycidyloxypropyltriethoxysilane (GOPTS), and subsequently, the resultant conjugates were directly immobilized onto the virgin glass surface by making use of silanization chemistry. The constructed microarrays were then used for discrimination of base mismatches. On subjecting to different pH and thermal conditions, the microarray showed sufficient stability. Application of this chemistry to manufacture oligonucleotide probe-based microarrays for detection of bacterial meningitis is demonstrated. Single-step reaction for the formation of conjugates with the commercially available reagent (GOPTS), omission of capping step and surface modification, and efficient immobilization of oligonucleotides onto the virgin glass surface are the key features of the proposed strategy.

  16. Nucleic acid sequence detection using multiplexed oligonucleotide PCR

    DOEpatents

    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.

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

    SciTech Connect

    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.

  18. Method for the preparation of size marker for synthetic oligonucleotides

    SciTech Connect

    Jing, G.Z.; Liu, A.; Leung, W.C.

    1986-01-01

    Terminal deoxynucleotidyltransferase was used for the addition of (..cap alpha..-/sup 32/P)dCTP to the 3'-OH termini of oligo(dT)/sub 12-18/. A collection of oligonucleotides with chain lengths ranging continuously from 13-mer to over 100-mer was generated. The reaction mixture was then mixed with oligo(dT)/sub 12-18/ labeled with (..gamma..-/sup 32/P)ATP by T/sub 4/ polynucleotide kinase. A sequence ladder with the bottom base as 12-mer was then formed. These oligonucleotides served as size marker for the purification and identification of oligonucleotides on polyacrylamide gel.

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

    PubMed

    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.

  20. Therapeutic oligonucleotides and delivery technologies: Research topics in Japan.

    PubMed

    Murakami, Masahiro

    2016-01-01

    Oligonucleotides have been gaining considerable attention as promising and effective candidate therapeutics against various diseases. This special issue is aimed at providing a better understanding of the recent progress in the development of oligonucleotide-based therapeutics to encourage further research and innovation in this field to achieve these advancements. Several Japanese scientists have been invited to contribute to this issue by describing their recent findings, overviews, insights, or commentaries on rational designing of therapeutic oligonucleotide molecules and their novel delivery technologies, especially nanocarrier systems.

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

    PubMed

    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.

  2. Oligonucleotide-Functionalized Anisotropic Gold Nanoparticles

    NASA Astrophysics Data System (ADS)

    Jones, Matthew Robert

    In this thesis, we describe the properties of oligonucleotide-functionalized gold colloids under the unique set of conditions where the particles are geometrically anisotropic and have nanometer-scale dimensions. While nearly two decades of previous work elucidated numerous unexpected and emergent phenomena arising from the combination of inorganic nanoparticles with surface-bound DNA strands, virtually nothing was known about how these properties are altered when the shape of the nanoparticle core is chosen to be non-spherical. In particular, we are interested in understanding, and ultimately controlling, the ways in which these DNA-conjugated anisotropic nanostructures interact when their attraction is governed by programmable DNA hybridization events. Chapter 1 introduces the field of DNA-based materials assembly by discussing how nanoscale building blocks which present rigid, directional interactions can be thought of as possessing artificial versions of the familiar chemical principles of "bonds" and "valency". In chapter 2 we explore the fundamental interparticle binding thermodynamics of DNA-functionalized spherical and anisotropic nanoparticles, which reveals enormous preferences for collective ligand interactions occurring between flat surfaces over those that occur between curved surfaces. Using these insights, chapter 3 demonstrates that when syntheses produce mixtures of different nanoparticle shapes, the tailorable nature of DNA-mediated interparticle association can be used to selectively crystallize and purify the desired anisotropic nanostructure products, leaving spherical impurity particles behind. Chapter 4 leverages the principle that the flat facets of anisotropic particles generate directional DNA-based hybridization interactions to assemble a variety of tailorable nanoparticle superlattices whose symmetry and dimensionality are a direct consequence of the shape of the nanoparticle building block used in their construction. Chapter 5 explores

  3. Polyphosphorylation and non-enzymatic template-directed ligation of oligonucleotides

    NASA Technical Reports Server (NTRS)

    Gao, K.; Orgel, L. E.; Bada, J. L. (Principal Investigator)

    2000-01-01

    Oligonucleotide 5'-polyphosphates are formed under potentially prebiotic conditions from oligonucleotide 5'-phosphates and sodium trimetaphosphate. Oligonucleotides activated as polyphosphates undergo template-directed ligation. We believe that these reactions could have produced longer oligonucleotide products from shorter substrates under prebiotic conditions.

  4. Sequence-dependent theory of oligonucleotide hybridization kinetics

    SciTech Connect

    Marimuthu, Karthikeyan; Chakrabarti, Raj E-mail: rajc@andrew.cmu.edu

    2014-05-07

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

  5. Micro- and nano-structure based oligonucleotide sensors.

    PubMed

    Ferrier, David C; Shaver, Michael P; Hands, Philip J W

    2015-06-15

    This paper presents a review of micro- and nano-structure based oligonucleotide detection and quantification techniques. The characteristics of such devices make them very attractive for Point-of-Care or On-Site-Testing biosensing applications. Their small scale means that they can be robust and portable, their compatibility with modern CMOS electronics means that they can easily be incorporated into hand-held devices and their suitability for mass production means that, out of the different approaches to oligonucleotide detection, they are the most suitable for commercialisation. This review discusses the advantages of micro- and nano-structure based sensors and covers the various oligonucleotide detection techniques that have been developed to date. These include: Bulk Acoustic Wave and Surface Acoustic Wave devices, micro- and nano-cantilever sensors, gene Field Effect Transistors, and nanowire and nanopore based sensors. Oligonucleotide immobilisation techniques are also discussed.

  6. Tritium labeling of antisense oligonucleotides by exchange with tritiated water.

    PubMed Central

    Graham, M J; Freier, S M; Crooke, R M; Ecker, D J; Maslova, R N; Lesnik, E A

    1993-01-01

    We describe a simple, efficient, procedure for labeling oligonucleotides to high specific activity (< 1 x 10(8) cpm/mumol) by hydrogen exchange with tritiated water at the C8 positions of purines in the presence of beta-mercaptoethanol, an effective radical scavenger. Approximately 90% of the starting material is recovered as intact, labeled oligonucleotide. The radiolabeled compounds are stable in biological systems; greater than 90% of the specific activity is retained after 72 hr incubation at 37 degrees C in serum-containing media. Data obtained from in vitro cellular uptake experiments using oligonucleotides labeled by this method are similar to those obtained using 35S or 14C-labeled compounds. Because this protocol is solely dependent upon the existence of purine residues, it should be useful for radiolabeling modified as well as unmodified phosphodiester oligonucleotides. Images PMID:8367289

  7. PRACTICAL STRATEGIES FOR PROCESSING AND ANALYZING SPOTTED OLIGONUCLEOTIDE MICROARRAY DATA

    EPA Science Inventory

    Thoughtful data analysis is as important as experimental design, biological sample quality, and appropriate experimental procedures for making microarrays a useful supplement to traditional toxicology. In the present study, spotted oligonucleotide microarrays were used to profile...

  8. Methods to Characterize the Oligonucleotide Functionalization of Quantum Dots.

    PubMed

    Weichelt, Richard; Leubner, Susanne; Henning-Knechtel, Anja; Mertig, Michael; Gaponik, Nikolai; Schmidt, Thorsten-Lars; Eychmüller, Alexander

    2016-09-01

    Currently, DNA nanotechnology offers the most programmable, scalable, and accurate route for the self-assembly of matter with nanometer precision into 1, 2, or 3D structures. One example is DNA origami that is well suited to serve as a molecularly defined "breadboard", and thus, to organize various nanomaterials such as nanoparticles into hybrid systems. Since the controlled assembly of quantum dots (QDs) is of high interest in the field of photonics and other optoelectronic applications, a more detailed view on the functionalization of QDs with oligonucleotides shall be achieved. In this work, four different methods are presented to characterize the functionalization of thiol-capped cadmium telluride QDs with oligonucleotides and for the precise quantification of the number of oligonucleotides bound to the QD surface. This study enables applications requiring the self-assembly of semiconductor-oligonucleotide hybrid materials and proves the conjugation success in a simple and straightforward manner.

  9. Photophysical deactivation pathways in adenine oligonucleotides.

    PubMed

    Spata, Vincent A; Matsika, Spiridoula

    2015-12-14

    In this work we study deactivation processes in adenine oligomers after absorption of UV radiation using Quantum Mechanics combined with Molecular Mechanics (QM/MM). Correlated electronic structure methods appropriate for describing the excited states are used to describe a π-stacked dimer of adenine bases incorporated into (dA)20(dT)20. The results of these calculations reveal three different types of excited state minima which play a role in deactivation processes. Within this set of minima there are minima where the excited state is localized on one adenine (monomer-like) as well as minima where the excited state is delocalized on two adenines, forming different types of excimers and bonded excimers of varying but inter-related character. The proximity of their energies reveals that the minima can decay into one another along a flat potential energy surface dependent on the interbase separation. Additionally, analysis of the emissive energies and other physical properties, including theoretical anisotropy calculations, and comparison with fluorescence experiments, provides evidence that excimers play an important role in long-lived signals in adenine oligonucleotides while the subpicosecond decay is attributed to monomer-like minima. The necessity for a close approach of the nucleobases reveals that the deactivation mechanism is tied to macro-molecular motion.

  10. Antisense oligonucleotide therapeutics for human leukemia.

    PubMed

    Gewirtz, A M

    1998-01-01

    The development of reliable gene disruption strategies, and their application in living cells, has proven to be an extraordinary important advance for cell and molecular biologists. Using the various available approaches, the specific functions of any given gene may now be investigated directly in the relevant cell type. Application of similar experimental tools in a clinical setting might prove to be equally valuable and could well form the basis of a monumental advance in the practice of clinical medicine. This seems particularly true at the present time because much progress has been made in understanding the molecular pathogenesis of many diseases, including cancer. For these reasons a tremendous amount of interest has been generated in the use of oligodeoxynucleotides to modify gene expression. However, in spite of some notable successes which are detailed in this review, oligonucleotides have generated controversy in regard to their mechanism of action, reliability, and ultimate therapeutic utility. Nevertheless, the potential power of the "antisense" approach remains undisputed, and its ultimate therapeutic utility is far reaching. Accordingly, the problems associated with the use of these compounds are clearly worth solving. It remains the hope of many laboratories that the day will soon come when these techniques will make an important contribution to the management of chronic myelogenous leukemia and other neoplastic disorders.

  11. Antisense oligonucleotide therapeutics for human leukemia.

    PubMed

    Gewirtz, A M

    1997-01-01

    The development of reliable gene disruption strategies, and their application in living cells, has proven to be an extraordinarily important advance for cell and molecular biologists. Using the various available approaches, the specific functions of any given gene may now be investigated directly in the relevant cell type. Application of similar experimental tools in a clinical setting might prove to be equally valuable and could well form the basis of a monumental advance in the practice of clinical medicine. This seems particularly true at the present time since much progress has been made in understanding the molecular pathogenesis of many diseases, including cancer. For these reasons a tremendous amount of interest has been generated in the use of oligodeoxynucleotides to modify gene expression. However, in spite of some notable successes which are detailed in this review, oligonucleotides have generated controversy in regards to their mechanism of action, reliability, and ultimate therapeutic utility. Nevertheless, the potential power of the "antisense" approach remains undisputed, and its ultimate therapeutic utility is far reaching. Accordingly, the problems associated with the use of these compounds are clearly worth solving. It remains the hope of many laboratories that the day will soon come when these techniques will make an important contribution to the management of CML and other neoplastic disorders.

  12. Antisense Oligonucleotide Therapy for Inherited Retinal Dystrophies.

    PubMed

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

    2016-01-01

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

  13. Oligonucleotide conjugates - Candidates for gene silencing therapeutics.

    PubMed

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

    2016-10-01

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

  14. An Integrated ISFET Sensor Array

    PubMed Central

    Nakazato, Kazuo

    2009-01-01

    A monolithically integrated ISFET sensor array and interface circuit are described. A new high-density, low-power source-drain follower was developed for the sensor array. ISFETs were formed by depositing Au/Ti extended-gate electrodes on standard MOSFETs, then thin silicon nitride layers using catalytic chemical vapor deposition and/or SU-8 protective layers were formed on the extended-gate electrodes. Applications for the array include: (1) pH detection by statistical distribution observing time and space fluctuations; (2) DNA detection using thiol-modified or silane-coupled oligonucleotides; (3) bio-image sensing by converting photons to electrons using Photosystem I of Thermosynechococcus elongatus, and sensing the converted electric charges by ISFETs. PMID:22291539

  15. Syntheses of oligonucleotide derivatives with P(V) porphyrin and their properties.

    PubMed

    Shimidzu, T; Segawa, H; Kitamura, M; Nimura, A

    1992-01-01

    Two types of oligonucleotide derivatives which are substituted by P(V) porphyrin at the phosphorus atom of an internucleotidic linkage and at the 5'-terminal internucleotidic linkage via a spacer were synthesized (Fig. 1), and hybridization capabilities of them with complementary oligonucleotides were evaluated. A novel method for a sensing of oligonucleotide by the fluorescence quenching via photo-induced electron transfer between the P(V) porphyrin labeled oligonucleotide and pyrene-labeled one on the oligonucleotide template is reported.

  16. Nanogels for Oligonucleotide Delivery to the Brain

    PubMed Central

    Vinogradov, Serguei V.; Batrakova, Elena V.; Kabanov, Alexander V.

    2009-01-01

    Systemic delivery of oligonucleotides (ODN) to the central nervous system is needed for development of therapeutic and diagnostic modalities for treatment of neurodegenerative disorders. Macromolecules injected in blood are poorly transported across the blood–brain barrier (BBB) and rapidly cleared from circulation. In this work we propose a novel system for ODN delivery to the brain based on nanoscale network of cross-linked poly(ethylene glycol) and polyethylenimine (“nanogel”). The methods of synthesis of nanogel and its modification with specific targeting molecules are described. Nanogels can bind and encapsulate spontaneously negatively charged ODN, resulting in formation of stable aqueous dispersion of polyelectrolyte complex with particle sizes less than 100 nm. Using polarized monolayers of bovine brain microvessel endothelial cells as an in vitro model this study demonstrates that ODN incorporated in nanogel formulations can be effectively transported across the BBB. The transport efficacy is further increased when the surface of the nanogel is modified with transferrin or insulin. Importantly the ODN is transported across the brain microvessel cells through the transcellular pathway; after transport, ODN remains mostly incorporated in the nanogel and ODN displays little degradation compared to the free ODN. Using mouse model for biodistribution studies in vivo, this work demonstrated that as a result of incorporation into nanogel 1 h after intravenous injection the accumulation of a phosphorothioate ODN in the brain increases by over 15 fold while in liver and spleen decreases by 2-fold compared to the free ODN. Overall, this study suggests that nanogel is a promising system for delivery of ODN to the brain. PMID:14733583

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

    SciTech Connect

    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.

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

    PubMed Central

    2009-01-01

    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 signal intensity were

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

    NASA Astrophysics Data System (ADS)

    Tripathy, Sushant

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

  20. High-density fiber optic biosensor arrays

    NASA Astrophysics Data System (ADS)

    Epstein, Jason R.; Walt, David R.

    2002-02-01

    Novel approaches are required to coordinate the immense amounts of information derived from diverse genomes. This concept has influenced the expanded role of high-throughput DNA detection and analysis in the biological sciences. A high-density fiber optic DNA biosensor was developed consisting of oligonucleotide-functionalized, 3.1 mm diameter microspheres deposited into the etched wells on the distal face of a 500 micrometers imaging fiber bundle. Imaging fiber bundles containing thousands of optical fibers, each associated with a unique oligonucleotide probe sequence, were the foundation for an optically connected, individually addressable DNA detection platform. Different oligonucleotide-functionalized microspheres were combined in a stock solution, and randomly dispersed into the etched wells. Microsphere positions were registered from optical dyes incorporated onto the microspheres. The distribution process provided an inherent redundancy that increases the signal-to-noise ratio as the square root of the number of sensors examined. The representative amount of each probe-type in the array was dependent on their initial stock solution concentration, and as other sequences of interest arise, new microsphere elements can be added to arrays without altering the existing detection capabilities. The oligonucleotide probe sequences hybridize to fluorescently-labeled, complementary DNA target solutions. Fiber optic DNA microarray research has included DNA-protein interaction profiles, microbial strain differentiation, non-labeled target interrogation with molecular beacons, and single cell-based assays. This biosensor array is proficient in DNA detection linked to specific disease states, single nucleotide polymorphism (SNP's) discrimination, and gene expression analysis. This array platform permits multiple detection formats, provides smaller feature sizes, and enables sensor design flexibility. High-density fiber optic microarray biosensors provide a fast

  1. Construction and evaluation of a Clostridium thermocellum ATCC 27405 whole-genome oligonucleotide microarray

    SciTech Connect

    Brown, Steven David; Raman, Babu; McKeown, Catherine K; Kale, Shubhangi P; He, Zhili; Mielenz, Jonathan R

    2007-04-01

    Clostridium thermocellum is an anaerobic, thermophilic bacterium that can directly convert cellulosic substrates into ethanol. Microarray technology is a powerful tool to gain insights into cellular processes by examining gene expression under various physiological states. Oligonucleotide microarray probes were designed for 96.7% of the 3163 C. thermocellum ATCC 27405 candidate protein-encoding genes and then a partial-genome microarray containing 70 C. thermocellum specific probes was constructed and evaluated. We detected a signal-to-noise ratio of three with as little as 1.0 ng of genomic DNA and only low signals from negative control probes (nonclostridial DNA), indicating the probes were sensitive and specific. In order to further test the specificity of the array we amplified and hybridized 10 C. thermocellum polymerase chain reaction products that represented different genes and found gene specific hybridization in each case. We also constructed a whole-genome microarray and prepared total cellular RNA from the same point in early-logarithmic growth phase from two technical replicates during cellobiose fermentation. The reliability of the microarray data was assessed by cohybridization of labeled complementary DNA from the cellobiose fermentation samples and the pattern of hybridization revealed a linear correlation. These results taken together suggest that our oligonucleotide probe set can be used for sensitive and specific C. thermocellum transcriptomic studies in the future.

  2. Predicting oligonucleotide-directed mutagenesis failures in protein engineering

    PubMed Central

    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. PMID:15585664

  3. Target mRNA inhibition by oligonucleotide drugs in man

    PubMed Central

    Lightfoot, Helen L.; Hall, Jonathan

    2012-01-01

    Oligonucleotide delivery in vivo is commonly seen as the principal hurdle to the successful development of oligonucleotide drugs. In an analysis of 26 oligonucleotide drugs recently evaluated in late-stage clinical trials we found that to date at least half have demonstrated suppression of the target mRNA and/or protein levels in the relevant cell types in man, including those present in liver, muscle, bone marrow, lung, blood and solid tumors. Overall, this strongly implies that the drugs are being delivered to the appropriate disease tissues. Strikingly we also found that the majority of the drug targets of the oligonucleotides lie outside of the drugable genome and represent new mechanisms of action not previously investigated in a clinical setting. Despite the high risk of failure of novel mechanisms of action in the clinic, a subset of the targets has been validated by the drugs. While not wishing to downplay the technical challenges of oligonucleotide delivery in vivo, here we demonstrate that target selection and validation are of equal importance for the success of this field. PMID:22989709

  4. Fast large scale oligonucleotide selection using the longest common factor approach.

    PubMed

    Rahmann, Sven

    2003-07-01

    We present a fast method that selects oligonucleotide probes (such as DNA 25-mers) for microarray experiments on a truly large scale. For example, reliable oligos for human genes can be found within four days, a speedup of one to two orders of magnitude compared to previous approaches. This speed is attained by using the longest common substring as a specificity measure for candidate oligos. We present a space- and time-efficient algorithm, based on a suffix array with additional information, to compute matching statistics (lengths of longest matches) between all candidate oligos and all remaining sequences. With the matching statistics available, we show how to incorporate constraints such as oligo length, melting temperature, and self-complementarity into the selection process at a postprocessing stage. As a result, we can now design custom oligos for any sequenced genome, just as the technology for on-site chip synthesis is becoming increasingly mature.

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

    PubMed Central

    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

  6. Safety of antisense oligonucleotide and siRNA-based therapeutics.

    PubMed

    Chi, Xuan; Gatti, Philip; Papoian, Thomas

    2017-01-31

    Oligonucleotide-based therapy is an active area of drug development designed to treat a variety of gene-specific diseases. Two of the more promising platforms are the antisense oligonucleotides (ASOs) and short interfering RNAs (siRNAs), both of which are often directed against similar targets. In light of recent reports on clinical trials of severe thrombocytopenia with two different ASO drugs and increased peripheral neuropathy with an siRNA drug, we compared and contrasted the specific safety characteristics of these two classes of oligonucleotide therapeutic. The objectives were to assess factors that could contribute to the specific toxicities observed with these two classes of promising drugs, and get a better understanding of the potential mechanism(s) responsible for these rare, but serious, adverse events.

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

    PubMed

    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.

  8. MIMAS: an innovative tool for network-based high density oligonucleotide microarray data management and annotation

    PubMed Central

    Hermida, Leandro; Schaad, Olivier; Demougin, Philippe; Descombes, Patrick; Primig, Michael

    2006-01-01

    Background The high-density oligonucleotide microarray (GeneChip) is an important tool for molecular biological research aiming at large-scale detection of small nucleotide polymorphisms in DNA and genome-wide analysis of mRNA concentrations. Local array data management solutions are instrumental for efficient processing of the results and for subsequent uploading of data and annotations to a global certified data repository at the EBI (ArrayExpress) or the NCBI (GeneOmnibus). Description To facilitate and accelerate annotation of high-throughput expression profiling experiments, the Microarray Information Management and Annotation System (MIMAS) was developed. The system is fully compliant with the Minimal Information About a Microarray Experiment (MIAME) convention. MIMAS provides life scientists with a highly flexible and focused GeneChip data storage and annotation platform essential for subsequent analysis and interpretation of experimental results with clustering and mining tools. The system software can be downloaded for academic use upon request. Conclusion MIMAS implements a novel concept for nation-wide GeneChip data management whereby a network of facilities is centered on one data node directly connected to the European certified public microarray data repository located at the EBI. The solution proposed may serve as a prototype approach to array data management between research institutes organized in a consortium. PMID:16597336

  9. Development of a short oligonucleotide microarray for the detection and identification of multiple potyviruses.

    PubMed

    Wei, Ting; Pearson, Michael N; Blohm, Dietmar; Nölte, Manfred; Armstrong, Karen

    2009-12-01

    The genus Potyvirus is the largest and one of the most economically important virus genera infecting plants. However, current diagnostic techniques are limited in their ability to identify multiple potyvirus infections. An assay that can identify multiple potyviruses simultaneously, with good specificity and sensitivity, is therefore highly desirable. To determine the feasibility of simultaneous detection of multiple potyviruses a 25-mer oligonucleotide microarray was developed targeting four distinct potyviruses: Dasheen mosaic virus (DsMV), Leek yellow stripe virus (LYSV), Potato virus Y (PVY) and Zucchini yellow mosaic virus (ZYMV). A total of 85 probes including 33 perfect-match and 52 mismatch probes were designed from conserved and variable sequence regions of the nuclear inclusion b (NIb) gene, RNA-dependent RNA polymerase (RdRp) gene, coat protein (CP) gene and the 3' untranslated region (UTR), representing the four targeted potyviruses at both species and strain levels. Each probe was synthesized with spacers of either 6 or 12 poly-cytosine or poly-thymine at the 5' terminus. The array showed high specificity when tested with nineteen different geographically diverse potyvirus isolates of the four target species, four distinct but closely related potyviruses, and four healthy plant species. The approaches and protocols developed in this study form a useful basis for developing other potyviruses arrays and the results also provide useful insights into generic issues for the development of arrays for detecting other pathogens.

  10. Manipulation of oligonucleotides immobilized on solid supports - DNA computations on surfaces

    NASA Astrophysics Data System (ADS)

    Liu, Qinghua

    The manipulation of DNA oligonucleotides immobilized on various solid supports has been studied intensively, especially in the area of surface hybridization. Recently, surface-based biotechnology has been applied to the area of molecular computing. These surface-based methods have advantages with regard to ease of handling, facile purification, and less interference when compared to solution methodologies. This dissertation describes the investigation of molecular approaches to DNA computing. The feasibility of encoding a bit (0 or 1) of information for DNA-based computations at the single nucleotide level was studied, particularly with regard to the efficiency and specificity of hybridization discrimination. Both gold and glass surfaces, with addressed arrays of 32 oligonucleotides, were employed with similar hybridization results. Although single-base discrimination may be achieved in the system, it is at the cost of a severe decrease in the efficiency of hybridization to perfectly matched sequences. This compromises the utility of single nucleotide encoding for DNA computing applications in the absence of some additional mechanism for increasing specificity. Several methods are suggested including a multiple-base encoding strategy. The multiple-base encoding strategy was employed to develop a prototype DNA computer. The approach was demonstrated by solving a small example of the Satisfiability (SAT) problem, an NP-complete problem in Boolean logic. 16 distinct DNA oligonucleotides, encoding all candidate solutions to the 4-variable-4-clause-3-SAT problem, were immobilized on a gold surface in the non-addressed format. Four cycles of MARK (hybridization), DESTROY (enzymatic destruction) and UNMARK (denaturation) were performed, which identified and eliminated members of the set which were not solutions to the problem. Determination of the answer was accomplished in the READOUT (sequence identification) operation by PCR amplification of the remaining molecules and

  11. Magnetic arrays

    SciTech Connect

    Trumper, David L.; Kim, Won-jong; Williams, Mark E.

    1997-05-20

    Electromagnet arrays which can provide selected field patterns in either two or three dimensions, and in particular, which can provide single-sided field patterns in two or three dimensions. These features are achieved by providing arrays which have current densities that vary in the windings both parallel to the array and in the direction of array thickness.

  12. Magnetic arrays

    DOEpatents

    Trumper, D.L.; Kim, W.; Williams, M.E.

    1997-05-20

    Electromagnet arrays are disclosed which can provide selected field patterns in either two or three dimensions, and in particular, which can provide single-sided field patterns in two or three dimensions. These features are achieved by providing arrays which have current densities that vary in the windings both parallel to the array and in the direction of array thickness. 12 figs.

  13. Oligonucleotide labelling using a fluorogenic "click" reaction with a hemicarboxonium salt.

    PubMed

    Maether, Marie-Pierre; Lapin, Kristie; Muntean, Andreea; Payrastre, Corinne; Escudier, Jean-Marc

    2013-10-17

    Two fluorescent streptocyanine labelled oligonucleotides have been synthesized by a simple "click" reaction between a non-fluorescent hemicarboxonium salt and aminoalkyl functionalized thymidines within the oligonucleotide and their spectrophotometric properties have been studied.

  14. Validation of the Swine Protein-Annotated Oligonucleotide Microarray

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The specificity and utility of the Swine Protein-Annotated Oligonucleotide Microarray, or Pigoligoarray (www.pigoligoarray.org), has been evaluated by profiling the expression of transcripts from four porcine tissues. Tools for comparative analyses of expression on the Pigoligoarray were developed i...

  15. Gene expression profiling in peanut using oligonucleotide microarrays

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Transcriptome expression analysis in peanut to date has been limited to a relatively small set of genes and only recently have a moderately significant number of ESTs been released into the public domain. Utilization of these ESTs for the oligonucleotide microarrays provides a means to investigate l...

  16. Oligonucleotide-directed mutagenesis for precision gene editing.

    PubMed

    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.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

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

    PubMed Central

    Belov, Sergey S; Tarasenko, Yulia V; Silnikov, Vladimir N

    2014-01-01

    Summary An efficient solid-phase-supported peptide synthesis (SPPS) of morpholinoglycine oligonucleotide (MorGly) mimics has been developed. The proposed strategy includes a novel specially designed labile linker group containing the oxalyl residue and the 2-aminomethylmorpholino nucleoside analogues as first subunits. PMID:24991266

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

    PubMed Central

    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

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

    PubMed

    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.

  1. Ratiometric detection of oligonucleotide stoichiometry on multifunctional gold nanoparticles by whispering gallery mode biosensing.

    PubMed

    Wu, F C; Wu, Y; Niu, Z; Vollmer, F

    2015-05-07

    A label-free method is developed to ratiometrically determine the stoichiometry of oligonucleotides attached to the surface of gold nanoparticle (GNP) by whispering gallery mode biosensing. Utilizing this scheme, it is furthermore shown that the stoichiometric ratio of GNP attached oligonucleotide species can be controlled by varying the concentration ratio of thiolated oligonucleotides that are used to modify the GNP.

  2. Synthesis and anti-HIV activity of thiocholesteryl-coupled phosphodiester antisense oligonucleotides incorporated into immunoliposomes.

    PubMed

    Zelphati, O; Wagner, E; Leserman, L

    1994-09-01

    Encapsulation of oligonucleotides in antibody-targeted liposomes (immunoliposomes) which bind to target cells permits intracellular delivery of the oligonucleotides. This approach circumvents problems of extracellular degradation by nucleases and poor membrane permeability which free phosphodiester oligonucleotides are subject to, but leaves unresolved the inefficiency of encapsulation of oligonucleotides in liposomes. We have coupled oligonucleotides to cholesterol via a reversible disulfide bond. This modification of oligonucleotides improved their association with immunoliposomes by a factor of about 10 in comparison to unmodified oligonucleotides. The presence of cholesteryl-modified oligonucleotides incorporated in the bilayer of liposomes did not interfere with the coupling of the targeting protein to the liposome surface. Free or cholesterol coupled oligonucleotides associated with liposomes and directed against the tat gene of HIV-1 were tested for inhibition of HIV-1 proliferation in acutely infected cells. We demonstrate that the cholesteryl-modified as well as unmodified oligonucleotides acquire the target specificity of the antibody on the liposome. Their antiviral activity when delivered into cells is sequence-specific. The activity of these modified or unmodified oligonucleotides to inhibit the replication of HIV was the same on an equimolar basis (EC50 around 0.1 microM). Cholesterol coupled oligonucleotides thus offer increased liposome association without loss of antiviral activity.

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

    PubMed

    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.

  4. Pentopyranosyl Oligonucleotide Systems. Part 11: Systems with Shortened Backbones: D)-beta-Ribopyranosyl-(4 yields 3 )- and (L)-alpha - Lyxopyranosyl-(4 yields 3 )-oligonucleotides

    NASA Technical Reports Server (NTRS)

    Wippo, Harald; Reck, Folkert; Kudick, Rene; Ramaseshan, Mahesh; Ceulemans, Griet; Bolli, Martin; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert

    2001-01-01

    The (L)-a-lyxopyranosyl-(4'yields 3')-oligonucleotide system-a member of a pentopyranosyl oligonucleotide family containing a shortened backbone-is capable of cooperative base-pairing and of cross-pairing with DNA and RNA. In contrast, corresponding (D)-beta-ribopyransoyl-(4' yields 3')-oligonucleotides do not show base-pairing under similar conditions. We conclude that oligonucleotide systems can violate the six-bonds-per-backbone-unit rule by having five bonds instead, if their vicinally bound phosphodiester bridges can assume an antiperiplanar conformation. An additional structural feature that seems relevant to the cross-pairing capability of the (L)-a-lyxopyranosyl-(4' yields 3')-oligonucleotide system is its (small) backbone/basepair axes inclination. An inclination which is similar to that in B-DNA seems to be a prerequisite for an oligonucleotide system s capability to cross-pair with DNA.

  5. Kokkos Array

    SciTech Connect

    Edwards Daniel Sunderland, Harold Carter

    2012-09-12

    The Kokkos Array library implements shared-memory array data structures and parallel task dispatch interfaces for data-parallel computational kernels that are performance-portable to multicore-CPU and manycore-accelerator (e.g., GPGPU) devices.

  6. Nanocylinder arrays

    DOEpatents

    Tuominen, Mark; Schotter, Joerg; Thurn-Albrecht, Thomas; Russell, Thomas P.

    2007-03-13

    Pathways to rapid and reliable fabrication of nanocylinder arrays are provided. Simple methods are described for the production of well-ordered arrays of nanopores, nanowires, and other materials. This is accomplished by orienting copolymer films and removing a component from the film to produce nanopores, that in turn, can be filled with materials to produce the arrays. The resulting arrays can be used to produce nanoscale media, devices, and systems.

  7. Nanocylinder arrays

    DOEpatents

    Tuominen, Mark; Schotter, Joerg; Thurn-Albrecht, Thomas; Russell, Thomas P.

    2009-08-11

    Pathways to rapid and reliable fabrication of nanocylinder arrays are provided. Simple methods are described for the production of well-ordered arrays of nanopores, nanowires, and other materials. This is accomplished by orienting copolymer films and removing a component from the film to produce nanopores, that in turn, can be filled with materials to produce the arrays. The resulting arrays can be used to produce nanoscale media, devices, and systems.

  8. Oligonucleotide Immobilization and Hybridization on Aldehyde-Functionalized Poly(2-hydroxyethyl methacrylate) Brushes.

    PubMed

    Bilgic, Tugba; Klok, Harm-Anton

    2015-11-09

    DNA biosensing requires high oligonucleotide binding capacity interface chemistries that can be tuned to maximize probe presentation as well as hybridization efficiency. This contribution investigates the feasibility of aldehyde-functionalized poly(2-hydroxyethyl methacrylate) (PHEMA) brush-based interfaces for oligonucleotide binding and hybridization. These polymer brushes, which allow covalent immobilization of oligonucleotides, are prepared by surface-initiated atom transfer radical polymerization (SI-ATRP) of HEMA followed by a postpolymerization oxidation step to generate side chain aldehyde groups. A series of polymer brushes covering a range of film thicknesses and grafting densities was investigated with regard to their oligonucleotide binding capacity as well as their ability to support oligonucleotide hybridization. Densely grafted brushes were found to have probe oligonucleotide binding capacities of up to ∼30 pmol/cm(2). Increasing the thickness of these densely grafted brush films, however, resulted in a decrease in the oligonucleotide binding capacity. Less densely grafted brushes possess binding capacities of ∼10 pmol/cm(2), which did not significantly depend on film thickness. The oligonucleotide hybridization efficiencies, however, were highest (93%) on those brushes that present the lowest surface concentration of the probe oligonucleotide. These results highlight the importance of optimizing the probe oligonucleotide surface concentration and binding interface chemistry. The versatility and tunability of the PHEMA-based brushes presented herein makes these films a very attractive platform for the immobilization and hybridization of oligonucleotides.

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

    PubMed Central

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

    PubMed

    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.

  11. Inhibition of HTLV-III by exogenous oligonucleotides

    SciTech Connect

    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.

  12. Palladium-catalyzed modification of unprotected nucleosides, nucleotides, and oligonucleotides.

    PubMed

    Shaughnessy, Kevin H

    2015-05-22

    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.

  13. Detection of high-resolution Raman spectra in short oligonucleotides

    NASA Astrophysics Data System (ADS)

    Bairamov, F. B.; Poloskin, E. D.; Chernev, A. L.; Toporov, V. V.; Dubina, M. V.; Lahderanta, E.; Lipsanen, H.; Bairamov, B. Kh.

    2014-06-01

    High-resolution spectra of single-chain short oligonucleotides d(20G, 20T), where d is a deoxyribonucleoside, G is guanine, and T is thymine, have been obtained by the highly sensitive nonresonant Raman scattering method of biomacromolecules. In addition to their own multifunctional significance, short oligonucleotides attract interest as ideal model objects for revealing poorly studied peculiarities of tertiary and quaternary structures of DNA. The detection of narrow spectral lines has allowed determining the characteristic time scale and makes it possible to study the dynamics of fast relaxation processes of vibrational motions of atoms in biomacromolecules. It has been found that the FWHM of the narrowest 1355.4 cm-1 spectral line attributed to the vibrations of the dT methyl group is 14.6 cm-1. The corresponding lifetime is 0.38 ps.

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

    PubMed

    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+).

  15. P-chiral oligonucleotides in biological recognition processes.

    PubMed

    Guga, Piotr

    2007-01-01

    Internucleotide phosphodiester linkages in non-modified oligonucleotides are quickly degraded by nucleolytic enzymes present in the cells and this feature practically eliminates natural DNA and RNA molecules from medical applications and from many structural and mechanistic studies. P-chiral oligonucleotide analogs, in which one of the non-bridging phosphate oxygen atoms is substituted with another heteroatom (e.g. S, Se) or a chemical group (e.g. CH3, BH3(-)), have significantly greater nuclease resistance and also offer important possibilities for detailed studies of interactions with other biomolecules at the molecular level. Notably, these substitutions do not disrupt hydrogen bonding between nucleobases and affect the overall geometry of the oligomers to only low or moderate extent, although important changes of hydration patterns and changes of interactions with metal ions are observed. Such the probes, including isotopomeric species labeled with a heavy oxygen isotope, possessing phosphorus atoms of selected absolute configurations, have been used for elucidation of the mode of action of many enzymes (nucleases, transferases, kinases), ribozymes and DNA-zymes, as well as for investigations on thermodynamic stability of nucleic acids complexes (duplexes, triplexes, i-motif) and for studies on a mechanism of conformational changes of B-Z type. They are also useful tools for analysis of interactions of the phosphoryl oxygen atoms in natural precursors with functional groups of proteins. The synthetic routes to stereodefined forms of selected types of P-chiral oligonucleotides are presented, as well as recently developed methods for their configurational analysis at micromolar concentration. Selected examples of application of diastereomerically pure P-chiral oligonucleotides for structural, biochemical and biological experiments are discussed.

  16. Induction of Radiosensitization by Antisense Oligonucleotide Gene Therapy

    DTIC Science & Technology

    2002-07-01

    Miraglia L and Strobl JS: Sensitization of breast cancer cells to ionizing radiation by protein kinase C inhibition. Proc. of the 9 0 ,h American Assoc...sensitizes human tumor cells to ionizing radiation . Radiat Res 129:345-350. O’Brian C, Vogel VG, Singletary SE and Ward NE (1989) Elevated protein...Antisense Oligonucleotides, Ionizing Radiation , Breast Cancer, Abbreviations: IR, ionizing radiation ; PKC, protein kinase C; MCF-7, Michigan Cancer

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

    NASA Astrophysics Data System (ADS)

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

    2015-02-01

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

  18. Serotyping of Human Group A Rotavirus with Oligonucleotide Probes

    DTIC Science & Technology

    1990-01-01

    Cold Spring Harbor , other oligonucleotides, HuG8Ac and HuG9Ac...HuG8Ac (5’ NY: Cold Spring Harbor Laboratory, 1988;5l1-159 CGA ACT ATC TUC TAT CTC TGT CTC T 3’) was based 9. Bastardo JW, McKimm-Bresckin JL, Sonza...Coulson BS, Unicomb LE, Pitson GA, Bishop RE Simple and specific manual. Cold Spring Harbor , NY Cold Spring Harbor Laboratory. enzyme

  19. The Design of Oligonucleotides Which Attack Specific Gene Targets

    DTIC Science & Technology

    1989-12-08

    identify by block number) FIELD GROUP SUB-GROUP ’" DNA Recognition; 06 03 Triplet helix formation, <r: " 19 ABSTRACT (Continue on reverse if necessary and...Such local triplet bonding schemes give rise to H bonding between the triplex forming oligonucleotide and the purine of the underlying Watson Crick ...identify by block number) During the first year of Navy support, we have refined our understanding of triple helix formation and in the process, have

  20. Oligonucleotide Frequencies of Barcoding Loci Can Discriminate Species across Kingdoms

    PubMed Central

    Shukla, Virendra; Tuli, Rakesh

    2010-01-01

    Background DNA barcoding refers to the use of short DNA sequences for rapid identification of species. Genetic distance or character attributes of a particular barcode locus discriminate the species. We report an efficient approach to analyze short sequence data for discrimination between species. Methodology and Principal Findings A new approach, Oligonucleotide Frequency Range (OFR) of barcode loci for species discrimination is proposed. OFR of the loci that discriminates between species was characteristic of a species, i.e., the maxima and minima within a species did not overlap with that of other species. We compared the species resolution ability of different barcode loci using p-distance, Euclidean distance of oligonucleotide frequencies, nucleotide-character based approach and OFR method. The species resolution by OFR was either higher or comparable to the other methods. A short fragment of 126 bp of internal transcribed spacer region in ribosomal RNA gene was sufficient to discriminate a majority of the species using OFR. Conclusions/Significance Oligonucleotide frequency range of a barcode locus can discriminate between species. Ability to discriminate species using very short DNA fragments may have wider applications in forensic and conservation studies. PMID:20808837

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

    PubMed

    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.

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

    PubMed

    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.

  3. Microarray oligonucleotide probe designer (MOPeD): A web service.

    PubMed

    Patel, Viren C; Mondal, Kajari; Shetty, Amol Carl; Horner, Vanessa L; Bedoyan, Jirair K; Martin, Donna; Caspary, Tamara; Cutler, David J; Zwick, Michael E

    2010-11-01

    Methods of genomic selection that combine high-density oligonucleotide microarrays with next-generation DNA sequencing allow investigators to characterize genomic variation in selected portions of complex eukaryotic genomes. Yet choosing which specific oligonucleotides to be use can pose a major technical challenge. To address this issue, we have developed a software package called MOPeD (Microarray Oligonucleotide Probe Designer), which automates the process of designing genomic selection microarrays. This web-based software allows individual investigators to design custom genomic selection microarrays optimized for synthesis with Roche NimbleGen's maskless photolithography. Design parameters include uniqueness of the probe sequences, melting temperature, hairpin formation, and the presence of single nucleotide polymorphisms. We generated probe databases for the human, mouse, and rhesus macaque genomes and conducted experimental validation of MOPeD-designed microarrays in human samples by sequencing the human X chromosome exome, where relevant sequence metrics indicated superior performance relative to a microarray designed by the Roche NimbleGen proprietary algorithm. We also performed validation in the mouse to identify known mutations contained within a 487-kb region from mouse chromosome 16, the mouse chromosome 16 exome (1.7 Mb), and the mouse chromosome 12 exome (3.3 Mb). Our results suggest that the open source MOPeD software package and website (http://moped.genetics.emory.edu/) will make a valuable resource for investigators in their sequence-based studies of complex eukaryotic genomes.

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

    NASA Astrophysics Data System (ADS)

    Moreno, Pedro; Pêgo, Ana

    2014-10-01

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

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

    Katebi, Samira; Esmaeili, Abolghasem; Ghaedi, Kamran

    2016-03-01

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

  7. Quantitative, multiplexed detection of Salmonella and other pathogens by Luminex xMAP suspension array.

    PubMed

    Dunbar, Sherry A; Jacobson, James W

    2007-01-01

    We describe a suspension array hybridization assay for rapid detection and identification of Salmonella and other bacterial pathogens using Luminex xMAP technology. The Luminex xMAP system allows simultaneous detection of up to 100 different targets in a single multiplexed reaction. Included in the method are the procedures for (1) design of species-specific oligonucleotide capture probes and PCR amplification primers, (2) coupling oligonucleotide capture probes to carboxylated microspheres, (3) hybridization of coupled microspheres to oligonucleotide targets, (4) production of targets from DNA samples by PCR amplification, and (5) detection of PCR-amplified targets by direct hybridization to probe-coupled microspheres. The Luminex xMAP suspension array hybridization assay is rapid, requires few sample manipulations, and provides adequate sensitivity and specificity to detect and differentiate Salmonella and nine other test organisms through direct detection of species-specific DNA sequences.

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

    SciTech Connect

    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

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

    PubMed

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2015-11-01

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

  11. Polyamine-oligonucleotide conjugates: a promising direction for nucleic acid tools and therapeutics.

    PubMed

    Menzi, Mirjam; Lightfoot, Helen L; Hall, Jonathan

    2015-01-01

    Chemical modification and/or the conjugation of small functional molecules to oligonucleotides have significantly improved their biological and biophysical properties, addressing issues such as poor cell penetration, stability to nucleases and low affinity for their targets. Here, the authors review the literature reporting on the biophysical, biochemical and biological properties of one particular class of modification - polyamine-oligonucleotide conjugates. Naturally derived and synthetic polyamines have been grafted onto a variety of oligonucleotide formats, including antisense oligonucleotides and siRNAs. In many cases this has had beneficial effects on their properties such as target hybridization, nuclease resistance, cellular uptake and activity. Polyamine-oligonucleotide conjugation, therefore, represents a promising direction for the further development of oligonucleotide-based therapeutics and tools.

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

    NASA Astrophysics Data System (ADS)

    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.

  13. Nanoparticle-bridge assay for amplification-free electrical detection of oligonucleotides

    NASA Astrophysics Data System (ADS)

    Teimouri, Manouchehr

    The aim of this research is to investigate a highly sensitive, fast, inexpensive, and field-applicable amplification-free nanoparticle-based oligonucleotide detection method which does not rely on any enzymatic or signal amplification process. In this approach, target oligonucleotide strands are detected through the formation of nanoparticle satellites which make an electrical path between two electrodes. This method enables an extremely sensitive oligonucleotide detection because even a few oligonucleotide strands can form a single nanoparticle satellite which can solely generates an electrical output signal. Results showed that this oligonucleotide detection method can detect oligonucleotide single strands at concentrations as low as 50 femtomolar without any amplification process. This detection method can be implemented in many fields such as biodefense, food safety, clinical research, and forensics.

  14. Construction of a versatile SNP array for pyramiding useful genes of rice.

    PubMed

    Kurokawa, Yusuke; Noda, Tomonori; Yamagata, Yoshiyuki; Angeles-Shim, Rosalyn; Sunohara, Hidehiko; Uehara, Kanako; Furuta, Tomoyuki; Nagai, Keisuke; Jena, Kshirod Kumar; Yasui, Hideshi; Yoshimura, Atsushi; Ashikari, Motoyuki; Doi, Kazuyuki

    2016-01-01

    DNA marker-assisted selection (MAS) has become an indispensable component of breeding. Single nucleotide polymorphisms (SNP) are the most frequent polymorphism in the rice genome. However, SNP markers are not readily employed in MAS because of limitations in genotyping platforms. Here the authors report a Golden Gate SNP array that targets specific genes controlling yield-related traits and biotic stress resistance in rice. As a first step, the SNP genotypes were surveyed in 31 parental varieties using the Affymetrix Rice 44K SNP microarray. The haplotype information for 16 target genes was then converted to the Golden Gate platform with 143-plex markers. Haplotypes for the 14 useful allele are unique and can discriminate among all other varieties. The genotyping consistency between the Affymetrix microarray and the Golden Gate array was 92.8%, and the accuracy of the Golden Gate array was confirmed in 3 F2 segregating populations. The concept of the haplotype-based selection by using the constructed SNP array was proofed.

  15. Array2BIO: A Comprehensive Suite of Utilities for the Analysis of Microarray Data

    SciTech Connect

    Loots, G G; Chain, P G; Mabery, S; Rasley, A; Garcia, E; Ovcharenko, I

    2006-02-13

    We have developed an integrative and automated toolkit for the analysis of Affymetrix microarray data, named Array2BIO. It identifies groups of coexpressed genes using two complementary approaches--comparative analysis of signal versus control microarrays and clustering analysis of gene expression across different conditions. The identified genes are assigned to functional categories based on the Gene Ontology classification, and a detection of corresponding KEGG protein interaction pathways. Array2BIO reliably handles low-expressor genes and provides a set of statistical methods to quantify the odds of observations, including the Benjamini-Hochberg and Bonferroni multiple testing corrections. Automated interface with the ECR Browser provides evolutionary conservation analysis of identified gene loci while the interconnection with Creme allows high-throughput analysis of human promoter regions and prediction of gene regulatory elements that underlie the observed expression patterns. Array2BIO is publicly available at http://array2bio.dcode.org.

  16. Development of an oligonucleotide-based DNA microarray for transcriptional analysis of Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) genes.

    PubMed

    Yang, Dan-Hui; Barari, Mehrnoosh; Arif, Basil M; Krell, Peter J

    2007-08-01

    A modified oligonucleotide-based two-channel DNA microarray was developed for characterization of temporal expression profiles of select Choristoneura fumiferana nucleopolyhedrovirus (CfMNPV) ORFs including its 7 unique ORFs. The microarray chip contained oligonucleotide probes for 23 CfMNPV ORFs and their complements as well as five host genes. Total RNA was isolated at different times post infection from Cf203 insect cells infected with CfMNPV. The cDNA was synthesized, fluorescent labelled with Cy3, and co-hybridized to the microarray chips along with Cy5-labelled viral genomic DNA, which served as equimolar reference standards for each probe. Transcription of the 7 CfMNPV unique ORFs was detected using DNA microarray analysis and their temporal expression profiles suggest that they are functional genes. The expression levels of three host genes varied throughout virus infection and therefore were unsuitable for normalization between microarrays. The DNA microarray results were compared to quantitative RT-PCR (qRT-PCR). Transcription of the non-coding (antisense) strands of some of the CfMNPV select genes including the polyhedrin gene, was also detected by array analysis and confirmed by qRT-PCR. The polyhedrin antisense transcript, based on long-range RT-PCR analysis, appeared to be a read-through product of an adjacent ORF in the same orientation as the antisense transcript.

  17. New strategies for cyclization and bicyclization of oligonucleotides by click chemistry assisted by microwaves.

    PubMed

    Lietard, Jory; Meyer, Albert; Vasseur, Jean-Jacques; Morvan, François

    2008-01-04

    The synthesis of cyclic, branched, and bicyclic oligonucleotides was performed by copper-catalyzed azide-alkyne cycloaddition assisted by microwaves in solution and on solid support. For that purpose, new phosphoramidite building blocks and new solid supports were designed to introduce alkyne and bromo functions into the same oligonucleotide by solid-phase synthesis on a DNA synthesizer. The bromine atom was then substituted by sodium azide to yield azide oligonucleotides. Cyclizations were found to be more efficient in solution than on solid support. This method allowed the efficient preparation of cyclic (6- to 20-mers), branched (with one or two dangling sequences), and bicyclic (2 x 10-mers) oligonucleotides.

  18. Properties of amphiphilic oligonucleotide films at the air/water interface and after film transfer.

    PubMed

    Keller, R; Kwak, M; de Vries, J W; Sawaryn, C; Wang, J; Anaya, M; Müllen, K; Butt, H-J; Herrmann, A; Berger, R

    2013-11-01

    The self-assembly of amphiphilic hybrid materials containing an oligonucleotide sequence at the air/water interface was investigated by means of pressure-molecular area (Π-A) isotherms. In addition, films were transferred onto solid substrates and imaged using scanning force microscopy. We used oligonucleotide molecules with lipid tails, which consisted of a single stranded oligonucleotide 11 mer containing two hydrophobically modified 5-(dodec-1-ynyl)uracil nucleobases (dU11) at the 5'-end of the oligonucleotide sequence. The air/water interface was used as confinement for the self-assembling process of dU11. Scanning force microscopy of films transferred via Langmuir-Blodgett technique revealed mono-, bi- (Π ≥ 2 mN/m) and multilayer formation (Π ≥ 30 mN/m). The first layer was 1.6 ± 0.1 nm thick. It was oriented with the hydrophilic oligonucleotide moiety facing the hydrophilic substrate while the hydrophobic alkyl chains faced air. In the second layer the oligonucleotide moiety was found to face the air. The second layer was found to cover up to 95% of the sample area. Our measurements indicated that the rearrangement of the molecules into bi- and multiple bilayers happened already at the air/water interface. Similar results were obtained with a second type of oligonucleotide amphiphile, an oligonucleotide block copolymer, which was composed of an oligonucleotide 11 mer covalently attached at the terminus to polypropyleneoxide (PPO).

  19. Diels-Alder cycloadditions in water for the straightforward preparation of peptide–oligonucleotide conjugates

    PubMed Central

    Marchán, Vicente; Ortega, Samuel; Pulido, Daniel; Pedroso, Enrique; Grandas, Anna

    2006-01-01

    The Diels-Alder reaction between diene-modified oligonucleotides and maleimide-derivatized peptides afforded peptide–oligonucleotide conjugates with high purity and yield. Synthesis of the reagents was easily accomplished by on-column derivatization of the corresponding peptides and oligonucleotides. The cycloaddition reaction was carried out in mild conditions, in aqueous solution at 37°C. The speed of the reaction was found to vary depending on the size of the reagents, but it can be completed in 8–10 h by reacting the diene-oligonucleotide with a small excess of maleimide-peptide. PMID:16478710

  20. Avian oncogenic virus differential diagnosis in chickens using oligonucleotide microarray.

    PubMed

    Wang, Lih-Chiann; Huang, Dean; Pu, Chang-En; Wang, Ching-Ho

    2014-12-15

    Avian oncogenic viruses include the avian leukosis virus (ALV), reticuloendotheliosis virus (REV) and Marek's disease virus (MDV). Multiple oncogenic viral infections are frequently seen, with even Marek's disease vaccines reported to be contaminated with ALV and REV. The gross lesions caused by avian oncogenic viruses often overlap, making differentiation diagnosis based on histopathology difficult. The objective of this study is to develop a rapid approach to simultaneously differentiate, subgroup and pathotype the avian oncogenic viruses. The oligonucleotide microarray was employed in this study. Particular DNA sequences were recognized using specific hybridization between the DNA target and probe on the microarray, followed with colorimetric development through enzyme reaction. With 10 designed probes, ALV-A, ALV-E, ALV-J, REV, MDV pathogenic and vaccine strains were clearly discriminated on the microarray with the naked eyes. The detection limit was 27 copy numbers, which was 10-100 times lower than multiplex PCR. Of 102 field samples screened using the oligonucleotide microarray, 32 samples were positive for ALV-E, 17 samples were positive for ALV-J, 6 samples were positive for REV, 4 samples were positive for MDV, 7 samples were positive for both ALV-A and ALV-E, 5 samples were positive for ALV-A, ALV-E and ALV-J, one sample was positive for both ALV-J and MDV, and 3 samples were positive for both REV and MDV. The oligonucleotide microarray, an easy-to-use, high-specificity, high-sensitivity and extendable assay, presents a potent technique for rapid differential diagnosis of avian oncogenic viruses and the detection of multiple avian oncogenic viral infections under field conditions.

  1. In vivo site-directed mutagenesis using oligonucleotides.

    PubMed

    Storici, F; Lewis, L K; Resnick, M A

    2001-08-01

    Functional characterization of the genes of higher eukaryotes has been aided by their expression in model organisms and by analyzing site-specific changes in homologous genes in model systems such as the yeast Saccharomyces cerevisiae. Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together with subcloning. PCR-based procedures that do not involve cloning are inefficient or require multistep reactions that increase the risk of additional mutations. An alternative approach, demonstrated in yeast, relies on transformation with an oligonucleotide, but the method is restricted to the generation of mutants with a selectable phenotype. Oligonucleotides, when combined with gap repair, have also been used to modify plasmids in yeast; however, this approach is limited by restriction-site availability. We have developed a mutagenesis approach in yeast based on transformation by unpurified oligonucleotides that allows the rapid creation of site-specific DNA mutations in vivo. A two-step, cloning-free process, referred to as delitto perfetto, generates products having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion, or even random mutations. The system provides for multiple rounds of mutation in a window up to 200 base pairs. The process is RAD52 dependent, is not constrained by the distribution of naturally occurring restriction sites, and requires minimal DNA sequencing. Because yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes such as yeast artificial chromosomes, the delitto perfetto strategy also provides an efficient way to create precise changes in mammalian or other DNA sequences.

  2. PCR amplification on microarrays of gel immobilized oligonucleotides

    DOEpatents

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

    2003-11-04

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

  3. Inhibition Of Molecular And Biological Processes Using Modified Oligonucleotides

    DOEpatents

    Kozyavkin, Sergei A.; Malykh, Andrei G.; Polouchine, Nikolai N.; Slesarev, Alexei I.

    2003-04-15

    A method of inhibiting at least one molecular process in a sample, comprising administering to the sample an oligonucleotide or polynucleotide containing at least one monomeric unit having formula (I): wherein A is an organic moiety, n is at least 1, and each X is independently selected from the group consisting of --NRCOCONu, --NHCOCR.sub.2 CR.sub.2 CONu, --NHCOCR.dbd.CRCONu, and --NHCOSSCONu, wherein each R independently represents H or a substituted or unsubstituted alkyl group, and Nu represents a nucleophile, or a salt of the compound.

  4. Oligonucleotide primers for PCR amplification of coelomate introns.

    PubMed

    Jarman, Simon N; Ward, Robert D; Elliott, Nicholas G

    2002-09-01

    Abstract Seven novel oligonucleotide primer pairs for polymerase chain reaction amplification of introns from nuclear genes in coelomates were designed and tested. Each pair bound to adjacent exons that are separated by a single intron in most coelomate species. The primer sets amplified introns in species as widely separated by the course of evolution as oysters (Mollusca: Protostoma) and salmon (Chordata: Deuterostoma). Each primer set was tested on a further 6 coelomate species and found to amplify introns in most cases. These primer sets may therefore be useful tools for developing nuclear DNA markers in diverse coelomate species for studies of population genetics, phylogenetics, or genome mapping.

  5. Oligonucleotide microarray for subtyping of influenza A viruses

    NASA Astrophysics Data System (ADS)

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

    2012-02-01

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

  6. Detection and validation of single feature polymorphisms using RNA expression data from a rice genome array

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A large number of genetic variations have been identified in rice. Such variations must in many cases control phenotypic differences in abiotic stress tolerance and other traits. A single feature polymorphism (SFP) is an oligonucleotide array-based polymorphism which can be used for identification o...

  7. Preparation of DNA and protein micro arrays on glass slides coated with an agarose film

    PubMed Central

    Afanassiev, Victor; Hanemann, Vera; Wölfl, Stefan

    2000-01-01

    A thin layered agarose film on microscope slides provides a versatile support for the preparation of arrayed molecular libraries. An activation step leading to the formation of aldehyde groups in the agarose creates reactive sites that allow covalent immobilization of molecules containing amino groups. Arrays of oligonucleotides and PCR products were prepared by tip printing. After hybridization with complementary fluorescence labeled nucleic acid probes strong fluorescence signals of sequence-specific binding to the immobilized probes were detected. The intensity of the fluorescence signals was proportional to the relative amount of immobilized oligonucleotides and to the concentration of the fluorescence labeled probe. We also used the agarose film-coated slides for the preparation of protein arrays. In combination with specific fluorescence labeled antibodies these protein arrays can be used for fluorescence linked immune assays. With this approach different protein tests can be performed in parallel in a single reaction with minimal amounts of the binding reagents. PMID:10871389

  8. SNP Arrays

    PubMed Central

    Louhelainen, Jari

    2016-01-01

    The papers published in this Special Issue “SNP arrays” (Single Nucleotide Polymorphism Arrays) focus on several perspectives associated with arrays of this type. The range of papers vary from a case report to reviews, thereby targeting wider audiences working in this field. The research focus of SNP arrays is often human cancers but this Issue expands that focus to include areas such as rare conditions, animal breeding and bioinformatics tools. Given the limited scope, the spectrum of papers is nothing short of remarkable and even from a technical point of view these papers will contribute to the field at a general level. Three of the papers published in this Special Issue focus on the use of various SNP array approaches in the analysis of three different cancer types. Two of the papers concentrate on two very different rare conditions, applying the SNP arrays slightly differently. Finally, two other papers evaluate the use of the SNP arrays in the context of genetic analysis of livestock. The findings reported in these papers help to close gaps in the current literature and also to give guidelines for future applications of SNP arrays. PMID:27792140

  9. Mapping RNase T1-resistant oligonucleotides of avian tumor virus RNAs: sarcoma-specific oligonucleotides are near the poly(A) end and oligonucleotides common to sarcoma and transformation-defective viruses are at the poly(A) end.

    PubMed Central

    Wang, L H; Duesberg, P; Beemon, K; Vogt, P K

    1975-01-01

    The large RNase T1-resistant oligonucleotides of the nondefective (nd) Rous sarcoma virus (RSV): Prague RSV of subgroup B (PR-B), PR-C and B77 of subgroup C; of their transformation-defective (td0 deletion mutants: td PR-B, td PR-C, and td B77; and of replication-defective (rd) RSV(-) were completely or partially mapped on the 30 to 40S viral RNAs. The location of a given oligonucleotide relative to the poly(A) terminus of the viral RNAs was directly deduced from the smallest size of the poly(A)-tagged RNA fragment from which it could be isolated. Identification of distinct oligonucleotides was based on their location in the electrophoretic/chromatographic fingerprint pattern and on analysis of their RNase A-resistant fragments. The following results were obtained. (i) The number of large oligonucleotides per poly(A)-tagged ffagment increased with increasing size of the fragment. This implies that the genetic map is linear and that a given RNase T1-resistant oligonucleotides has, relative to the poly(A) end, the same location on all 30 to 40S RNA subunits of a given 60 to 70S viral RNA complex, (ii) Three sarcoma-specific oligonucleotides were identified in the RNAs of Pr-B, PR-C and B77 by comparison with the RNAs of the corresponding td viruses... Images PMID:170411

  10. Direct Detection of 16S rRNA in Soil Extracts by Using Oligonucleotide Microarrays

    PubMed Central

    Small, Jack; Call, Douglas R.; Brockman, Fred J.; Straub, Timothy M.; Chandler, Darrell P.

    2001-01-01

    We report on the development and validation of a simple microarray method for the direct detection of intact 16S rRNA from unpurified soil extracts. Total RNAs from Geobacter chapellei and Desulfovibrio desulfuricans were hybridized to an oligonucleotide array consisting of universal and species-specific 16S rRNA probes. PCR-amplified products from Geobacter and Desulfovibrio were easily and specifically detected under a range of hybridization times, temperatures, and buffers. However, reproducible, specific hybridization and detection of intact rRNA could be accomplished only by using a chaperone-detector probe strategy. With this knowledge, assay conditions were developed for rRNA detection using a 2-h hybridization time at room temperature. Hybridization specificity and signal intensity were enhanced using fragmented RNA. Formamide was required in the hybridization buffer in order to achieve species-specific detection of intact rRNA. With the chaperone detection strategy, we were able to specifically hybridize and detect G. chapellei 16S rRNA directly from a total-RNA soil extract, without further purification or removal of soluble soil constituents. The detection sensitivity for G. chapellei 16S rRNA in soil extracts was at least 0.5 μg of total RNA, representing approximately 7.5 × 106 Geobacter cell equivalents of RNA. These results suggest that it is now possible to apply microarray technology to the direct detection of microorganisms in environmental samples, without using PCR. PMID:11571176

  11. Identification of Upper Respiratory Tract Pathogens Using Electrochemical Detection on an Oligonucleotide Microarray

    PubMed Central

    Lodes, Michael J.; Suciu, Dominic; Wilmoth, Jodi L.; Ross, Marty; Munro, Sandra; Dix, Kim; Bernards, Karen; Stöver, Axel G.; Quintana, Miguel; Iihoshi, Naomi; Lyon, Wanda J.; Danley, David L.; McShea, Andrew

    2007-01-01

    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, parainfluinza 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. PMID:17895966

  12. Transcription profiling of the early gravitropic response in Arabidopsis using high-density oligonucleotide probe microarrays

    NASA Technical Reports Server (NTRS)

    Moseyko, Nick; Zhu, Tong; Chang, Hur-Song; Wang, Xun; Feldman, Lewis J.

    2002-01-01

    Studies of plant tropisms, the directed growth toward or away from external stimuli such as light and gravity, began more than a century ago. Yet biochemical, physiological, and especially molecular mechanisms of plant tropic responses remain for the most part unclear. We examined expression of 8,300 genes during early stages of the gravitropic response using high-density oligonucleotide probe microarrays. Approximately 1.7% of the genes represented on the array exhibited significant expression changes within the first 30 min of gravity stimulation. Among gravity-induced genes were a number of genes previously implicated to be involved in gravitropism. However, a much larger number of the identified genes have not been previously associated with gravitropism. Because reorientation of plants may also expose plants to mechanical perturbations, we also compared the effects of a gentle mechanical perturbation on mRNA levels during the gravity response. It was found that approximately 39% of apparently gravity-regulated genes were also regulated by the mechanical perturbation caused by plant reorientation. Our study revealed the induction of complex gene expression patterns as a consequence of gravitropic reorientation and points to an interplay between the gravitropic and mechanical responses and to the extreme sensitivity of plants to even very gentle mechanical perturbations.

  13. Periostin antisense oligonucleotide prevents adhesion formation after surgery in mice.

    PubMed

    Takai, Shinji; Yoshino, Masafumi; Takao, Kazumasa; Yoshikawa, Kazunori; Jin, Denan

    2017-02-09

    To study the role of periostin in adhesion formation, the effect of periostin antisense oligonucleotide (PAO) on adhesion formation was evaluated in mice. Under anesthesia, the serous membrane of the cecum was abraded, and the adhesion score and mRNA levels of periostin and its related factors were determined after surgery. Saline, 40 mg/kg of negative sense oligonucleotide (NSO), or 40 mg/kg of PAO were injected into the abdomen after surgery, and the adhesion score and mRNA levels were evaluated 14 days later. Filmy adhesion formation was observed 1 day after surgery, and the adhesion score increased gradually to 14 days. The mRNA levels of periostin, transforming growth factor (TGF)-β, and collagen I increased gradually from 3 days to 14 days. The adhesion score of PAO was significantly lower than of saline or NSO 14 days after surgery. The mRNA levels of periostin, TGF-β, and collagen I were also significantly attenuated by treatment with PAO compared with saline or NSO. Thus, these results demonstrated that the periostin mRNA level increased in the abraded cecum, and PAO prevented adhesion formation along with attenuation of the periostin mRNA level.

  14. Oligonucleotide bias in Bacillus subtilis: general trends and taxonomic comparisons.

    PubMed Central

    Rocha, E P; Viari, A; Danchin, A

    1998-01-01

    We present a general analysis of oligonucleotide usage in the complete genome of Bacillus subtilis . Several datasets were built in order to assign various biological contexts to the biased use of words and to reveal local asymmetries in word usage that may be coupled with replication, the control of gene expression and the restriction/modification system. This analysis was complemented by cross-comparisons with the complete genomes of Escherichia coli , Haemophilus influenzae and Methanococcus jannaschii . We have observed a large number of biased oligonucleotides for words of size up to 8, throughout the datasets and species, indicating that such long strict words play an important role as biological signals. We speculate that some of them are involved in interactions with DNA and/or RNA polymerases. An extensive analysis of palindrome abundances and distributions provides the surprising result that prophage-like elements embedded in the genome exhibit a smaller avoidance of restriction sites. This may reinforce a recently proposed hypothesis of a selfish gene phenomena in the transfer of restriction/modification systems in bacteria. PMID:9611243

  15. DOTAP/UDCA vesicles: novel approach in oligonucleotide delivery.

    PubMed

    Ruozi, Barbara; Battini, Renata; Montanari, Monica; Mucci, Adele; Tosi, Giovanni; Forni, Flavio; Vandelli, Maria Angela

    2007-03-01

    The relatively hydrophilic bile acid, ursodeoxycholic acid (UDCA), was used as an additive to DOTAP cationic liposomes to evaluate the effect on the cellular uptake of an oligonucleotide. Nuclear magnetic resonance studies were applied to estimate the relative amount of incorporated UDCA into the lipidic bilayers. DOTAP or DOTAP-UDCA vesicles (MixVes; DOTAP/UDCA molar ratios 1:0.25, 1:0.5, 1:1, and 1:2) formed complexes with 5'-fluorescein conjugated 29-mer phosphorothioate oligonucleotides (PS-ODNs) and studied using gel electrophoresis. In addition, the complexes were tested after transfection to assess the cellular uptake and the localization of the oligo in a HaCaT cell line by the use of cytofluorimetric and confocal microscopic analysis. DOTAP lipid formulated in the presence of a defined amount of UDCA forms more stable, flexible, and active MixVes. In particular, the MixVes at 1:0.25 and 1:0.5 molar ratios increase and modify the cellular uptake of PS-ODNs if compared with DOTAP liposomes 3 hours after the transfection studies. Moreover, the in vitro data suggest that these new formulations are not toxic.

  16. Gas-phase Dissociation of homo-DNA Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Stucki, Silvan R.; Désiron, Camille; Nyakas, Adrien; Marti, Simon; Leumann, Christian J.; Schürch, Stefan

    2013-12-01

    Synthetic modified oligonucleotides are of interest for diagnostic and therapeutic applications, as their biological stability, pairing selectivity, and binding strength can be considerably increased by the incorporation of unnatural structural elements. Homo-DNA is an oligonucleotide homologue based on dideoxy-hexopyranosyl sugar moieties, which follows the Watson-Crick A-T and G-C base pairing system, but does not hybridize with complementary natural DNA and RNA. Homo-DNA has found application as a bioorthogonal element in templated chemistry applications. The gas-phase dissociation of homo-DNA has been investigated by ESI-MS/MS and MALDI-MS/MS, and mechanistic aspects of its gas-phase dissociation are discussed. Experiments revealed a charge state dependent preference for the loss of nucleobases, which are released either as neutrals or as anions. In contrast to DNA, nucleobase loss from homo-DNA was found to be decoupled from backbone cleavage, thus resulting in stable products. This renders an additional stage of ion activation necessary in order to generate sequence-defining fragment ions. Upon MS3 of the primary base-loss ion, homo-DNA was found to exhibit unspecific backbone dissociation resulting in a balanced distribution of all fragment ion series.

  17. Oligonucleotide Aptamers: New Tools for Targeted Cancer Therapy

    PubMed Central

    Sun, Hongguang; Zhu, Xun; Lu, Patrick Y; Rosato, Roberto R; Tan, Wen; Zu, Youli

    2014-01-01

    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. PMID:25093706

  18. In vivo delivery of transcription factors with multifunctional oligonucleotides

    NASA Astrophysics Data System (ADS)

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

    2015-07-01

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

  19. Enthalpy arrays

    PubMed Central

    Torres, Francisco E.; Kuhn, Peter; De Bruyker, Dirk; Bell, Alan G.; Wolkin, Michal V.; Peeters, Eric; Williamson, James R.; Anderson, Gregory B.; Schmitz, Gregory P.; Recht, Michael I.; Schweizer, Sandra; Scott, Lincoln G.; Ho, Jackson H.; Elrod, Scott A.; Schultz, Peter G.; Lerner, Richard A.; Bruce, Richard H.

    2004-01-01

    We report the fabrication of enthalpy arrays and their use to detect molecular interactions, including protein–ligand binding, enzymatic turnover, and mitochondrial respiration. Enthalpy arrays provide a universal assay methodology with no need for specific assay development such as fluorescent labeling or immobilization of reagents, which can adversely affect the interaction. Microscale technology enables the fabrication of 96-detector enthalpy arrays on large substrates. The reduction in scale results in large decreases in both the sample quantity and the measurement time compared with conventional microcalorimetry. We demonstrate the utility of the enthalpy arrays by showing measurements for two protein–ligand binding interactions (RNase A + cytidine 2′-monophosphate and streptavidin + biotin), phosphorylation of glucose by hexokinase, and respiration of mitochondria in the presence of 2,4-dinitrophenol uncoupler. PMID:15210951

  20. Array tomography: imaging stained arrays.

    PubMed

    Micheva, Kristina D; O'Rourke, Nancy; Busse, Brad; Smith, Stephen J

    2010-11-01

    Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time-consuming and require some practice to perfect. In this protocol, tissue arrays are imaged using conventional wide-field fluorescence microscopy. Images can be captured manually or, with the appropriate software and hardware, the process can be automated.

  1. Array tomography: production of arrays.

    PubMed

    Micheva, Kristina D; O'Rourke, Nancy; Busse, Brad; Smith, Stephen J

    2010-11-01

    Array tomography is a volumetric microscopy method based on physical serial sectioning. Ultrathin sections of a plastic-embedded tissue are cut using an ultramicrotome, bonded in an ordered array to a glass coverslip, stained as desired, and imaged. The resulting two-dimensional image tiles can then be reconstructed computationally into three-dimensional volume images for visualization and quantitative analysis. The minimal thickness of individual sections permits high-quality rapid staining and imaging, whereas the array format allows reliable and convenient section handling, staining, and automated imaging. Also, the physical stability of the arrays permits images to be acquired and registered from repeated cycles of staining, imaging, and stain elution, as well as from imaging using multiple modalities (e.g., fluorescence and electron microscopy). Array tomography makes it possible to visualize and quantify previously inaccessible features of tissue structure and molecular architecture. However, careful preparation of the tissue is essential for successful array tomography; these steps can be time consuming and require some practice to perfect. This protocol describes the sectioning of embedded tissues and the mounting of the serial arrays. The procedures require some familiarity with the techniques used for ultramicrotome sectioning for electron microscopy.

  2. Infrared Arrays

    NASA Astrophysics Data System (ADS)

    McLean, I.; Murdin, P.

    2000-11-01

    Infrared arrays are small electronic imaging devices subdivided into a grid or `array' of picture elements, or pixels, each of which is made of a material sensitive to photons (ELECTROMAGNETIC RADIATION) with wavelengths much longer than normal visible light. Typical dimensions of currently available devices are about 27-36 mm square, and formats now range from 2048×2048 pixels for the near-infra...

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

    DOEpatents

    Golova, Julia B.; Chernov, Boris K.

    2010-04-27

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

  4. In vivo generation of highly abundant sequence-specific oligonucleotides for antisense and triplex gene regulation.

    PubMed Central

    Noonberg, S B; Scott, G K; Garovoy, M R; Benz, C C; Hunt, C A

    1994-01-01

    Antisense and triplex oligonucleotides continue to demonstrate potential as mediators of gene-specific repression of protein synthesis. However, inefficient and heterogeneous cellular uptake, intracellular sequestration, and rapid intracellular and extracellular degradation represent obstacles to their eventual clinical utility. Efficient cellular delivery of targeted ribozymes can present similar problems. In this report we describe a system for circumventing these obstacles and producing large quantities of short, sequence-specific RNA oligonucleotides for use in these gene regulation strategies. The oligonucleotides are generated from a vector containing promoter, capping, and termination sequences from the human small nuclear U6 gene, surrounding a synthetic sequence incorporating the oligonucleotide of interest. In vivo, these oligonucleotides are produced constitutively and without cell type specificity in levels up to 5 x 10(6) copies per cell, reach steady-state levels of expression within 9 hours post-transfection, and are still readily detectable 7 days post-transfection. In addition, these oligonucleotides are retained in the nucleus, obtain a 5' gamma-monomethyl phosphate cap, and have an intracellular half-life of approximately one hour. This expression vector provides a novel and efficient method of intracellular delivery of antisense or triplex RNA oligonucleotides (and/or ribozymes) for gene regulation, as well as a cost-effective means of comparing the biological activity arising from a variety of different potential oligonucleotide sequences. Images PMID:8052538

  5. Ex vivo regulation of specific gene expression by nanomolar concentration of double-stranded dumbbell oligonucleotides.

    PubMed Central

    Clusel, C; Ugarte, E; Enjolras, N; Vasseur, M; Blumenfeld, M

    1993-01-01

    Inhibition of specific transcriptional regulatory proteins is a new approach to control gene expression. Transcriptional activity of DNA-binding proteins can be inhibited by the use of double-stranded (ds) oligodeoxynucleotides that compete for the binding to their specific target sequences in promoters and enhancers. As a model, we used phosphodiester dumbbell oligonucleotides containing a binding site for the liver-enriched transcription factor HNF-1 (Hepatocyte Nuclear Factor 1). Binding affinity of HNF-1 to dumbbell oligonucleotides was the same as that to ds oligonucleotides, as determined by gel retardation assays. HNF-1 dumbbells specifically inhibited in vitro transcription driven by the albumin promoter by more than 90%. HNF-1-dependent activation of a CAT reporter plasmid was specifically inhibited when the HNF-1 dumbbell oligonucleotide was added at nM concentration to transiently transfected C33 cells. On the contrary, HNF-1 ds oligonucleotides, which displayed the same activity as the dumbbell oligonucleotides in the in vitro assays, were no more effective in the ex vivo experiments. These results might reflect the increased stability of the circular dumbbell oligonucleotides towards cellular nuclease degradation, as shown in vitro with nucleolytic enzymes. Dumbbell oligonucleotides containing unmodified phosphodiester bonds may efficiently compete for binding of specific transcription factors within cells, then providing a potential therapeutic tool to control disease-causing genes. Images PMID:7688452

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

    PubMed

    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.

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

    PubMed

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

    2010-01-01

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

  8. 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.

    PubMed

    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.

  9. Optical detection and discrimination of cystic fibrosis-related genetic mutations using oligonucleotide-nanoparticle conjugates.

    PubMed

    Murphy, Deirdre; Redmond, Gareth

    2005-03-01

    Novel methods for application of oligonucleotide-gold nanoparticle conjugates to selective colorimetric detection and discrimination of cystic fibrosis (CF) related genetic mutations in model oligonucleotide systems are presented. Three-strand oligonucleotide complexes are employed, wherein two probe oligonucleotide-gold nanoparticle conjugates are linked together by a third target oligonucleotide strand bearing the CF-related mutation(s). By monitoring the temperature dependence of the optical properties of the complexes, either in solution or on silica gel plates, melting behaviors may be accurately and reproducibly compared. Using this approach, fully complementary sequences are successfully distinguished from mismatched sequences, with single base mismatch resolution, for Delta F 508, M470V, R74W and R75Q mutations.

  10. [Array technology in skin pharmacology and allergology].

    PubMed

    Baron, J M; Merk, H F; Heise, R

    2003-04-01

    Because of their variable application, microarrays are currently used in different areas of research and development, such as skin pharmacology and allergology. Microarrays are plane carriers, on whose surface a variety of known DNA-molecules and proteins were immobilised. Transcripts can be detected by cDNA- and oligonucleotid-arrays and proteins of activated genes can be discovered using antibody microarrays. Detection of allergen-specific IgE from human serum can be performed using allergen chips. Since many details of the molecular mechanism and pathogenesis of skin cancer and inflammatory skin diseases and the effect of xenobiotics on cells of the human skin are still not known, array-technologies are a powerful tool to identify novel marker genes and offer the possibility of develop new therapeutic strategies as well as prognosis- and diagnosis-systems.

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

    PubMed

    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

  12. Monitoring integrity and localization of modified single-stranded RNA oligonucleotides using ultrasensitive fluorescence methods

    PubMed Central

    Hadwiger, Philipp; Wagner, Ernst; Lamb, Don C.

    2017-01-01

    Short single-stranded oligonucleotides represent a class of promising therapeutics with diverse application areas. Antisense oligonucleotides, for example, can interfere with various processes involved in mRNA processing through complementary base pairing. Also RNA interference can be regulated by antagomirs, single-stranded siRNA and single-stranded microRNA mimics. The increased susceptibility to nucleolytic degradation of unpaired RNAs can be counteracted by chemical modification of the sugar phosphate backbone. In order to understand the dynamics of such single-stranded RNAs, we investigated their fate after exposure to cellular environment by several fluorescence spectroscopy techniques. First, we elucidated the degradation of four differently modified, dual-dye labeled short RNA oligonucleotides in HeLa cell extracts by fluorescence correlation spectroscopy, fluorescence cross-correlation spectroscopy and Förster resonance energy transfer. We observed that the integrity of the oligonucleotide sequence correlates with the extent of chemical modifications. Furthermore, the data showed that nucleolytic degradation can only be distinguished from unspecific effects like aggregation, association with cellular proteins, or intramolecular dynamics when considering multiple measurement and analysis approaches. We also investigated the localization and integrity of the four modified oligonucleotides in cultured HeLa cells using fluorescence lifetime imaging microscopy. No intracellular accumulation could be observed for unmodified oligonucleotides, while completely stabilized oligonucleotides showed strong accumulation within HeLa cells with no changes in fluorescence lifetime over 24 h. The integrity and accumulation of partly modified oligonucleotides was in accordance with their extent of modification. In highly fluorescent cells, the oligonucleotides were transported to the nucleus. The lifetime of the RNA in the cells could be explained by a balance between

  13. Microlens arrays

    NASA Astrophysics Data System (ADS)

    Hutley, Michael C.; Stevens, Richard F.; Daly, Daniel J.

    1992-04-01

    Microlenses have been with us for a long time as indeed the very word lens reminds us. Many early lenses,including those made by Hooke and Leeuwenhoek in the 17th century were small and resembled lentils. Many languages use the same word for both (French tilentillelt and German "Linse") and the connection is only obscure in English because we use the French word for the vegetable and the German for the optic. Many of the applications for arrays of inicrolenses are also well established. Lippmann's work on integral photography at the turn of the century required lens arrays and stimulated an interest that is very much alive today. At one stage, lens arrays played an important part in high speed photography and various schemes have been put forward to take advantage of the compact imaging properties of combinations of lens arrays. The fact that many of these ingenious schemes have not been developed to their full potential has to a large degree been due to the absence of lens arrays of a suitable quality and cost.

  14. Comparative immune responses of pigs to infection with Salmonella enterica serovars of food safety (Typhimurium) and animal health (Choleraesuis) importance

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Salmonella infections cause food safety concerns for humans as well as production problems for swine. Our team has used suppression hybridization (SSH), long oligonucleotide Qiagen and Affymetrix porcine GeneChip® arrays, and real time gene expression (Q-PCR) to understand the host response to, and ...

  15. transcriptional response of pigs to Salmonella infection: Comparison of responses to infection with Salmonella eimerica serotype Typhimurium as that observed in S. choleraesuis infection.

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Swine responses to, and control of, Salmonella enterica serotype Typhimurium (ST) infection have been compared to S. enterica serotype Choleraesuis (SC) infection. Using subtractive suppression hybridization (SSH), long oligonucleotide Qiagen and Affymetrix porcine GeneChip® arrays, and real time ge...

  16. Interdigitated microelectrode array-coupled bipolar semiconductor photodiode array (IMEA-PDA) microchip for on-chip electrochemiluminescence detection.

    PubMed

    Pal, Sukdeb; Kim, Min Jung; Tak, Yu Kyung; Kwon, Ho Taik; Song, Joon Myong

    2009-10-01

    This paper reports the design, fabrication and testing of a microchip wherein interdigitated microelectrode arrays (IMEA) were integrated with bipolar semiconductor photodiode array (PDA) chip to fabricate a highly compact embodiment for on-chip handling of solutions and electrochemiluminescence (ECL) detection. A 12 x 12 micro array of photodiodes, each coupled with an interdigitated microelectrode array (IMEA), an array of current amplifiers, and a photodiode element-addressing circuit were integrated into a single 2 x 2 cm² IC chip. Each photodiode had dimensions of 300 x 300 μm² and the photodiode-to-photodiode distance was 100 μm. The chip was successfully applied to the on-chip quantification of electro-chemiluminescing probe-labeled single stranded oligonucleotides. The minimum detectable limit at signal/noise ≥ 3 was found to be 5 x 10⁻¹⁴ moles of oligonucleotides with a sample volume as low as 5 microl (i.e., 10 fmole/μl). The attractive features of the developed IMEA-PDA microchip are that a plurality of samples can be analyzed simultaneously using a chip and that for a given sample the data can be averaged from values obtained from multiple, individually addressed pixels. These in turn bring in speed and statistical confidence in analysis. The IMEA-PDA microchip system has the potential to be used as a versatile and highly compact chemical analysis tool for chemical sensing and metrology applications.

  17. DNA Oligonucleotide Fragment Ion Rearrangements Upon Collision-Induced Dissociation

    NASA Astrophysics Data System (ADS)

    Harper, Brett; Neumann, Elizabeth K.; Solouki, Touradj

    2015-08-01

    Collision-induced dissociation (CID) of m/z-isolated w type fragment ions and an intact 5' phosphorylated DNA oligonucleotide generated rearranged product ions. Of the 21 studied w ions of various nucleotide sequences, fragment ion sizes, and charge states, 18 (~86%) generated rearranged product ions upon CID in a Synapt G2-S HDMS (Waters Corporation, Manchester, England, UK) ion mobility-mass spectrometer. Mass spectrometry (MS), ion mobility spectrometry (IMS), and theoretical modeling data suggest that purine bases can attack the free 5' phosphate group in w type ions and 5' phosphorylated DNA to generate sequence permuted [phosphopurine]- fragment ions. We propose and discuss a potential mechanism for generation of rearranged [phosphopurine]- and complementary y-B type product ions.

  18. Targeted gene correction with 5' acridine-oligonucleotide conjugates.

    PubMed

    de Piédoue, G; Andrieu-Soler, C; Concordet, J P; Maurisse, R; Sun, J-S; Lopez, B; Kuzniak, I; Leboulch, P; Feugeas, J-P

    2007-01-01

    Single-stranded oligonucleotides (SSOs) mediate gene repair of punctual chromosomal mutations at a low frequency. We hypothesized that enhancement of DNA binding affinity of SSOs by intercalating agents may increase the number of corrected cells. Several biochemical modifications of SSOs were tested for their capability to correct a chromosomally integrated and mutated GFP reporter gene in human 293 cells. SSOs of 25 nucleotide length conjugated with acridine at their 5' end increased the efficiency of gene correction up to 10-fold compared to nonmodified SSOs. Acridine and psoralen conjugates were both evaluated, and acridine-modified SSOs were the most effective. Conjugation with acridine at the 3' end of the SSO inhibited gene correction, whereas flanking the SSO by acridine on both sides provided an intermediate level of correction. These results suggest that increasing the stability of hybridization between SSO and its target without hampering a 3' extension improves gene targeting, in agreement with the "annealing-integration" model of DNA repair.

  19. Association of branched oligonucleotides into the i-motif.

    PubMed

    Robidoux, S; Klinck, R; Gehring, K; Damha, M J

    1997-12-01

    The unique architecture of branched oligonucleotides mimicking lariat RNA introns [Wallace and Edmons, Proc. Natl. Acad. Sci. USA 80, 950-954 (1983)] was exploited to study compounds that associate as two parallel duplexes with intercalating C/C+ base pairs (i-motif DNA) [Gehring et al. Nature 363, 561-565 (1993)]. The formation of a branched cytosine tetrad was induced by joining the 5'-ends of pair of pentadeoxycytidine strands with a branching riboadenosine (rA) linker. This arrangement causes the orientation of the dC strands to be parallel, and forces the formation of a C/C+ duplex that self-associates into i-DNA. Presence of the i-motif in this structure is supported by thermal denaturation, native gel electrophoresis, CD, and NMR spectroscopy.

  20. Recent Methods for Purification and Structure Determination of Oligonucleotides

    PubMed Central

    Zhang, Qiulong; Lv, Huanhuan; Wang, Lili; Chen, Man; Li, Fangfei; Liang, Chao; Yu, Yuanyuan; Jiang, Feng; Lu, Aiping; Zhang, Ge

    2016-01-01

    Aptamers are single-stranded DNA or RNA oligonucleotides that can interact with target molecules through specific three-dimensional structures. The excellent features, such as high specificity and affinity for target proteins, small size, chemical stability, low immunogenicity, facile chemical synthesis, versatility in structural design and engineering, and accessible for site-specific modifications with functional moieties, make aptamers attractive molecules in the fields of clinical diagnostics and biopharmaceutical therapeutics. However, difficulties in purification and structural identification of aptamers remain a major impediment to their broad clinical application. In this mini-review, we present the recently attractive developments regarding the purification and identification of aptamers. We also discuss the advantages, limitations, and prospects for the major methods applied in purifying and identifying aptamers, which could facilitate the application of aptamers. PMID:27999357

  1. Predicting the Kinetics of RNA Oligonucleotides Using Markov State Models.

    PubMed

    Pinamonti, Giovanni; Zhao, Jianbo; Condon, David E; Paul, Fabian; Noè, Frank; Turner, Douglas H; Bussi, Giovanni

    2017-02-14

    Nowadays different experimental techniques, such as single molecule or relaxation experiments, can provide dynamic properties of biomolecular systems, but the amount of detail obtainable with these methods is often limited in terms of time or spatial resolution. Here we use state-of-the-art computational techniques, namely, atomistic molecular dynamics and Markov state models, to provide insight into the rapid dynamics of short RNA oligonucleotides, to elucidate the kinetics of stacking interactions. Analysis of multiple microsecond-long simulations indicates that the main relaxation modes of such molecules can consist of transitions between alternative folded states, rather than between random coils and native structures. After properly removing structures that are artificially stabilized by known inaccuracies of the current RNA AMBER force field, the kinetic properties predicted are consistent with the time scales of previously reported relaxation experiments.

  2. OligoCalc: an online oligonucleotide properties calculator

    PubMed Central

    Kibbe, Warren A.

    2007-01-01

    We developed OligoCalc as a web-accessible, client-based computational engine for reporting DNA and RNA single-stranded and double-stranded properties, including molecular weight, solution concentration, melting temperature, estimated absorbance coefficients, inter-molecular self-complementarity estimation and intra-molecular hairpin loop formation. OligoCalc has a familiar ‘calculator’ look and feel, making it readily understandable and usable. OligoCalc incorporates three common methods for calculating oligonucleotide-melting temperatures, including a nearest-neighbor thermodynamic model for melting temperature. Since it first came online in 1997, there have been more than 900 000 accesses of OligoCalc from nearly 200 000 distinct hosts, excluding search engines. OligoCalc is available at http://basic.northwestern.edu/biotools/OligoCalc.html, with links to the full source code, usage patterns and statistics at that link as well. PMID:17452344

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

    PubMed Central

    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 the genome allows us to demonstrate that the largest relative concentration of TTS is found in regulatory regions, especially in promoter zones, which suggests a tremendous potentiality for triplex strategy in the control of gene expression. The dependence of the stability and selectivity of the triplexes on the length of the TTS is also analysed using knowledge-based rules. PMID:14726484

  4. Insights to primitive replication derived from structures of small oligonucleotides

    NASA Technical Reports Server (NTRS)

    Smith, G. K.; Fox, G. E.

    1995-01-01

    Available information on the structure of small oligonucleotides is surveyed. It is observed that even small oligomers typically exhibit defined structures over a wide range of pH and temperature. These structures rely on a plethora of non-standard base-base interactions in addition to the traditional Watson-Crick pairings. Stable duplexes, though typically antiparallel, can be parallel or staggered and perfect complementarity is not essential. These results imply that primitive template directed reactions do not require high fidelity. Hence, the extensive use of Watson-Crick complementarity in genes rather than being a direct consequence of the primitive condensation process, may instead reflect subsequent selection based on the advantage of accuracy in maintaining the primitive genetic machinery once it arose.

  5. Oligonucleotide synthesis catalyzed by the Zn/2+/ ion

    NASA Technical Reports Server (NTRS)

    Sawai, H.; Orgel, L. E.

    1975-01-01

    Results of experiments are reported in which Zn(2+) ion catalyzed the formation of oligonucleotides from nucleoside phosphorimidazolides in aqueous solution, even in the absence of a template. Specifically, the imidazolides (ImpU or ImpA) polymerized to form ImpApA, and pApA, pApApA, and pApApApA, or the analogous uracil compounds. In addition, the expected hydrolysis products of the hydrolysis of ImpA were formed (pA, imidazole). Judging from the ratio of pA(n) over pA (with and without zinc ion), this ion increased the efficiency of phosphodiester-bond formation by up to 10 times. Possible mechanisms for the reaction are tentatively proposed.

  6. Patterns of oligonucleotide distribution within DNA and RNA functional sites

    SciTech Connect

    Kolchanov, N.A.; Kel, A.E.; Ponomarenko, M.P.; Romachenko, A.G.; Likchachev, J.; Milanesi, L.; Lim, H.

    1993-12-31

    Patterns of short oligonucleotide distribution within DNA and RNA functional sites have been analyzed using ``Site-Video`` computer system. The group of DNA functional sites involved nucleosome binding sites, gyrase cleavage sites, promoters of E. coli and men. The group of RNA functional sites involved donor and acceptor splice sites of men, translation initiation sites of E. coli and men and translation frame shift site sites. Analysis of these samples of nucleotide sequences have been carried out by the ``Site-Video`` computer system. For each type of site specific set of patterns of oligonucleotide distribution important for the functioning and recognition have been revealed. At the same time, the number of specific patterns revealed in RNA sites was significantly higher than those in DNA sites. On the base of the results obtained, the script of functional sites for evolutionary emergency have been prompted. According to it, two types of context feature selection took place: (1) positive selection targeted to the appearance of the definite types of context features in particular regions of functional sites;and (2) negative selection targeted to the elimination of definite types of context features in particular regions of functional sites. The authors suppose that evolutionary formation of any functional site is a multistep process realized via combination of positive and negative selections. Negative selection, via fixation of a specific pattern of mutations, eliminates false signals of regulatory proteins binding with the functional site. Positive selection leads to the appearance of local context features (signals) which provide for the specificity and efficiency of the site functioning.

  7. Antisense oligonucleotide-based therapy in human erythropoietic protoporphyria.

    PubMed

    Oustric, Vincent; Manceau, Hana; Ducamp, Sarah; Soaid, Rima; Karim, Zoubida; Schmitt, Caroline; Mirmiran, Arienne; Peoc'h, Katell; Grandchamp, Bernard; Beaumont, Carole; Lyoumi, Said; Moreau-Gaudry, François; Guyonnet-Dupérat, Véronique; de Verneuil, Hubert; Marie, Joëlle; Puy, Herve; Deybach, Jean-Charles; Gouya, Laurent

    2014-04-03

    In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315-48T>C) localized in intron 3. The 315-48C allele increases usage of the 3' cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals.

  8. Antisense Oligonucleotide-Based Therapy in Human Erythropoietic Protoporphyria

    PubMed Central

    Oustric, Vincent; Manceau, Hana; Ducamp, Sarah; Soaid, Rima; Karim, Zoubida; Schmitt, Caroline; Mirmiran, Arienne; Peoc’h, Katell; Grandchamp, Bernard; Beaumont, Carole; Lyoumi, Said; Moreau-Gaudry, François; Guyonnet-Dupérat, Véronique; de Verneuil, Hubert; Marie, Joëlle; Puy, Herve; Deybach, Jean-Charles; Gouya, Laurent

    2014-01-01

    In 90% of people with erythropoietic protoporphyria (EPP), the disease results from the inheritance of a common hypomorphic FECH allele, encoding ferrochelatase, in trans to a private deleterious FECH mutation. The activity of the resulting FECH enzyme falls below the critical threshold of 35%, leading to the accumulation of free protoporphyrin IX (PPIX) in bone marrow erythroblasts and in red cells. The mechanism of low expression involves a biallelic polymorphism (c.315−48T>C) localized in intron 3. The 315−48C allele increases usage of the 3′ cryptic splice site between exons 3 and 4, resulting in the transcription of an unstable mRNA with a premature stop codon, reducing the abundance of wild-type FECH mRNA, and finally reducing FECH activity. Through a candidate-sequence approach and an antisense-oligonucleotide-tiling method, we identified a sequence that, when targeted by an antisense oligonucleotide (ASO-V1), prevented usage of the cryptic splice site. In lymphoblastoid cell lines derived from symptomatic EPP subjects, transfection of ASO-V1 reduced the usage of the cryptic splice site and efficiently redirected the splicing of intron 3 toward the physiological acceptor site, thereby increasing the amount of functional FECH mRNA. Moreover, the administration of ASO-V1 into developing human erythroblasts from an overtly EPP subject markedly increased the production of WT FECH mRNA and reduced the accumulation of PPIX to a level similar to that measured in asymptomatic EPP subjects. Thus, EPP is a paradigmatic Mendelian disease in which the in vivo correction of a common single splicing defect would improve the condition of most affected individuals. PMID:24680888

  9. Empirical Establishment of Oligonucleotide Probe Design Criteria; Use of Microarrays with Different Probe Sizes for Monitoring Gene Expression; Temporal Transcriptomic Analysis as Desulfovibrio vulgaris Hildenborough Transitions into Stationary Phase during Electron Donor Depletion

    SciTech Connect

    He, Q.; He, Z.; Huang, K. H.; Alm, E. J.; Wan, X. F.; Hazen, T. C.; Arkin, A. P.; Wall, J. D.; Zhou, J. Z.; Fields, M. W.

    2005-07-15

    In order to experimentally establish the criteria for designing gene-specific and group-specific oligonucleotide probes, an oligonucleotide array was constructed that contained perfect match (PM) and mismatch (MM) probes (50mers and 70mers) based upon 4 genes. The effects of probe-target identity, continuous stretch, mismatch position, and hybridization free energy on specificity were examined. Little hybridization was observed at a probe-target identity of <85% for both 50mer and 70mer probes........Based on the experimental results, a set of criteria are suggested for the design of gene-specific and group-specific oligonucleotide probes, and these criteria should provide valuable information for the development of new software and algorithms for microarray-based studies.; Microarrays with oligonucleotides of different lengths were used to monitor gene expression at a wholegenome level. To determine what length of oligonucleotide is a better alternative to PCR-generated probes, the performance of oligonucleotide probes was systematically compared to that of their PCR-generated counterparts for 96 genes from Shewanella oneidensis MR-1 in terms of overall signal intensity, numbers of genes detected, specificity, sensitivity, and differential gene expression under experimental conditions. .......To evaluate differential gene expression under experimental conditions, S. oneidensis MR-1 cells were exposed to low- or high-pH conditions for 30 and 60 min, and the transcriptional profiles detected by oligonucleotide probes (50-mer, 60-mer, and 70-mer) were closely correlated with those detected by the PCR probes. The results demonstrated that 70-mer oligonucleotides can provide the performance most comparable to the performance obtained with PCR-generated probes.; Desulfovibrio vulgaris was cultivated in a defined medium, and biomass was sampled for approximately 70 h to characterize the shifts in gene expression as cells transitioned from the exponential to the

  10. Pacific Array

    NASA Astrophysics Data System (ADS)

    Kawakatsu, H.; Takeo, A.; Isse, T.; Nishida, K.; Shiobara, H.; Suetsugu, D.

    2014-12-01

    Based on our recent results on broadband ocean bottom seismometry, we propose a next generation large-scale array experiment in the ocean. Recent advances in ocean bottom broadband seismometry (e.g., Suetsugu & Shiobara, 2014, Annual Review EPS), together with advances in the seismic analysis methodology, have now enabled us to resolve the regional 1-D structure of the entire lithosphere/asthenosphere system, including seismic anisotropy (both radial and azimuthal), with deployments of ~10-15 broadband ocean bottom seismometers (BBOBSs) (namely "ocean-bottom broadband dispersion survey"; Takeo et al., 2013, JGR; Kawakatsu et al., 2013, AGU; Takeo, 2014, Ph.D. Thesis; Takeo et al., 2014, JpGU). Having ~15 BBOBSs as an array unit for 2-year deployment, and repeating such deployments in a leap-frog way (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, 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 might be sought.

  11. Genome-wide copy number profiling on high-density bacterial artificial chromosomes, single-nucleotide polymorphisms, and oligonucleotide microarrays: a platform comparison based on statistical power analysis.

    PubMed

    Hehir-Kwa, Jayne Y; Egmont-Petersen, Michael; Janssen, Irene M; Smeets, Dominique; van Kessel, Ad Geurts; Veltman, Joris A

    2007-02-28

    Recently, comparative genomic hybridization onto bacterial artificial chromosome (BAC) arrays (array-based comparative genomic hybridization) has proved to be successful for the detection of submicroscopic DNA copy-number variations in health and disease. Technological improvements to achieve a higher resolution have resulted in the generation of additional microarray platforms encompassing larger numbers of shorter DNA targets (oligonucleotides). Here, we present a novel method to estimate the ability of a microarray to detect genomic copy-number variations of different sizes and types (i.e. deletions or duplications). We applied our method, which is based on statistical power analysis, to four widely used high-density genomic microarray platforms. By doing so, we found that the high-density oligonucleotide platforms are superior to the BAC platform for the genome-wide detection of copy-number variations smaller than 1 Mb. The capacity to reliably detect single copy-number variations below 100 kb, however, appeared to be limited for all platforms tested. In addition, our analysis revealed an unexpected platform-dependent difference in sensitivity to detect a single copy-number loss and a single copy-number gain. These analyses provide a first objective insight into the true capacities and limitations of different genomic microarrays to detect and define DNA copy-number variations.

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

    PubMed Central

    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

  13. Oligonucleotides designed to inhibit TLR9 block Herpes simplex virus type 1 infection at multiple steps.

    PubMed

    Sauter, Monica M; Gauger, Joshua J L; Brandt, Curtis R

    2014-09-01

    Herpes simplex virus type 1 (HSV-1) is an important human pathogen which requires activation of nuclear factor-kappa B (NFκB) during its replication cycle. The persistent nature of HSV-1 infection, and the emergence of drug-resistant strains, highlights the importance of research to develop new antiviral agents. Toll-like receptors (TLRs) play a prominent role during the early antiviral response by recognizing viral nucleic acid and gene products, activating NFκB, and stimulating the production of inflammatory cytokines. We demonstrate a significant effect on HSV-1 replication in ARPE-19 and Vero cells when oligonucleotides designed to inhibit TLR9 are added 2h prior to infection. A greater than 90% reduction in the yield of infectious virus was achieved at oligonucleotide concentrations of 10-20 μM. TLR9 inhibitory oligonucleotides prevented expression of essential immediate early herpes gene products as determined by immunofluorescence microscopy and Western blotting. TLR9 oligonucleotides also interfered with viral attachment and entry. A TLR9 inhibitory oligonucleotide containing five adjacent guanosine residues (G-ODN) exhibited virucidal activity and inhibited HSV-1 replication when added post-infection. The antiviral effect of the TLR9 inhibitory oligonucleotides did not depend on the presence of TLR9 protein, suggesting a mechanism of inhibition that is not TLR9 specific. TLR9 inhibitory oligonucleotides also reduced NFκB activity in nuclear extracts. Studies using these TLR inhibitors in the context of viral infection should be interpreted with caution.

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

    PubMed

    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.

  15. Resonant raman scattering in complexes of nc-Si/SiO2 quantum dots and oligonucleotides

    NASA Astrophysics Data System (ADS)

    Bairamov, F. B.; Poloskin, E. D.; Kornev, A. A.; Chernev, A. L.; Toporov, V. V.; Dubina, M. V.; Röder, C.; Sprung, C.; Lipsanen, H.; Bairamov, B. Kh.

    2014-11-01

    We report on the functionalization of nanocrystalline nc-Si/SiO2 semiconductor quantum dots (QDs) by short d(20G, 20T) oligonucleotides. The obtained complexes have been studied by Raman spectroscopy techniques with high spectral and spatial resolution. A new phenomenon of multiband resonant light scattering on single oligonucleotide molecules has been discovered, and peculiarities of this effect related to the nonradiative transfer of photoexcitation from nc-Si/SiO2 quantum dots to d(20G, 20T) oligonucleotide molecules have been revealed.

  16. Method for promoting specific alignment of short oligonucleotides on nucleic acids

    DOEpatents

    Studier, F. William; Kieleczawa, Jan; Dunn, John J.

    1996-01-01

    Disclosed is a method for promoting specific alignment of short oligonucleotides on a nucleic acid polymer. The nucleic acid polymer is incubated in a solution containing a single-stranded DNA-binding protein and a plurality of oligonucleotides which are perfectly complementary to distinct but adjacent regions of a predetermined contiguous nucleotide sequence in the nucleic acid polymer. The plurality of oligonucleotides anneal to the nucleic acid polymer to form a contiguous region of double stranded nucleic acid. Specific application of the methods disclosed include priming DNA synthesis and template-directed ligation.

  17. Oligonucleotide microchips as genosensors for determinative and environmental studies in microbiology.

    PubMed Central

    Guschin, D Y; Mobarry, B K; Proudnikov, D; Stahl, D A; Rittmann, B E; Mirzabekov, A D

    1997-01-01

    The utility of parallel hybridization of environmental nucleic acids to many oligonucleotides immobilized in a matrix of polyacrylamide gel pads on a glass slide (oligonucleotide microchip) was evaluated. Oligonucleotides complementary to small-subunit rRNA sequences of selected microbial groups, encompassing key genera of nitrifying bacteria, were shown to selectively retain labeled target nucleic acid derived from either DNA or RNA forms of the target sequences. The utility of varying the probe concentration to normalize hybridization signals and the use of multicolor detection for simultaneous quantitation of multiple probe-target populations were demonstrated. PMID:9172361

  18. Oligonucleotide-mediated gene repair at DNA level: the potential applications for gene therapy.

    PubMed

    Liu, Chang-Mei; Liu, De-Pei; Liang, Chih-Chuan

    2002-10-01

    Mutations in gene sequence can cause many genetic disorders, and researchers have attempted to develop treatments or cures at the DNA level for these diseases. Several strategies including triple-helix-forming oligonucleotides (TFOs), chimeric RNA/DNA oligonucleotide (RDO), and short single-stranded oligodeoxynucleotide (ODN) have been used to correct the dysfunctional genes in situ in the chromosome. Experimental data from cells and animal models suggest that all these strategies can repair the mutations in situ at DNA level. More effective structures of oligonucleotide, efficient delivery systems, and gene correction efficiency should be improved. Development of these strategies holds great potentials for treatments of genetic defects and other disorders.

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

    NASA Technical Reports Server (NTRS)

    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.

  20. Carbon Nanotube Nanoelectrode Array for Ultrasensitive DNA Detection

    NASA Technical Reports Server (NTRS)

    Li, Jun; Koehne, Jessica; Chen, Hua; Cassell, Alan; Ng, Hou Tee; Fan, Wendy; Ye, Qi; Han, Jie; Meyyappan, M.

    2003-01-01

    A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in SiO2 is used for ultrasensitive DNA detection. Characteristic nanoelectrode behavior is observed using low-density MWNT arrays for measuring both bulk and surface immobilized redox species such as K4Fe(CN)6. The open-end of MWNTs present similar properties as graphite edge-plane electrodes with wide potential window, flexible chemical functionalities, and good biocompatibility. Oligonucleotide probes are selectively functionalized at the open ends cf the nanotube array and specifically hybridized with oligonucleotide targets. The guanine groups are employed as the signal moieties in the electrochemical measurements. Ru(bpy)3(2+) mediator is used to further amplify the guanine oxidation signal. The hybridization of subattomoles of PCR amplified DNA targets is detected electrochemically by combining the MWNT nanoelectrode array with the Ru(bpy)32' amplification mechanism. This system provides a general platform of molecular diagnostics for applications requiring ultrahigh sensitivity, high-degree of miniaturization, and simple sample preparations.

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

    PubMed

    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.

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

    SciTech Connect

    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

  3. Indel arrays: an affordable alternative for genotyping.

    PubMed

    Salathia, Neeraj; Lee, Hana N; Sangster, Todd A; Morneau, Keith; Landry, Christian R; Schellenberg, Kurt; Behere, Aditi S; Gunderson, Kevin L; Cavalieri, Duccio; Jander, Georg; Queitsch, Christine

    2007-08-01

    Natural variation and induced mutations are important resources for gene discovery and the elucidation of genetic circuits. Mapping such polymorphisms requires rapid and cost-efficient methods for genome-wide genotyping. Here we report the development of a microarray-based method that assesses 240 unique markers in a single hybridization experiment at a cost of less than US$50 in materials per line. Our genotyping array is built with 70-mer oligonucleotide elements representing insertion/deletion (indel) polymorphisms between the Arabidopsis thaliana accessions Columbia-0 (Col) and Landsberg erecta (Ler). These indel polymorphisms are recognized with great precision by comparative genomic hybridization, eliminating the need for array replicates and complex statistical analysis. Markers are present genome-wide, with an average spacing of approximately 500 kb. PCR primer information is provided for all array indels, allowing rapid single-locus inquiries. Multi-well chips allow groups of 16 lines to be genotyped in a single experiment. We demonstrate the utility of the array for accurately mapping recessive mutations, RIL populations and mixed genetic backgrounds from accessions other than Col and Ler. Given the ease of use of shotgun sequencing to generate partial genomic sequences of unsequenced species, this approach is readily transferable to non-model organisms.

  4. Orthogonal ion pairing reversed phase liquid chromatography purification of oligonucleotides with bulky fluorophores.

    PubMed

    Anacleto, Concordio; Ouye, Randall; Schoenbrunner, Nancy

    2014-02-14

    A dual labeled oligonucleotide used as TaqMan® or 5' nuclease probe for in vitro diagnostic has been purified through orthogonal ion-pairing reversed phase chromatography, using polymeric semi-preparative and preparative PRP-1 column. We studied the mechanism of separation of oligonucleotides using ion-pairing reversed phase chromatography. We found that elution profiles of dye labeled oligonucleotides can be controlled by use of specific ion-pairing reagents. Here, we report a method for purification of an oligonucleotide containing an internally positioned rhodamine dye using two orthogonal chromatographic steps, in which the primary step resolves mostly by differences in hydrophobicity by using a weak ion-pairing reagent, and a secondary step uses a strong ion-pairing reagent for separation of length variants. Purification is demonstrated for both 1 and 15μmol scale syntheses, and amenable to further scale up for commercial lot production.

  5. The Fidelity of Template-Directed Oligonucleotide Ligation and the Inevitability of Polymerase Function

    NASA Astrophysics Data System (ADS)

    James, Kenneth D.; Ellington, Andrew D.

    1999-08-01

    The first living systems may have employed template-directed oligonucleotide ligation for replication. The utility of oligonucleotide ligation as a mechanism for the origin and evolution of life is in part dependent on its fidelity. We have devised a method for evaluating ligation fidelity in which ligation substrates are selected from random sequence libraries. The fidelities of chemical and enzymatic ligation are compared under a variety of conditions. While reaction conditions can be found that promote high fidelity copying, departure from these conditions leads to error-prone copying. In particular, ligation reactions with shorter oligonucleotide substrates are less efficient but more faithful. These results support a model for origins in which there was selective pressure for template-directed oligonucleotide ligation to be gradually supplanted by mononucleotide polymerization.

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

    PubMed

    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.

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

    PubMed

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

    2007-05-15

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

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

    SciTech Connect

    Plesner, P.; Goodchild, J.; Kalckar, H.M.; Zamecnik, P.C.

    1987-04-01

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

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

    SciTech Connect

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

    1988-04-01

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

  10. Elasticity of the transition state for oligonucleotide hybridization

    PubMed Central

    Whitley, Kevin D.; Comstock, Matthew J.; Chemla, Yann R.

    2017-01-01

    Despite its fundamental importance in cellular processes and abundant use in biotechnology, we lack a detailed understanding of the kinetics of nucleic acid hybridization. In particular, the identity of the transition state, which determines the kinetics of the two-state reaction, remains poorly characterized. Here, we used optical tweezers with single-molecule fluorescence to observe directly the binding and unbinding of short oligonucleotides (7–12 nt) to a complementary strand held under constant force. Binding and unbinding rate constants measured across a wide range of forces (1.5–20 pN) deviate from the exponential force dependence expected from Bell's equation. Using a generalized force dependence model, we determined the elastic behavior of the transition state, which we find to be similar to that of the pure single-stranded state. Our results indicate that the transition state for hybridization is visited before the strands form any significant amount of native base pairs. Such a transition state supports a model in which the rate-limiting step of the hybridization reaction is the alignment of the two strands prior to base pairing. PMID:27903889

  11. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

    PubMed Central

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

    2016-01-01

    We present a microfluidic approach to integrated isolation of DNA aptamers via systematic evolution of ligands by exponential enrichment (SELEX). The approach employs a microbead-based protocol for the processes of affinity selection and amplification of target-binding oligonucleotides, and an electrophoretic DNA manipulation scheme for the coupling of these processes, which are required to occur in different buffers. This achieves the full microfluidic integration of SELEX, thereby enabling 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. PMID:27217242

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

    PubMed Central

    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

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

    PubMed

    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.

  14. Integrated Microfluidic Isolation of Aptamers Using Electrophoretic Oligonucleotide Manipulation

    NASA Astrophysics Data System (ADS)

    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.

  15. Splice-switching antisense oligonucleotides as therapeutic drugs

    PubMed Central

    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 manipulate protein production from a gene. Splicing modulation is particularly valuable in cases of disease caused by mutations that lead to disruption of normal splicing or when interfering with the normal splicing process of a gene transcript may be therapeutic. SSOs offer an effective and specific way to target and alter splicing in a therapeutic manner. Here, we discuss the different approaches used to target and alter pre-mRNA splicing with SSOs. We detail the modifications to the nucleic acids that make them promising therapeutics and discuss the challenges to creating effective SSO drugs. We highlight the development of SSOs designed to treat Duchenne muscular dystrophy and spinal muscular atrophy, which are currently being tested in clinical trials. PMID:27288447

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

    PubMed

    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.

  17. Molecular architectures for electrocatalytic amplification of oligonucleotide hybridization.

    PubMed

    Mir, Mònica; Alvarez, Marta; Azzaroni, Omar; Tiefenauer, Louis; Knoll, Wolfgang

    2008-09-01

    In this work, we describe a new platform suitable for electrocatalytic amplification of oligonucleotide hybridization based on the use of supramolecular bioconjugates incorporating ferrocene-labeled streptavidin. Our goals were aimed at designing a biosensing platform which could support highly reproducible and stable electrocatalytic amplification with maximum efficiency. The use of nonlabeled streptavidin as an underlying layer promotes a major improvement on the characteristics of the amplified electrochemical signal. In addition, the electrocatalytic current can be easily amplified by tuning the concentration of electron donor species in solution. Because of the fact that the redox labels are bioconjugated to the DNA strands, increasing the ionic strength does not lead to the loss of redox labels. More importantly, increasing the concentration of donors only involves the magnification of the signal without implying the permeation of donors (thus reducing the efficient electrocatalysis). This approach represents a major improvement on the use of electrocatalytically amplified DNA-sensing platforms, thus overcoming any possible limitation in connection with the reproducibility and reliability of this well-established method.

  18. Skipping multiple exons of dystrophin transcripts using cocktail antisense oligonucleotides.

    PubMed

    Echigoya, Yusuke; Yokota, Toshifumi

    2014-02-01

    Duchenne muscular dystrophy (DMD) is one of the most common and lethal genetic disorders, with 20,000 children per year born with DMD globally. DMD is caused by mutations in the dystrophin (DMD) gene. Antisense-mediated exon skipping therapy is a promising therapeutic approach that uses short DNA-like molecules called antisense oligonucleotides (AOs) to skip over/splice out the mutated part of the gene to produce a shortened but functional dystrophin protein. One major challenge has been its limited applicability. Multiple exon skipping has recently emerged as a potential solution. Indeed, many DMD patients need exon skipping of multiple exons in order to restore the reading frame, depending on how many base pairs the mutated exon(s) and adjacent exons have. Theoretically, multiple exon skipping could be used to treat approximately 90%, 80%, and 98% of DMD patients with deletion, duplication, and nonsense mutations, respectively. In addition, multiple exon skipping could be used to select deletions that optimize the functionality of the truncated dystrophin protein. The proof of concept of systemic multiple exon skipping using a cocktail of AOs has been demonstrated in dystrophic dog and mouse models. Remaining challenges include the insufficient efficacy of systemic treatment, especially for therapies that target the heart, and limited long-term safety data. Here we review recent preclinical developments in AO-mediated multiple exon skipping and discuss the remaining challenges.

  19. Template-directed synthesis of oligonucleotides under eutectic conditions

    NASA Technical Reports Server (NTRS)

    Stribling, R.; Miller, S. L.

    1991-01-01

    One of the most important sets of model prebiotic experiments consists of reactions that synthesize complementary oligonucleotides from preformed templates under nonenzymatic conditions. Most of these experiments are conducted at 4 degrees C using 0.01-0.1 M concentrations of activated nucleotide monomer and template (monomer equivalent). In an attempt to extend the conditions under which this type of reaction can occur, we have concentrated the reactants by freezing at -18 degrees C, which is close to the NaCl-H2O eutectic at -21 degrees C. The results from this set of experiments suggest that successful syntheses can occur with poly(C) concentrations as low at 5 x 10(-4) M and 2MeImpG concentrations at 10(-3) M. It was also anticipated that this mechanism might allow the previously unsuccessful poly(A)-directed synthesis of oligo(U)s to occur. However, no template effect was seen with the poly(A) and ImpU system. The failure of these conditions to allow template-directed synthesis of oligo(U)s supports the previously proposed idea that pyrimidines may not have been part of the earliest genetic material. Because of the low concentrations of monomer and template that would be expected from prebiotic syntheses, this lower temperature could be considered a more plausible geologic setting for template-directed synthesis than the standard reaction conditions.

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

    PubMed

    Kupriushkin, M S; Pyshnyĭ, D V

    2012-01-01

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

  1. In vitro characterization of two novel biodegradable vectors for the delivery of radiolabeled antisense oligonucleotides.

    PubMed

    von Guggenberg, Elisabeth; Shahhosseini, Soraya; Koslowsky, Ingrid; Lavasanifar, Afsaneh; Murray, David; Mercer, John

    2010-12-01

    The development of antisense oligonucleotides suitable for tumor targeting applications is hindered by low stability and bioavailability of oligonucleotides in vivo and by the absence of efficient and safe vectors for oligonucleotide delivery. Stabilization in vivo has been achieved through chemical modification of oligonucleotides by various means, but effective approaches to enhance their intracellular delivery are lacking. This study reports on the characterization in vitro of a fully phosphorothioated 20-mer oligonucleotide, complementary to p21 mRNA, radiolabeled with fluorine-18 using a thiol reactive prosthetic group. The potential of two novel synthetic block copolymers containing grafted polyamines on their hydrophobic blocks for vector-assisted cell delivery was studied in vitro. Extensive cellular uptake studies were performed in human colon carcinoma cell lines with enhanced or deficient p21 expression to evaluate and compare the uptake mechanism of naked and vectorized radiolabeled formulations. Uptake studies with the two novel biodegradable vectors showed a moderate increase in cell uptake of the radiofluorinated antisense oligonucleotide. The two vectors show, however, promising advantages over conventional lipidic vectors regarding their biocompatibility and subcellular distribution.

  2. Analysis of copy number variations in Mexican Holstein cattle using axiom genome-wide Bos 1 array

    PubMed Central

    Salomon-Torres, Ricardo; Villa-Angulo, Rafael; Villa-Angulo, Carlos

    2015-01-01

    Recently, for copy number variation (CNV) analysis, bovine researchers have focused mainly on the use of genome-wide SNP genotyping arrays. One of the highest densities commercially available SNPchips for cattle is the Affymetrix axiom genome-wide Bos 1, which assays 648,315 informative SNPs across the whole bovine genome. Here, we describe the microarray data, quality controls and validation implemented in a study published in Genetics and Molecular Research Journal in 2015 [1]. The microarray raw data has been deposited into Gene Expression Omnibus under accession #GSE54813. PMID:26981375

  3. Global Arrays

    SciTech Connect

    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.

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

    PubMed Central

    Stomakhin, Andrey A.; Vasiliskov, Vadim A.; Timofeev, Edward; Schulga, Dennis; Cotter, Richard J.; Mirzabekov, Andrei D.

    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°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. PMID:10666462

  5. 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

    SciTech Connect

    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.

  6. Oligonucleotide facilitators may inhibit or activate a hammerhead ribozyme.

    PubMed Central

    Jankowsky, E; Schwenzer, B

    1996-01-01

    Facilitators are oligonucleotides capable of affecting hammerhead ribozyme activity by interacting with the substrate at the termini of the ribozyme. Facilitator effects were determined in vitro using a system consisting of a ribozyme with 7 nucleotides in every stem sequence and two substrates with inverted facilitator binding sequences. The effects of 9mer and 12mer RNA as well as DNA facilitators which bind either adjacent to the 3'- or 5'-end of the ribozyme were investigated. A kinetic model was developed which allows determination of the apparent dissociation constant of the ribozyme-substrate complex from single turnover reactions. We observed a decreased dissociation constant of the ribozyme-substrate complex due to facilitator addition corresponding to an additional stabilization energy of delta delta G=-1.7 kcal/mol with 3'-end facilitators. The cleavage rate constant was increased by 3'-end facilitators and decreased by 5'-end facilitators. Values for Km were slightly lowered by all facilitators and kcat was increased by 3'-end facilitators and decreased by 5'-end facilitators in our system. Generally the facilitator effects increased with the length of the facilitators and RNA provided greater effects than DNA of the same sequence. Results suggest facilitator influences on several steps of the hammerhead reaction, substrate association, cleavage and dissociation of products. Moreover, these effects are dependent in different manners on ribozyme and substrate concentration. This leads to the conclusion that there is a concentration dependence whether activation or inhibition is caused by facilitators. Conclusions are drawn with regard to the design of hammerhead ribozyme facilitator systems. PMID:8602353

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

    PubMed

    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.

  8. Application of heteronuclear couplings to conformational analysis of oligonucleotides

    SciTech Connect

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

    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.

  9. Development of LNA oligonucleotide-PCR clamping technique in investigating the community structures of plant-associated bacteria.

    PubMed

    Ikenaga, Makoto; Tabuchi, Masakazu; Oyama, Takuya; Akagi, Isao; Sakai, Masao

    2015-01-01

    Simultaneous extraction of plant organelle (mitochondria and plastid) genes during the DNA extraction step is major limitation in investigating the community structures of plant-associated bacteria. Although locked nucleic acid (LNA) oligonucleotides was designed to selectively amplify the bacterial small subunit rRNA genes by applying the PCR clamping technique, those for plastids were applicable only for particular plants, while those for mitochondria were available throughout most plants. To widen the applicable range, new LNA oligonucleotides specific for plastids were designed, and the efficacy was investigated. PCR without LNA oligonucleotides predominantly amplified the organelle genes, while bacterial genes were predominantly observed in having applied the LNA oligonucleotides. Denaturing gradient gel electrophoresis (DGGE) analysis displayed additional bacterial DGGE bands, the amplicons of which were prepared using the LNA oligonucleotides. Thus, new designed LNA oligonucleotides specific for plastids were effective and have widened the scope in investigating the community structures of plant-associated bacteria.

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

    PubMed

    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.

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

    PubMed

    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.

  12. Array-based electrical detection of DNA with nanoparticle probes.

    PubMed

    Park, So-Jung; Taton, T Andrew; Mirkin, Chad A

    2002-02-22

    A DNA array detection method is reported in which the binding of oligonucleotides functionalized with gold nanoparticles leads to conductivity changes associated with target-probe binding events. The binding events localize gold nanoparticles in an electrode gap; silver deposition facilitated by these nanoparticles bridges the gap and leads to readily measurable conductivity changes. An unusual salt concentration-dependent hybridization behavior associated with these nanoparticle probes was exploited to achieve selectivity without a thermal-stringency wash. Using this method, we have detected target DNA at concentrations as low as 500 femtomolar with a point mutation selectivity factor of approximately 100,000:1.

  13. Microfluidic gene arrays for rapid genomic profiling

    NASA Astrophysics Data System (ADS)

    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.

  14. 50 nm DNA nanoarrays generated from uniform oligonucleotide films.

    PubMed

    Noh, Hyunwoo; Hung, Albert M; Choi, Chulmin; Lee, Ju Hun; Kim, Jin-Yeol; Jin, Sungho; Cha, Jennifer N

    2009-08-25

    One of the most challenging but potentially rewarding goals in nanoscience is the ability to direct the assembly of nanoscale materials into functional architectures with high yields, minimal steps, and inexpensive procedures. Despite their unique physical properties, the inherent difficulties of engineering wafer-level arrays of useful devices from nanoscale materials in a cost-effective manner have provided serious roadblocks toward technological impact. To address nanoscale features while still maintaining low fabrication costs, we demonstrate here an inexpensive printing method that enables repeated patterning of large-area arrays of nanoscale materials. DNA strands were patterned over 4 mm areas with 50 nm resolution by a soft-lithographic subtraction printing process, and DNA hybridization was used to direct the assembly of sub-20 nm materials to create highly ordered two-dimensional nanoparticle arrays. The entire printing and assembly process was accomplished in as few as three fabrication steps and required only a single lithographically templated silicon master that could be used repeatedly. The low-cost procedures developed to generate nanoscale DNA patterns can be easily extended toward roll-to-roll assembly of nanoscale materials with sub-50 nm resolution and fidelity.

  15. Comparison of Nanostring nCounter® Data on FFPE Colon Cancer Samples and Affymetrix Microarray Data on Matched Frozen Tissues.

    PubMed

    Chen, Xi; Deane, Natasha G; Lewis, Keeli B; Li, Jiang; Zhu, Jing; Washington, M Kay; Beauchamp, R Daniel

    2016-01-01

    The prognosis of colorectal cancer (CRC) stage II and III patients remains a challenge due to the difficulties of finding robust biomarkers suitable for testing clinical samples. The majority of published gene signatures of CRC have been generated on fresh frozen colorectal tissues. Because collection of frozen tissue is not practical for routine surgical pathology practice, a clinical test that improves prognostic capabilities beyond standard pathological staging of colon cancer will need to be designed for formalin-fixed paraffin-embedded (FFPE) tissues. The NanoString nCounter® platform is a gene expression analysis tool developed for use with FFPE-derived samples. We designed a custom nCounter® codeset based on elements from multiple published fresh frozen tissue microarray-based prognostic gene signatures for colon cancer, and we used this platform to systematically compare gene expression data from FFPE with matched microarray array data from frozen tissues. Our results show moderate correlation of gene expression between two platforms and discovery of a small subset of genes as candidate biomarkers for colon cancer prognosis that are detectable and quantifiable in FFPE tissue sections.

  16. Effect of non-specific species competition from total RNA on the static mode hybridization response of nanomechanical assays of oligonucleotides

    NASA Astrophysics Data System (ADS)

    Mishra, Rohit; Hegner, Martin

    2014-06-01

    We investigate here the nanomechanical response of microcantilever sensors in real-time for detecting a range of ultra-low concentrations of oligonucleotides in a complex background of total cellular RNA extracts from cell lines without labeling or amplification. Cantilever sensor arrays were functionalized with probe single stranded DNA (ssDNA) and reference ssDNA to obtain a differential signal. They were then exposed to complementary target ssDNA strands that were spiked in a fragmented total cellular RNA background in biologically relevant concentrations so as to provide clinically significant analysis. We present a model for prediction of the sensor behavior in competitive backgrounds with parameters that are indicators of the change in nanomechanical response with variation in the target and background concentration. For nanomechanical assays to compete with current technologies it is essential to comprehend such responses with eventual impact on areas like understanding non-coding RNA pharmacokinetics, nucleic acid biomarker assays and miRNA quantification for disease monitoring and diagnosis to mention a few. Additionally, we also achieved a femtomolar sensitivity limit for online oligonucleotide detection in a non-competitive environment with these sensors.

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

    PubMed

    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.

  18. Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy

    PubMed Central

    Hazell, Gareth; Shabanpoor, Fazel; Saleh, Amer F.; Bowerman, Melissa; Meijboom, Katharina E.; Zhou, Haiyan; Muntoni, Francesco; Talbot, Kevin; Gait, Michael J.; Wood, Matthew J. A.

    2016-01-01

    The development of antisense oligonucleotide therapy is an important advance in the identification of corrective therapy for neuromuscular diseases, such as spinal muscular atrophy (SMA). Because of difficulties of delivering single-stranded oligonucleotides to the CNS, current approaches have been restricted to using invasive intrathecal single-stranded oligonucleotide delivery. Here, we report an advanced peptide-oligonucleotide, Pip6a-morpholino phosphorodiamidate oligomer (PMO), which demonstrates potent efficacy in both the CNS and peripheral tissues in severe SMA mice following systemic administration. SMA results from reduced levels of the ubiquitously expressed survival motor neuron (SMN) protein because of loss-of-function mutations in the SMN1 gene. Therapeutic splice-switching oligonucleotides (SSOs) modulate exon 7 splicing of the nearly identical SMN2 gene to generate functional SMN protein. Pip6a-PMO yields SMN expression at high efficiency in peripheral and CNS tissues, resulting in profound phenotypic correction at doses an order-of-magnitude lower than required by standard naked SSOs. Survival is dramatically extended from 12 d to a mean of 456 d, with improvement in neuromuscular junction morphology, down-regulation of transcripts related to programmed cell death in the spinal cord, and normalization of circulating insulin-like growth factor 1. The potent systemic efficacy of Pip6a-PMO, targeting both peripheral as well as CNS tissues, demonstrates the high clinical potential of peptide-PMO therapy for SMA. PMID:27621445

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

    PubMed

    Ko, Young Tag; Bickel, Ulrich

    2012-06-01

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

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

    PubMed

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

    2011-11-01

    A general procedure, based on a new activated alkyne linker, for the preparation of peptide-oligonucleotide conjugates (POCs) on solid support has been developed. With this linker, conjugation is effective at room temperature (RT) in millimolar concentration and submicromolar amounts. This is made possible since the use of a readily attachable activated triple bond linker enhances the Cu(I) catalyzed 1,3-dipolar cycloaddition ('click' reaction). The preferred scheme for conjugate preparation involves sequential conjugation to oligonucleotides on solid support of (i) an H-phosphonate-based aminolinker; (ii) the triple bond donor p-(N-propynoylamino)toluic acid (PATA); and (iii) azido-functionalized peptides. The method gives conversion of oligonucleotide to the POC on solid support, and only involves a single purification step after complete assembly. The synthesis is flexible and can be carried out without the need for specific automated synthesizers since it has been designed to utilize commercially available oligonucleotide and peptide derivatives on solid support or in solution. Methodology for the ready conversion of peptides into 'clickable' azidopeptides with the possibility of selecting either N-terminus or C-terminus connection also adds to the flexibility and usability of the method. Examples of synthesis of POCs include conjugates of oligonucleotides with peptides known to be membrane penetrating and nuclear localization signals.

  1. A novel, one-step amplification and oligonucleotide ligation procedure for multiplex genetic typing

    SciTech Connect

    Eggerding, F.A.

    1994-09-01

    A new technique, coupled amplification and oligonucleotide ligation (CAL), has been developed for simultaneous multiplex amplification and genotyping of DNA. CAL is a biphasic method which combines in one assay DNA amplification by the polymerase chain reaction (PCR) with DNA genotyping by the oligonucleotide ligation assay (OLA). By virtue of a difference in the melting temperatures of PCR primer-target DNA and OLA probe-target DNA hybrids, the method allows preferential amplification of DNA during stage I and oligonucleotide ligation during stage II of the reaction. In stage I target DNA is amplified using high-melting primers in a two-step PCR cycle that employs a 72{degrees}C anneal-elongation step. In stage II genotyping of PCR products by competitive oligonucleotide ligation with oligonucleotide probes located between PCR primers is accomplished by several cycles of denaturation at 94{degrees}C followed by anneal-ligation at 55{degrees}C. Ligation products are fluorochrome-labeled at their 3{prime}-ends and analyzed electrophoretically on a fluorescent DNA sequencer. The CAL procedure has been used for multiplex detection of 30 cystic fibrosis mutations and for analysis of ras gene point mutations. Because mutation detection occurs concurrently with target amplification, the technique is rapid, highly sensitive and specific, easily automatable, and requires minimal sample processing.

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

    PubMed

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

    PubMed Central

    Abel, Biebele; Aslan, Kadir

    2013-01-01

    This study demonstrates the proof-of-principle of rapid surface modification of plasmonic nanostructured materials with oligonucleotides using low power microwave heating. Due to their interesting optical and electronic properties, silver nanoparticle films (SNFs, 2 nm thick) deposited onto glass slides were used as the model plasmonic nanostructured materials. Rapid surface modification of SNFs with oligonucleotides was carried out using two strategies (1) Strategy 1: for ss-oligonucleotides, surface hybridization and (2) Strategy 2: for ds-oligonucleotides, solution hybridization), where the samples were exposed to 10, 15, 30 and 60 seconds microwave heating. To assess the efficacy of our new rapid surface modification technique, identical experiments carried out without the microwave heating (i.e., conventional method), which requires 24 hours for the completion of the identical steps. It was found that SNFs can be modified with ss- and ds-oligonucleotides in 10 seconds, which typically requires several hours of incubation time for the chemisorption of thiol groups on to the planar metal surface using conventional techniques. PMID:23645933

  4. In vivo potentialities of EWS-Fli-1 targeted antisense oligonucleotides-nanospheres complexes.

    PubMed

    Maksimenko, Andrei; Polard, Valerie; Villemeur, Marie; Elhamess, Hind; Couvreur, Patrick; Bertrand, Jean-Remi; Aboubakar, Malam; Gottikh, Marina; Malvy, Claude

    2005-11-01

    The EWS/FLI-1 fusion gene, resulting from a t(11;22) translocation, plays a key role in the pathogenesis of Ewing sarcoma. Previously, we have shown that antisense oligonucleotides designed against EWS-Fli-1 inhibited tumor growth in nude mice provided they were delivered intratumorally by nanocapsules or by CTAB-coated nanospheres. In this study, we have used two types of nanospheres (designated as type 1 and type 2 nanospheres) stabilized with chitosan for both intratumoral and systemic administration of oligonucleotides. Inhibition of the tumor growth in vivo was found to be dependent on the carrier type as well as on antisense oligonucleotide modification. Indeed, whereas both types of nanospheres were efficient in reducing tumor growth after intratumoral injection, we have obtained only with type 2 nanospheres an antitumoral effect after intravenous injection in a preliminary experiment. Additionally, the anticancer efficacy of a localized modification of the EWS-Fli-1 phosphodiester/phosphorothioate chimeric antisense oligonucleotide was demonstrated. In cell culture the oligonucleotides inhibit cell growth by their antisense activity. Further investigations are needed in vivo to learn the mechanism of action of the complexes.

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

    PubMed

    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.

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

    PubMed Central

    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.

  7. Characterization of fecal microbiota from a Salmonella endemic cattle herd as determined by oligonucleotide fingerprinting of rDNA genes.

    PubMed

    Patton, Toni G; Scupham, Alexandra J; Bearson, Shawn M D; Carlson, Steve A

    2009-05-12

    The gastrointestinal (GI) tract microbiota is composed of complex communities. For all species examined thus far, culture and molecular analyses show that these communities are highly diverse and individuals harbor unique consortia. The objective of the current work was to examine inter-individual diversity of cattle fecal microbiota and determine whether Salmonella shedding status correlated with community richness or evenness parameters. Using a ribosomal gene array-based approach, oligonucleotide fingerprinting of ribosomal genes (OFRG), we analyzed 1440 16S genes from 19 fecal samples obtained from a cattle herd with a history of salmonellosis. Identified bacteria belonged to the phyla Firmicutes (53%), Bacteroidetes (17%), and Proteobacteria (17%). Sequence analysis of 16S rDNA gene clones revealed that Spirochaetes and Verrucomicrobia were also present in the feces. The majority of Firmicutes present in the feces belonged to the order Clostridiales, which was verified via dot blot analysis. beta-Proteobacteria represented 1.5% of the bacterial community as determined by real-time PCR. Statistical analysis of the 16S libraries from the 19 animals indicated very high levels of species richness and evenness, such that individual libraries represented unique populations. Finally, this study did not identify species that prevented Salmonella colonization or resulted from Salmonella colonization.

  8. Detection of Single-Nucleotide Polymorphism on uidA Gene of Escherichia coli by a Multiplexed Electrochemical DNA Biosensor with Oligonucleotide-Incorporated Nonfouling Surface

    PubMed Central

    Liu, Gang; Lao, Ruojun; Xu, Li; Xu, Qin; Li, Lanying; Zhang, Min; Shen, Hao; Mathur, Sanjay; Fan, Chunhai; Song, Shiping

    2011-01-01

    We report here a practical application of a multiplexed electrochemical DNA sensor for highly specific single-nucleotide polymorphism (SNP) detection. In this work, a 16-electrode array was applied with an oligonucleotide-incorporated nonfouling surfaces (ONS) on each electrode for the resistance of unspecific absorption. The fully matched target DNA templated the ligation between the capture probe assembled on gold electrodes and the tandem signal probe with a biotin moiety, which could be transduced to peroxidase-based catalyzed amperometric signals. A mutant site (T93G) in uidA gene of E. coli was analyzed in PCR amplicons. 10% percentage of single mismatched mutant gene was detected, which clearly proved the selectivity of the multiplexed electrochemical DNA biosensor when practically applied. PMID:22164059

  9. Toward a new paradigm of DNA writing using a massively parallel sequencing platform and degenerate oligonucleotide

    PubMed Central

    Hwang, Byungjin; Bang, Duhee

    2016-01-01

    All synthetic DNA materials require prior programming of the building blocks of the oligonucleotide sequences. The development of a programmable microarray platform provides cost-effective and time-efficient solutions in the field of data storage using DNA. However, the scalability of the synthesis is not on par with the accelerating sequencing capacity. Here, we report on a new paradigm of generating genetic material (writing) using a degenerate oligonucleotide and optomechanical retrieval method that leverages sequencing (reading) throughput to generate the desired number of oligonucleotides. As a proof of concept, we demonstrate the feasibility of our concept in digital information storage in DNA. In simulation, the ability to store data is expected to exponentially increase with increase in degenerate space. The present study highlights the major framework change in conventional DNA writing paradigm as a sequencer itself can become a potential source of making genetic materials. PMID:27876825

  10. Using Fluorophore-labeled Oligonucleotides to Measure Affinities of Protein-DNA Interactions

    PubMed Central

    Anderson, Brian J.; Larkin, Chris; Guja, Kip; Schildbach, Joel F.

    2011-01-01

    Changes in fluorescence emission intensity and anisotropy can reflect changes in the environment and molecular motion of a fluorophore. Researchers can capitalize on these characteristics to assess the affinity and specificity of DNA-binding proteins using fluorophore-labeled oligonucleotides. While there are many advantages to measuring binding using fluorescent oligonucleotides, there are also some distinct disadvantages. Here we describe some of the relevant issues for the novice, illustrating key points using data collected with the F plasmid relaxase domain and a variety of labeled oligonucleotides. Topics include selection of a fluorophore, experimental design using a fluorometer equipped with an automatic titrating unit, and analysis of direct binding and competition assays. PMID:19152864

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

    PubMed Central

    D'Agata, Roberta; Palladino, Pasquale

    2017-01-01

    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. PMID:28144559

  12. Stable triple helices formed by oligonucleotide N3'-->P5' phosphoramidates inhibit transcription elongation.

    PubMed Central

    Escudé, C; Giovannangeli, C; Sun, J S; Lloyd, D H; Chen, J K; Gryaznov, S M; Garestier, T; Hélène, C

    1996-01-01

    Oligonucleotide analogs with N3'-->P5' phosphoramidate linkages bind to the major groove of double-helical DNA at specific oligopurine.oligopyrimidine sequences. These triple-helical complexes are much more stable than those formed by oligonucleotides with natural phosphodiester linkages. Oligonucleotide phosphoramidates containing thymine and cytosine or thymine, cytosine, and guanine bind strongly to the polypurine tract of human immunodeficiency virus proviral DNA under physiological conditions. Site-specific cleavage by the Dra I restriction enzyme at the 5' end of the polypurine sequence was inhibited by triplex formation. A eukaryotic transcription assay was used to investigate the effect of oligophosphoramidate binding to the polypurine tract sequence on transcription of the type 1 human immunodeficiency virus nef gene under the control of a cytomegalovirus promoter. An efficient arrest of RNA polymerase II was observed at the specific triplex site at submicromolar concentrations. Images Fig. 2 Fig. 3 PMID:8633072

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

    NASA Astrophysics Data System (ADS)

    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.

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

    PubMed

    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.

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

    SciTech Connect

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

    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.

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

    NASA Astrophysics Data System (ADS)

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

    2004-02-01

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

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

    PubMed

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

    2015-07-07

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

    NASA Astrophysics Data System (ADS)

    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.

  19. Myelin Basic Protein and a Multiple Sclerosis-related MBP-peptide Bind to Oligonucleotides

    PubMed Central

    Rozenblum, Guido Tomás; Kaufman, Tomás; Vitullo, Alfredo Daniel

    2014-01-01

    Aptamer ligands for myelin basic protein (MBP) were obtained using the systematic evolution of ligand by exponential enrichment (SELEX) method. Two clones were isolated from a pool of oligonucleotides and tested for MBP targeting. Using purified MBP, we demonstrated the binding activity of the aptamers and we also showed the affinity of MBP for oligonucleotides of specific length. Moreover, one selected aptamer competitively inhibited the binding of an MBP-specific antibody to MBP and the aptamer was found more sensitive than a commercial antibody. In addition, we showed the ability of the aptamer to detect myelin-rich regions in paraffin-embedded mouse brain tissue. Therefore, the MBP-binding activity of the selected oligonucleotide may prove useful as a tool for life science and medical research for myelin detection and might be a good lead for testing it in autoimmune diseases such as multiple sclerosis. PMID:25202925

  20. Oligonucleotide primers, probes and molecular methods for the environmental monitoring of methanogenic archaea

    PubMed Central

    Narihiro, Takashi; Sekiguchi, Yuji

    2011-01-01

    Summary For the identification and quantification of methanogenic archaea (methanogens) in environmental samples, various oligonucleotide probes/primers targeting phylogenetic markers of methanogens, such as 16S rRNA, 16S rRNA gene and the gene for the α‐subunit of methyl coenzyme M reductase (mcrA), have been extensively developed and characterized experimentally. These oligonucleotides were designed to resolve different groups of methanogens at different taxonomic levels, and have been widely used as hybridization probes or polymerase chain reaction primers for membrane hybridization, fluorescence in situ hybridization, rRNA cleavage method, gene cloning, DNA microarray and quantitative polymerase chain reaction for studies in environmental and determinative microbiology. In this review, we present a comprehensive list of such oligonucleotide probes/primers, which enable us to determine methanogen populations in an environment quantitatively and hierarchically, with examples of the practical applications of the probes and primers. PMID:21375721

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

    PubMed Central

    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

  2. Sedimentation analysis of novel DNA structures formed by homo-oligonucleotides.

    PubMed

    Hatters, D M; Wilson, L; Atcliffe, B W; Mulhern, T D; Guzzo-Pernell, N; Howlett, G J

    2001-07-01

    Sedimentation velocity analysis has been used to examine the base-specific structural conformations and unusual hydrogen bonding patterns of model oligonucleotides. Homo-oligonucleotides composed of 8-28 residues of dA, dT, or dC nucleotides in 100 mM sodium phosphate, pH 7.4, at 20 degrees C behave as extended monomers. Comparison of experimentally determined sedimentation coefficients with theoretical values calculated for assumed helical structures show that dT and dC oligonucleotides are more compact than dA oligonucleotides. For dA oligonucleotides, the average width (1.7 nm), assuming a cylindrical model, is smaller than for control duplex DNA whereas the average rise per base (0.34 nm) is similar to that of B-DNA. For dC and dT oligonucleotides, there is an increase in the average widths (1.8 nm and 2.1 nm, respectively) whereas the average rise per base is smaller (0.28 nm and 0.23 nm, respectively). A significant shape change is observed for oligo dC(28) at lower temperatures (10 degrees C), corresponding to a fourfold decrease in axial ratio. Optical density, circular dichroism, and differential scanning calorimetry data confirm this shape change, attributable from nuclear magnetic resonance analysis to i-motif formation. Sedimentation equilibrium studies of oligo dG(8) and dG(16) reveal extensive self-association and the formation of G-quadruplexes. Continuous distribution analysis of sedimentation velocity data for oligo dG(16) identifies the presence of discrete dimers, tetramers, and dodecamers. These studies distinguish the conformational and colligative properties of the individual bases in DNA and their inherent capacity to promote specific folding pathways.

  3. Sedimentation analysis of novel DNA structures formed by homo-oligonucleotides.

    PubMed Central

    Hatters, D M; Wilson, L; Atcliffe, B W; Mulhern, T D; Guzzo-Pernell, N; Howlett, G J

    2001-01-01

    Sedimentation velocity analysis has been used to examine the base-specific structural conformations and unusual hydrogen bonding patterns of model oligonucleotides. Homo-oligonucleotides composed of 8-28 residues of dA, dT, or dC nucleotides in 100 mM sodium phosphate, pH 7.4, at 20 degrees C behave as extended monomers. Comparison of experimentally determined sedimentation coefficients with theoretical values calculated for assumed helical structures show that dT and dC oligonucleotides are more compact than dA oligonucleotides. For dA oligonucleotides, the average width (1.7 nm), assuming a cylindrical model, is smaller than for control duplex DNA whereas the average rise per base (0.34 nm) is similar to that of B-DNA. For dC and dT oligonucleotides, there is an increase in the average widths (1.8 nm and 2.1 nm, respectively) whereas the average rise per base is smaller (0.28 nm and 0.23 nm, respectively). A significant shape change is observed for oligo dC(28) at lower temperatures (10 degrees C), corresponding to a fourfold decrease in axial ratio. Optical density, circular dichroism, and differential scanning calorimetry data confirm this shape change, attributable from nuclear magnetic resonance analysis to i-motif formation. Sedimentation equilibrium studies of oligo dG(8) and dG(16) reveal extensive self-association and the formation of G-quadruplexes. Continuous distribution analysis of sedimentation velocity data for oligo dG(16) identifies the presence of discrete dimers, tetramers, and dodecamers. These studies distinguish the conformational and colligative properties of the individual bases in DNA and their inherent capacity to promote specific folding pathways. PMID:11423421

  4. Customized oligonucleotide microchips that convert multiple genetic information to simple patterns, are portable and reusable

    DOEpatents

    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.

  5. Purification of specific chromatin regions using oligonucleotides: capture hybridization analysis of RNA targets (CHART).

    PubMed

    Davis, Christopher P; West, Jason A

    2015-01-01

    Identification of genomic binding sites and proteins associated with noncoding RNAs will lead to more complete mechanistic characterization of the regulatory activities of noncoding RNAs. Capture hybridization analysis of RNA targets (CHART) is a powerful technique wherein specific RNA molecules are isolated from cross-linked nuclear extracts using complementary, biotinylated capture oligonucleotides, allowing subsequent identification of genomic DNA and proteins cross-linked to the RNA of interest. Here, we describe the procedure for CHART and list strategies to optimize nuclear extract preparation, capture oligonucleotide design, and isolation of nucleic acids and proteins enriched through CHART.

  6. Evaluation of an rRNA-derived oligonucleotide probe for culture confirmation of Neisseria gonorrhoeae.

    PubMed Central

    Rossau, R; Duhamel, M; Van Dyck, E; Piot, P; Van Heuverswyn, H

    1990-01-01

    The reliability of an rRNA-derived oligonucleotide probe for Neisseria gonorrhoeae was tested with 187 N. gonorrhoeae isolates, 81 Neisseria meningitidis isolates, and several strains of other bacterial species. The probe proved to be 100% specific and 100% sensitive. N. gonorrhoeae cells could also be reliably identified in contaminated cultures with the oligonucleotide probe. The 2.6-megadalton cryptic plasmid used as a probe for N. gonorrhoeae was shown to be less sensitive, detecting 179 of 181 N. gonorrhoeae isolates. Images PMID:1693630

  7. Transcriptional inhibition of the bacteriophage T7 early promoter region by oligonucleotide triple helix formation.

    PubMed

    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.

  8. Complementary addressed modification and cleavage of a single stranded DNA fragment with alkylating oligonucleotide derivatives.

    PubMed Central

    Vlassov, V V; Zarytova, V F; Kutiavin, I V; Mamaev, S V; Podyminogin, M A

    1986-01-01

    A single stranded DNA fragment was modified with alkylating derivatives of oligonucleotides complementary to a certain nucleotide sequences in the fragment. The derivatives carried aromatic 2-chloroethylamino groups at their 3'- or 5'-terminal nucleotide residues. Some of the derivatives carried both alkylating group and intercalating phenazine group which stabilized complementary complexes. It was found that these oligonucleotide derivatives modify the DNA fragment in a specific way near the target complementary nucleotide sequences, and the DNA fragment can be cleaved at the alkylated nucleotides positions. Alkylating derivatives carrying phenazine groups were found to be the most efficient in reaction with the DNA fragment. Images PMID:3714471

  9. [Oligonucleotide analogues bearing an acyclonucleoside linked by an internucleotide amide bond].

    PubMed

    Kochetkova, S V; Fillipova, E A; Kolganova, N A; Timofeev, E N; Florent'ev, V L

    2008-01-01

    Oligonucleotide analogues bearing an acyclocytidine linked to thymidine by an amide (3'-O-CH2-CO-N-5') bond were synthesized. Melting curves of duplexes formed by modified oligonucleotides and complementary natural oligomers were obtained and thermodynamic parameters of their formation were measured. Replacement of dCpT by a modified dinucleotide only moderately decreased the melting temperature of these modified duplexes in comparison with unmodified duplexes containing complementary natural bases. CD spectra of modified duplexes were studied, and the duplex spatial structures are discussed. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2008, vol. 34, no. 2; see also http://www.maik.ru.

  10. Oligonucleotide-templated chemical reactions: pushing the boundaries of a nature-inspired process.

    PubMed

    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.

  11. Sequence selective naked-eye detection of DNA harnessing extension of oligonucleotide-modified nucleotides.

    PubMed

    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.

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

    PubMed

    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.

  13. Diode Laser Arrays

    NASA Astrophysics Data System (ADS)

    Botez, Dan; Scifres, Don R.

    2005-11-01

    Contributors; 1. Monolithic phase-locked semiconductor laser arrays D. Botez; 2. High power coherent, semiconductor laser master oscillator power amplifiers and amplifier arrays D. F. Welch and D. G. Mehuys; 3. Microoptical components applied to incoherent and coherent laser arrays J. R. Leger; 4. Modeling of diode laser arrays G. R. Hadley; 5. Dynamics of coherent semiconductor laser arrays H. G. Winfuland and R. K. Defreez; 6. High average power semiconductor laser arrays and laser array packaging with an emphasis for pumping solid state lasers R. Solarz; 7. High power diode laser arrays and their reliability D. R. Scifres and H. H. Kung; 8. Strained layer quantum well heterostructure laser arrays J. J. Coleman; 9. Vertical cavity surface emitting laser arrays C. J. Chang-Hasnain; 10. Individually addressed arrays of diode lasers D. Carlin.

  14. Azide-alkyne "click" reaction performed on oligonucleotides with the universal nucleoside 7-octadiynyl-7-deaza-2'-deoxyinosine.

    PubMed

    Ming, Xin; Leonard, Peter; Heindl, Dieter; Seela, Frank

    2008-01-01

    Oligonucleotides containing 7-substituted 7-deaza-2'- deoxyinosine derivatives bearing alkynyl groups were prepared. The octa-1,7-diynyl derivative was functionalized with the non-fluorescent 3- azidocoumarin by the Huisgen-Sharpless-Meldal cycloaddition to afford a highly fluorescent oligonucleotide conjugate. The ambiguous base pairing character and the clickable side chain allows the incorporation of almost any reporter molecule to DNA.

  15. Oligonucleotides Containing Aminated 2'-Amino-LNA Nucleotides: Synthesis and Strong Binding to Complementary DNA and RNA.

    PubMed

    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.

  16. A reusable sensor for the label-free detection of specific oligonucleotides by surface plasmon fluorescence spectroscopy.

    PubMed

    Nöll, Gilbert; Su, Qiang; Heidel, Björn; Yu, Yaming

    2014-01-01

    The development of a reusable molecular beacon (MB)-based sensor for the label-free detection of specific oligonucleotides using surface plasmon fluorescence spectroscopy (SPFS) as the readout method is described. The MBs are chemisorbed at planar gold surfaces serving as fluorescence quenching units. Target oligonucleotides of 24 bases can be detected within a few minutes at high single-mismatch discrimination rates.

  17. Noncoder: a web interface for exon array-based detection of long non-coding RNAs

    PubMed Central

    Gellert, Pascal; Ponomareva, Yuliya; Braun, Thomas; Uchida, Shizuka

    2013-01-01

    Due to recent technical developments, a high number of long non-coding RNAs (lncRNAs) have been discovered in mammals. Although it has been shown that lncRNAs are regulated differently among tissues and disease statuses, functions of these transcripts are still unknown in most cases. GeneChip Exon 1.0 ST Arrays (exon arrays) from Affymetrix, Inc. have been used widely to profile genome-wide expression changes and alternative splicing of protein-coding genes. Here, we demonstrate that re-annotation of exon array probes can be used to profile expressions of tens of thousands of lncRNAs. With this annotation, a detailed inspection of lncRNAs and their isoforms is possible. To allow for a general usage to the research community, we developed a user-friendly web interface called ‘noncoder’. By uploading CEL files from exon arrays and with a few mouse clicks and parameter settings, exon array data will be normalized and analysed to identify differentially expressed lncRNAs. Noncoder provides the detailed annotation information of lncRNAs and is equipped with unique features to allow for an efficient search for interesting lncRNAs to be studied further. The web interface is available at http://noncoder.mpi-bn.mpg.de. PMID:23012263

  18. Quantitative Oligonucleotide Microarray Fingerprinting of Salmonella enterica isolates

    SciTech Connect

    Willse, Alan R.; Straub, Tim M.; Wunschel, Sharon C.; Small, Jack A.; Call, Douglas R.; Daly, Don S.; Chandler, Darrell P.

    2004-03-22

    We report on a genome-independent microbial fingerprinting method using nucleic acid microarrays for microbial forensics and epidemiology applications. We demonstrate that the microarray method provides high-resolution differentiation between closely related microorganisms using Salmonella enterica strains. In replicate trials we used a simple 192-probe nonamer array to construct a fingerprint library of 25 closely related Salmonella isolates. Controlling false discovery rate for multiple testing at alpha =.05, at least 295 of 300 pairs of S. enterica isolate fingerprints were found to be statistically distinct using a modified Hotelling Tsquared test. Although we find most pairs of Salmonella fingerprints to be distinct, forensic applications will also require a protocol for library construction and reliable microbial classification against a fingerprint library. We outline additional steps required to produce a protocol for library construction and reliable classification of unknown organisms.

  19. Development of a 37K high-density oligo-nucleotide microarray for rainbow trout

    Technology Transfer Automated Retrieval System (TEKTRAN)

    We have constructed a rainbow trout high-density oligonucleotide microarray by using all the available tentative consensus (TC) sequences from the Rainbow Trout Gene Index database (The Computational Biology and Functional Genomics Lab., Dana Farber Cancer Institute and Harvard School of Public Heal...

  20. Identification of enterotoxigenic Escherichia coli isolates with enzyme-labeled synthetic oligonucleotide probes.

    PubMed Central

    Medon, P P; Lanser, J A; Monckton, P R; Li, P; Symons, R H

    1988-01-01

    Commercially available kits containing alkaline phosphatase-labeled oligonucleotide probes for Escherichia coli heat-stable enterotoxins (STI-H, STI-P, and STII) and the heat-labile enterotoxin were compared with bioassays and radiolabeled recombinant DNA probes to identify enterotoxigenic E. coli from 100 clinical isolates. There was very good agreement between the three methods. PMID:3053766

  1. Methods for the Preparation of Large Quantities of Complex Single-Stranded Oligonucleotide Libraries

    PubMed Central

    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. PMID:24733454

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

    PubMed

    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.

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

    PubMed

    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.

  4. Tetrahedron-structured DNA and functional oligonucleotide for construction of an electrochemical DNA-based biosensor.

    PubMed

    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.

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

    PubMed

    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.

  6. Drug evaluation: ISIS-301012, an antisense oligonucleotide for the treatment of hypercholesterolemia.

    PubMed

    Burnett, John R

    2006-10-01

    ISIS-301012 is an antisense oligonucleotide inhibitor of apolipoprotein B-100, which is being developed by Isis Pharmaceuticals Inc for the potential treatment of hypercholesterolemia. A subcutaneous injectable formulation is currently undergoing phase 11 clinical trials, while phase I trials are underway with an oral formulation of the drug.

  7. Investigating Synthetic Oligonucleotide Targeting of Mir31 in Duchenne Muscular Dystrophy

    PubMed Central

    Hildyard, John CW; Wells, Dominic J

    2016-01-01

    Exon-skipping via synthetic antisense oligonucleotides represents one of the most promising potential therapies for Duchenne muscular dystrophy (DMD), yet this approach is highly sequence-specific and thus each oligonucleotide is of benefit to only a subset of patients. The discovery that dystrophin mRNA is subject to translational suppression by the microRNA miR31, and that miR31 is elevated in the muscle of DMD patients, raises the possibility that the same oligonucleotide chemistries employed for exon skipping could be directed toward relieving this translational block. This approach would act synergistically with exon skipping where possible, but by targeting the 3’UTR it would further be of benefit to the many DMD patients who express low levels of in-frame transcript. We here present investigations into the feasibility of combining exon skipping with several different strategies for miR31-modulation, using both in vitro models and the mdx mouse (the classical animal model of DMD), and monitoring effects on dystrophin at the transcriptional and translational level. We show that despite promising results from our cell culture model, our in vivo data failed to demonstrate similarly reproducible enhancement of dystrophin translation, suggesting that miR31-modulation may not be practical under current oligonucleotide approaches. Possible explanations for this disappointing outcome are discussed, along with suggestions for future investigations. PMID:27525173

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

    PubMed

    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.

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

    PubMed

    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. Chimeric RNase H-competent oligonucleotides directed to the HIV-1 Rev response element.

    PubMed

    Prater, Chrissy E; Saleh, Anthony D; Wear, Maggie P; Miller, Paul S

    2007-08-15

    Chimeric oligo-2'-O-methylribonucleotides containing centrally located patches of contiguous 2'-deoxyribonucleotides and terminating in a nuclease resistant 3'-methylphosphonate internucleotide linkage were prepared. The oligonucleotides were targeted to the 3'-side of HIV Rev response element (RRE) stem-loop IIB RNA, which is adjacent to the high affinity Rev protein binding site and is critical to virus function. Thermal denaturation experiments showed that chimeric oligonucleotides form very stable duplexes with a complementary single-stranded RNA, and gel electrophoretic mobility shift assays (EMSA) showed that they bind with high affinity and specificity to RRE stem-loop II RNA (K(D) approximately 200 nM). The chimeric oligonucleotides promote RNase H-mediated hydrolysis of RRE stem-loop II RNA and have half-lives exceeding 24h when incubated in cell culture medium containing 10% fetal calf serum. One of the chimeric oligonucleotides inhibited RRE mediated expression of chloramphenicol acetyl transferase (CAT) approximately 60% at a concentration of 300 nM in HEK 293T cells co-transfected with p-RRE/CAT and p-Rev mammalian expression vectors.

  11. Base-Pairing Systems Related to TNA: alpha-Threofuranosyl Oligonucleotides Containing Phosphoramidate Linkages

    NASA Technical Reports Server (NTRS)

    Meyer, Michael (Technical Monitor); Wu, Xiaolin; Guntha, Sreenivasulu; Ferenclc, Mathias; Krishnamurthy, Ramanarayanan; Eschenmoser, Albert

    2002-01-01

    (3'NH)- and (2'NH)-TNA, two isomeric phosphoramidate analogues of TNA (alpha-threofuranosyl-(3'-2') oligonucleotides), are shown to be efficient Watson-Crick base-pairing systems and to undergo intersystem crosspairing with TNA, RNA, and DNA.

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

    PubMed

    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.

  13. Discrimination of base differences in oligonucleotides using mid-infrared spectroscopy and multivariate analysis.

    PubMed

    Kelly, Jemma G; Martin-Hirsch, Pierre L; Martin, Francis L

    2009-07-01

    Attenuated total reflection Fourier transform-infrared (ATR-FTIR) spectroscopy was employed to interrogate a panel of simple oligonucleotides designed to contain various base differences; combined with subsequent multivariate analysis, we set out to determine whether the specificity of this approach would point to a novel means for mutation detection. Oligonucleotides were designed that were 15 bases in length and contained various combinations of purines (adenine, guanine) or pyrimidines (cytosine, thymine). These were applied to 1 cm x 1 cm low-E reflective glass slides, and triplicate samples were interrogated using ATR-FTIR spectroscopy. Per oligonucleotide sample, 10 independent spectral acquisitions were obtained. Prior to multivariate analysis, infrared spectra were baseline-corrected and vector normalized over the 1750-760 cm(-1) region specific to the chemical bonds of organic molecules. Spectral categories were then analyzed using principal component analysis (PCA) followed by linear discriminant analysis (LDA). Scores plots revealed that PCA-LDA clearly segregated different oligonucleotide sequences, even in the presence of a single base difference. Loadings plots confirmed the chemical entities associated with distinguishing base differences. These results suggest that mid-IR spectroscopy might have future roles in interrogating polymorphic forms of a DNA template.

  14. Genomic arrays in chronic lymphocytic leukemia routine clinical practice: are we ready to substitute conventional cytogenetics and fluorescence in situ hybridization techniques?

    PubMed

    Puiggros, Anna; Puigdecanet, Eulàlia; Salido, Marta; Ferrer, Ana; Abella, Eugènia; Gimeno, Eva; Nonell, Lara; Herranz, María José; Galván, Ana Belén; Rodríguez-Rivera, María; Melero, Carme; Pairet, Silvia; Bellosillo, Beatriz; Serrano, Sergi; Florensa, Lourdes; Solé, Francesc; Espinet, Blanca

    2013-05-01

    Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course. Del(11q) and del(17p), routinely studied by conventional G-banding cytogenetics (CGC) and fluorescence in situ hybridization (FISH), have been related to progression and shorter overall survival. Recently, array-based karyotyping has gained acceptance as a high-resolution new tool for detecting genomic imbalances. The aim of the present study was to compare genomic arrays with CGC and FISH to ascertain whether the current techniques could be substituted in routine procedures. We analyzed 70 patients with CLL using the Cytogenetics Whole-Genome 2.7M Array and CytoScan HD Array (Affymetrix), CGC and FISH with the classical CLL panel. Whereas 31.4% and 68.6% of patients presented abnormalities when studied by CGC and FISH, respectively, these rates increased when arrays were also analyzed (78.6% and 80%). Although abnormality detection is higher when arrays are applied, one case with del(11q) and three with del(17p) were missed by genomic arrays due to their limited sensitivity. We consider that the complete substitution of CGC and FISH by genomic arrays in routine laboratories could negatively affect the management of some patients harboring 11q or 17p deletions. In conclusion, genomic arrays are valid to detect known and novel genomic imbalances in CLL, but should be maintained as a complementary tool to the current techniques.

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

    PubMed

    Ito, Yuko; Sone, Yumiko; Mizutani, Takaharu

    2004-03-01

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

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

    PubMed Central

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

    2012-01-01

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

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

    PubMed Central

    2013-01-01

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

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

    PubMed

    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.

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

    PubMed Central

    Skogen, Michael; Roth, Jennifer; Yerkes, Sarah; Parekh-Olmedo, Hetal; Kmiec, Eric

    2006-01-01

    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. PMID:17014717

  20. Human leukocyte antigen typing using a knowledge base coupled with a high-throughput oligonucleotide probe array analysis.

    PubMed

    Zhang, Guang Lan; Keskin, Derin B; Lin, Hsin-Nan; Lin, Hong Huang; DeLuca, David S; Leppanen, Scott; Milford, Edgar L; Reinherz, Ellis L; Brusic, Vladimir

    2014-01-01

    Human leukocyte antigens (HLA) are important biomarkers because multiple diseases, drug toxicity, and vaccine responses reveal strong HLA associations. Current clinical HLA typing is an elimination process requiring serial testing. We present an alternative in situ synthesized DNA-based microarray method that contains hundreds of thousands of probes representing a complete overlapping set covering 1,610 clinically relevant HLA class I alleles accompanied by computational tools for assigning HLA type to 4-digit resolution. Our proof-of-concept experiment included 21 blood samples, 18 cell lines, and multiple controls. The method is accurate, robust, and amenable to automation. Typing errors were restricted to homozygous samples or those with very closely related alleles from the same locus, but readily resolved by targeted DNA sequencing validation of flagged samples. High-throughput HLA typing technologies that are effective, yet inexpensive, can be used to analyze the world's populations, benefiting both global public health and personalized health care.

  1. Triple helix-forming oligonucleotides conjugated to indolocarbazole poisons direct topoisomerase I-mediated DNA cleavage to a specific site.

    PubMed

    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.

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

    PubMed

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

    2016-01-20

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

  3. Design of a tobacco exon array with application to investigate the differential cadmium accumulation property in two tobacco varieties

    PubMed Central

    2012-01-01

    Background For decades the tobacco plant has served as a model organism in plant biology to answer fundamental biological questions in the areas of plant development, physiology, and genetics. Due to the lack of sufficient coverage of genomic sequences, however, none of the expressed sequence tag (EST)-based chips developed to date cover gene expression from the whole genome. The availability of Tobacco Genome Initiative (TGI) sequences provides a useful resource to build a whole genome exon array, even if the assembled sequences are highly fragmented. Here, the design of a Tobacco Exon Array is reported and an application to improve the understanding of genes regulated by cadmium (Cd) in tobacco is described. Results From the analysis and annotation of the 1,271,256 Nicotiana tabacum fasta and quality files from methyl filtered genomic survey sequences (GSS) obtained from the TGI and ~56,000 ESTs available in public databases, an exon array with 272,342 probesets was designed (four probes per exon) and tested on two selected tobacco varieties. Two tobacco varieties out of 45 accumulating low and high cadmium in leaf were identified based on the GGE biplot analysis, which is analysis of the genotype main effect (G) plus analysis of the genotype by environment interaction (GE) of eight field trials (four fields over two years) showing reproducibility across the trials. The selected varieties were grown under greenhouse conditions in two different soils and subjected to exon array analyses using root and leaf tissues to understand the genetic make-up of the Cd accumulation. Conclusions An Affymetrix Exon Array was developed to cover a large (~90%) proportion of the tobacco gene space. The Tobacco Exon Array will be available for research use through Affymetrix array catalogue. As a proof of the exon array usability, we have demonstrated that the Tobacco Exon Array is a valuable tool for studying Cd accumulation in tobacco leaves. Data from field and greenhouse

  4. Optimization and scale-up of oligonucleotide synthesis in packed bed reactors using computational fluid dynamics modeling.

    PubMed

    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).

  5. Characterization of the nanostructure of complexes formed by single- or double-stranded oligonucleotides with a cationic surfactant.

    PubMed

    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

  6. OligoPrep PVA support for oligonucleotide synthesis in columns on a scale up to 10 micromol.

    PubMed

    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.

  7. Base Composition-Independent Hybridization in Tetramethylammonium Chloride: A Method for Oligonucleotide Screening of Highly Complex Gene Libraries

    NASA Astrophysics Data System (ADS)

    Wood, William I.; Gitschier, Jane; Lasky, Laurence A.; Lawn, Richard M.

    1985-03-01

    An oligonucleotide hybridization procedure has been developed that eliminates the preferential melting of A\\cdot T versus G\\cdot C base pairs, allowing the stringency of the hybridization to be controlled as a function of probe length only. This technique, which uses tetramethylammonium chloride, is especially helpful whenever a highly complex library is screened with a pool of oligonucleotide probes, which usually vary widely in base composition. The procedure can also be applied advantageously whenever an exact match to an oligonucleotide probe is desired, such as in screening for clones having as little as a single-base alteration generated by in vitro mutagenesis.

  8. The development and characterization of a 57K single nucleotide polymorphism array for rainbow trout.

    PubMed

    Palti, Y; Gao, G; Liu, S; Kent, M P; Lien, S; Miller, M R; Rexroad, C E; Moen, T

    2015-05-01

    In this study, we describe the development and characterization of the first high-density single nucleotide polymorphism (SNP) genotyping array for rainbow trout. The SNP array is publically available from a commercial vendor (Affymetrix). The SNP genotyping quality was high, and validation rate was close to 90%. This is comparable to other farm animals and is much higher than previous smaller scale SNP validation studies in rainbow trout. High quality and integrity of the genotypes are evident from sample reproducibility and from nearly 100% agreement in genotyping results from other methods. The array is very useful for rainbow trout aquaculture populations with more than 40 900 polymorphic markers per population. For wild populations that were confounded by a smaller sample size, the number of polymorphic markers was between 10 577 and 24 330. Comparison between genotypes from individual populations suggests good potential for identifying candidate markers for populations' traceability. Linkage analysis and mapping of the SNPs to the reference genome assembly provide strong evidence for a wide distribution throughout the genome with good representation in all 29 chromosomes. A total of 68% of the genome scaffolds and contigs were anchored through linkage analysis using the SNP array genotypes, including ~20% of the genome assembly that has not been previously anchored to chromosomes.

  9. HNA and ANA high-affinity arrays for detections of DNA and RNA single-base mismatches.

    PubMed

    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.

  10. Development and characterization of a microheater array device for real-time DNA mutation detection

    NASA Astrophysics Data System (ADS)

    Williams, Layne; Okandan, Murat; Chagovetz, Alex; Blair, Steve

    2008-02-01

    DNA analysis, specifically single nucleotide polymorphism (SNP) detection, is becoming increasingly important in rapid diagnostics and disease detection. Temperature is often controlled to help speed reaction rates and perform melting of hybridized oligonucleotides. The difference in melting temperatures, Tm, between wild-type and SNP sequences, respectively, to a given probe oligonucleotide, is indicative of the specificity of the reaction. We have characterized Tm's in solution and on a solid substrate of three sequences from known mutations associated with Cystic Fibrosis. Taking advantage of Tm differences, a microheater array device was designed to enable individual temperature control of up to 18 specific hybridization events. The device was fabricated at Sandia National Laboratories using surface micromachining techniques. The microheaters have been characterized using an IR camera at Sandia and show individual temperature control with minimal thermal cross talk. Development of the device as a real-time DNA detection platform, including surface chemistry and associated microfluidics, is described.

  11. Development and characterization of a microheater array device for real-time DNA mutation detection

    NASA Astrophysics Data System (ADS)

    Williams, Layne; Okandan, Murat; Chagovetz, Alex; Blair, Steve

    2008-04-01

    DNA analysis, specifically single nucleotide polymorphism (SNP) detection, is becoming increasingly important in rapid diagnostics and disease detection. Temperature is often controlled to help speed reaction rates and perform melting of hybridized oligonucleotides. The difference in melting temperatures, Tm, between wild-type and SNP sequences, respectively, to a given probe oligonucleotide, is indicative of the specificity of the reaction. We have characterized Tm's in solution and on a solid substrate of three sequences from known mutations associated with Cystic Fibrosis. Taking advantage of Tm differences, a microheater array device was designed to enable individual temperature control of up to 18 specific hybridization events. The device was fabricated at Sandia National Laboratories using surface micromachining techniques. The microheaters have been characterized using an IR camera at Sandia and show individual temperature control with minimal thermal cross talk. Development of the device as a real-time DNA detection platform, including surface chemistry and associated microfluidics, is described.

  12. An Affymetrix Microarray Design for Microbial Genotyping

    DTIC Science & Technology

    2009-10-01

    Clostridium botulinum APRT Okra 5 Clostridium botulinum A str. ATCC 19397 5 Clostridium botulinum ATCC 3502 40 Clostridium botulinum B str. Eklund 17B 5...Clostridium botulinum SNP B1 str. Okra plasmid pCLD 20 Clostridium botulinum B1 str. Okra plasmid pCLD 5 Clostridium botulinum Bf 5 Clostridium...botulinum HPT Eklund 17B 10 Clostridium botulinum HPT Loch Maree 20 Clostridium botulinum HPT Okra 5 Clostridium botulinum A3 str. Loch Maree 5

  13. Carbon Nanotube Nanoelectrode Array as an Electronic Chip for Ultrasensitive Label-free DNA Detection

    NASA Technical Reports Server (NTRS)

    Li, Jun; Koehne, Jessica; Chen, Hua; Cassell, Alan; Ng, Hou Tee; Fan, Wendy; Ye, Qi; Han, Jie; Meyyappan, M.

    2003-01-01

    A reliable nanoelectrode array based on vertically aligned multi-walled carbon nanotubes (MWNTs) embedded in SiO2 is used for ultrasensitive DNA detection. Characteristic nanoelectrode behavior is observed using low-density MWNT arrays for measuring both bulk and surface immobilized redox species such as K4Fe(CN)6 and ferrocene derivatives. The open-end of MWNTs are found to present similar properties as graphite edge-plane electrodes with wide potential window, flexible chemical functionalities, and good biocompatibility. BRCA1 related oligonucleotide probes with 18 bp are selectively functionalized at the open ends of the nanotube array and specifically hybridized with oligonucleotide targets incorporated with a polyG tag. The guanine groups are employed as the signal moieties in the electrochemical measurements. R(bpy)(sup 2+, sub 3) mediator is used to further amplify the guanine oxidation signal. The hybridization of sub-attomoles of DNA targets is detected electrochemically by combining the MWNT nanoelectrode array with the R(bpy)(sup 2+, sub 3) amplification mechanism. This technique was employed for direct electrochemical detection of label-free PCR amplicon from a healthy donor through specific hybridization with the BRCA1 probe. The detection limit is estimated to be less than 1000 DNA molecules since abundant guanine bases in the PCR amplicon provides a large signal. This system provides a general platform for rapid molecular diagnostics in applications requiring ultrahigh sensitivity, high-degree of miniaturization, and simple sample preparation, and low-cost operation.

  14. Novel Targeted Therapy for Precursor B-Cell Acute Lymphoblastic Leukemia: Anti-CD22 Antibody-MXD3 Antisense Oligonucleotide Conjugate

    PubMed Central

    Satake, Noriko; Duong, Connie; Yoshida, Sakiko; Oestergaard, Michael; Chen, Cathy; Peralta, Rachael; Guo, Shuling; Seth, Punit P; Li, Yueju; Beckett, Laurel; Chung, Jong; Nolta, Jan; Nitin, Nitin; Tuscano, Joseph M

    2016-01-01

    The exponential rise in molecular and genomic data has generated a vast array of therapeutic targets. Oligonucleotide-based technologies to down regulate these molecular targets have promising therapeutic efficacy. However, there is relatively limited success in translating this into effective in vivo cancer therapeutics. The primary challenge is the lack of effective cancer cell-targeted delivery methods, particularly for a systemic disease such as leukemia. We developed a novel leukemia- targeting compound composed of a monoclonal antibody directly conjugated to an antisense oligonucleotide (ASO). Our compound uses an ASO that specifically targets the transcription factor MYC-associated factor X (MAX) dimerization protein 3 (MXD3), which was previously identified to be critical for precursor B-cell (preB) acute lymphoblastic leukemia (ALL) cell survival. The MXD3 ASO was conjugated to an anti-cluster of differentiation-22 (CD22) antibody (αCD22 Ab) that specifically targets most preB ALL. We demonstrated that the αCD22 Ab-ASO conjugate treatment showed MXD3 protein knockdown and leukemia cell apoptosis in vitro. We also demonstrated that the conjugate treatment showed cytotoxicity in normal B cells, but not in other hematopoietic cells, including hematopoietic stem cells. Furthermore, the conjugate treatment at the lowest dose tested (0.2 mg/kg Ab for 6 doses - twice a week for 3 wks) more than doubled the mouse survival time in both Reh (median survival time 20.5 versus 42.5 d, p < 0.001) and primary preB ALL (median survival time 29.3 versus 63 d, p < 0.001) xenograft models. Our conjugate that uses αCD22 Ab to target the novel molecule MXD3, which is highly expressed in preB ALL cells, appears to be a promising novel therapeutic approach. PMID:27455414

  15. A new approach to the synthesis of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotide analogues.

    PubMed Central

    Szabó, T; Kers, A; Stawinski, J

    1995-01-01

    A new synthetic method for the preparation of the 5'-deoxy-5'-methylphosphonate linked thymidine oligonucleotides (5'-methylenephosphonate analogues) was developed. The method is based on the use of a phosphonate protecting group, 4-methoxy-1-oxido-2-picolyl, enabling intramolecular nucleophilic catalysis which together with the condensing agent, 2,4,6-triisopropylbenzenesulfonyl chloride, secures fast and efficient formation of the 5'-methylenephosphonate internucleosidic bonds. The produced protected oligomers were treated with thiophenol and triethylamine to remove the phosphonate protecting groups, cleaved from the solid support using concentrated aqueous ammonia, and purified by HPLC. Several thymidine oligonucleotide analogues with the chain length of up to 20 nucleotidic units, in which all internal 5'-oxygen atoms have been replaced by methylene groups directly bound to phosphorus, were synthesised using this methodology. PMID:7731801

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

    PubMed

    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.

  17. The Role of Fluorinated Alcohols as Mobile Phase Modifiers for LC-MS Analysis of Oligonucleotides

    NASA Astrophysics Data System (ADS)

    Basiri, Babak; van Hattum, Hilde; van Dongen, William D.; Murph, Mandi M.; Bartlett, Michael G.

    2017-01-01

    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.

  18. Improving the understanding of oligonucleotide-nanoparticle conjugates using DNA-binding fluorophores

    NASA Astrophysics Data System (ADS)

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

    2013-05-01

    A novel fluorescent-based method for characterisation of oligonucleotide-nanoparticle conjugates (ONPCs) is reported. We exploit the ability of the double-stranded DNA-binding dye, SYBR Green I, to develop a powerful analytical tool to investigate the melting properties and hybridisation behavior of ONPCs as well as their corresponding DNA-mediated assemblies.A novel fluorescent-based method for characterisation of oligonucleotide-nanoparticle conjugates (ONPCs) is reported. We exploit the ability of the double-stranded DNA-binding dye, SYBR Green I, to develop a powerful analytical tool to investigate the melting properties and hybridisation behavior of ONPCs as well as their corresponding DNA-mediated assemblies. Electronic supplementary information (ESI) available: Schematic description of the ONPC system. Extinction spectra of Ag and Au colloids, together with the absorption and emission spectra of SYBR Green I. Extinction and melting properties of the ONPCs. See DOI: 10.1039/c3nr01197f

  19. Evaluating bacterial activity from cell-specific ribosomal RNA content measured with oligonucleotide probes

    SciTech Connect

    Kemp, P.F.; Lee, S.; LaRoche, J.

    1992-10-01

    We describe a procedure for measuring the cell-specific quantity of ribosomal RNA (rRNA) and DNA in order to evaluate the frequency distribution of activity among cells. The procedure is inherently quantitative, does not require sample incubation and potentially can be taxon-specific. Fluorescently-labelled oligonucleotide probes are hybridized to the complementary 16S rRNA sequences in preserved, intact cells. The resulting cell fluorescence is proportional to cellular rRNA content and can be measured with a microscope-mounted photometer system, by image analysis, or by flow cytometry. Similarly, DNA content is measured as fluorescence of cells stained with the DNA specific fluorochrome DAPI. These are either prepared as separate samples for purposes of enumeration and DNA measurements, or are dual-labelled cells which are also hybridized with oligonucleotide probes.

  20. Evaluating bacterial activity from cell-specific ribosomal RNA content measured with oligonucleotide probes

    SciTech Connect

    Kemp, P.F.; Lee, S.; LaRoche, J.

    1992-01-01

    We describe a procedure for measuring the cell-specific quantity of ribosomal RNA (rRNA) and DNA in order to evaluate the frequency distribution of activity among cells. The procedure is inherently quantitative, does not require sample incubation and potentially can be taxon-specific. Fluorescently-labelled oligonucleotide probes are hybridized to the complementary 16S rRNA sequences in preserved, intact cells. The resulting cell fluorescence is proportional to cellular rRNA content and can be measured with a microscope-mounted photometer system, by image analysis, or by flow cytometry. Similarly, DNA content is measured as fluorescence of cells stained with the DNA specific fluorochrome DAPI. These are either prepared as separate samples for purposes of enumeration and DNA measurements, or are dual-labelled cells which are also hybridized with oligonucleotide probes.

  1. Synthesis of the tellurium-derivatized phosphoramidites and their incorporation into DNA oligonucleotides.

    PubMed

    Jiang, Sibo; Sheng, Jia; Huang, Zhen

    2011-12-01

    In this unit, an efficient method for the synthesis of 2'-tellerium-modified phosphoramidite and its incorporation into oligonucleotide are presented. We choose 5'-O-DMTr-2,2'-anhydro-uridine and -thymidine nucleosides (S.1, S.2) as starting materials due to their easy preparation. The 5'-O-DMTr-2,2'-anhydro-uridine and -thymidine can be converted to the corresponding 2'-tellerium-derivatized nucleosides by treating with the telluride nucleophiles. Subsequently, the 2'-Te-nucleosides can be transformed into 3'-phosphoramidites, which are the building blocks for DNA/RNA synthesis. The DNA synthesis, purification, and applications of oligonucleotides containing 2'-Te-U or 2'-Te-T are described in the protocol.

  2. Synthesis of the first tellurium-derivatized oligonucleotides for structural and functional studies.

    PubMed

    Sheng, Jia; Hassan, Abdalla E A; Huang, Zhen

    2009-10-05

    We report here the first synthesis of Te-nucleoside phosphoramidites and Te-modified oligonucleotides. We protected the 2'-tellurium functionality by alkylation and found that the Te functionality is compatible with solid-phase synthesis and that the Te oligonucleotides are stable during deprotection and purification. In addition, the redox properties of the Te functionalities have been explored. We found that the telluride and telluoxide DNAs are interchangeable by redox reactions. At elevated temperature, the Te-DNA can also be site-specifically fragmented oxidatively or reductively when 2'-TePh functionality is present, whereas elimination of the nucleobase is observed in the presence of 2'-TeMe. Moreover, the stability of the DNA duplexes derivatized with the Te functionalities has been investigated. Our Te derivatization of nucleic acids provides a novel approach for investigating DNA damage as well as for structure and function studies of nucleic acids and their protein complexes.

  3. Recognition and sensing of low-epitope targets via ternary complexes with oligonucleotides and synthetic receptors

    NASA Astrophysics Data System (ADS)

    Yang, Kyung-Ae; Barbu, Mihaela; Halim, Marlin; Pallavi, Payal; Kim, Benjamin; Kolpashchikov, Dmitry M.; Pecic, Stevan; Taylor, Steven; Worgall, Tilla S.; Stojanovic, Milan N.

    2014-11-01

    Oligonucleotide-based receptors or aptamers can interact with small molecules, but the ability to achieve high-affinity and specificity of these interactions depends strongly on functional groups or epitopes displayed by the binding targets. Some classes of targets are particularly challenging: for example, monosaccharides have scarce functionalities and no aptamers have been reported to recognize, let alone distinguish from each other, glucose and other hexoses. Here we report aptamers that differentiate low-epitope targets such as glucose, fructose or galactose by forming ternary complexes with high-epitope organic receptors for monosaccharides. In a follow-up example, we expand this method to isolate high-affinity oligonucleotides against aromatic amino acids complexed in situ with a nonspecific organometallic receptor. The method is general and enables broad clinical use of aptamers for the detection of small molecules in mix-and-measure assays, as demonstrated by monitoring postprandial waves of phenylalanine in human subjects.

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

    PubMed

    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.

  5. Copper(II)-Thymine Coordination Polymer Nanoribbons as Potential Oligonucleotide Nanocarriers.

    PubMed

    Vegas, Verónica G; Lorca, Romina; Latorre, Ana; Hassanein, Khaled; Gómez-García, Carlos J; Castillo, Oscar; Somoza, Álvaro; Zamora, Félix; Amo-Ochoa, Pilar

    2017-01-19

    The direct reaction between copper nitrate, thymine-1-acetic acid, and 4,4'-bipyridine in water leads to the formation of a blue colloid comprising uniform crystalline nanoribbons (length >1 μm; width ca. 150-185 nm; diameter ca. 15-60 nm) of a coordination polymer. The polymer displays a thymine-based structure freely available for supramolecular interactions. These nanostructures show significant selective interaction with single-stranded oligonucleotides based on adenine. Remarkably, they present low cell toxicity in three cell lines-despite the copper(II) content-and can be used as nanocarriers of oligonucleotides. These results suggest the potential of these types of nanostructures in several biological applications.

  6. Wash-free magnetic oligonucleotide probes-based NMR sensor for detecting the Hg ion.

    PubMed

    Ma, Wenwei; Hao, Changlong; Ma, Wei; Xing, Changrui; Yan, Wenjing; Kuang, Hua; Wang, Libing; Xu, Chuanlai

    2011-12-14

    An easily applied and sensitive sensor for the detection of heavy metal ion residues based entirely on magnetic nanoparticle and oligonucleotide was developed. The tool is established on the relaxation of magnetic nanoparticles with different dispersion states. The target analyte, Hg ions, induce the aggregation of the MNP oligonucleotide probes. Accordingly, the light produced by the magnetic relaxation image and the transverse relaxation time (T(2)) all change due to the effect of the aggregation. The limit of qualitative detection of the sensor is 0.15 ppt. The recoveries from test samples range between 97.1-101.8%. Using the nuclear resonance instrument, the method is a high throughput and sensitive sensor.

  7. A Solution to the Common Problem of the Synthesis and Applications of Hexachlorofluorescein Labeled Oligonucleotides

    PubMed Central

    Chuvilin, Andrey N.; Smirnov, Igor P.; Mosina, Alena G.; Varizhuk, Anna M.; Pozmogova, Galina E.

    2016-01-01

    A common problem of the preparation of hexachlorofluorescein labeled oligonucleotides is the transformation of the fluorophore to an arylacridine derivative under standard ammonolysis conditions. We show here that the arylacridine byproduct with distinct optical characteristics cannot be efficiently separated from the major product by HPLC or electrophoretic methods, which hampers precise physicochemical experiments with the labeled oligonucleotides. Studies of the transformation mechanism allowed us to select optimal conditions for avoiding the side reaction. The novel method for the post-synthetic deblocking of hexachlorofluorescein-labeled oligodeoxyribonucleotides described in this paper prevents the formation of the arylacridine derivative, enhances the yield of target oligomers, and allows them to be proper real-time PCR probes. PMID:27861573

  8. Computer selection of oligonucleotide probes from amino acid sequences for use in gene library screening.

    PubMed

    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.

  9. Sequence-specific photoinduced c-fos gene damage mediated by triple stranded-forming oligonucleotide conjugated to psoralen

    NASA Astrophysics Data System (ADS)

    Cao, En-Hua; Wang, Ju-jun; Ma, Wenjian; Qin, Jingfen

    1999-09-01

    A psoralen-oligonucleotide conjugate was designed to photoinduce a cross-link at a specific sequence of c-fos oncogene. Psoralen was attached to its C-3 position of a 20-base mer oligonucleotide, which binds to a synthetic 49 bp duplex containing the c-fos gene polypurine site, where it forms a triple stranded DNA. Upon near-UV-irradiation, the two strand of DNA are crosslinked at the TpA step present at the triple-duplex junction. Results show that the yield of the photoinduce cross- linking reaction is quite high. We treated HeLa cells with above 2-mer oligonucleotide conjugated to psoralen. The expression of c-fos oncogene was significant reduced, no significant effect on the level of c-myc mRNA. These data indicate that such psoralen- oligonucleotide conjugates could be used to selectively control gene expression or to induce sequence-specific damages.

  10. Synthesis, thermal stability and reactivity towards 9-aminoellipticine of double-stranded oligonucleotides containing a true abasic site.

    PubMed Central

    Bertrand, J R; Vasseur, J J; Rayner, B; Imbach, J L; Paoletti, J; Paoletti, C; Malvy, C

    1989-01-01

    A 13 mers abasic oligonucleotide was synthetized. It was therefore possible to compare thermal stability and reactivity of duplex oligonucleotides either with an apurinic/apyrimidinic site or without any lesion. An important decrease in the melting temperature appeared for duplexes with an abasic site. The chemical reaction of these modified oligonucleotides with the intercalating agent 9-aminoellipticine was studied by gel electrophoresis and by fluorescence. The formation of a Schiff base between 9-aminoellipticine and abasic sites was rapid and complete with duplexes at 11 degrees C. Schiff base related fluorescence and beta-elimination cleavage were more important with the apyrimidinic sites than with the apurinic ones. When compared to previous results obtained with the model d(TprpT) some unexpected behaviours appeared with longer and duplex oligonucleotides. For instance only partial beta-elimination cleavage was observed. It is likely that stacking parameters in the double helix play a great role in the studied reaction. Images PMID:2602153

  11. Non-random frequencies of short oligonucleotide sequences within poliomyelitis and encephalomyocarditis virus RNAs.

    PubMed Central

    Hughes, D G; Maden, B E

    1975-01-01

    In poliomyelitis and encephalomyocarditis virus RNAs the relative frequencies of several short oligonucleotide sequences differ substantially from those expected on a random basis. The frequencies of CpGp-containing sequences are low, and the frequency of GpUpApGp is considerably lower than that of GpApUpGp. The relationship of these findings to those obtained from host cell RNAs is descussed. Images PMID:168559

  12. Nonenzymatic template-directed synthesis on hairpin oligonucleotides. III - Incorporation of adenosine and uridine residues

    NASA Technical Reports Server (NTRS)

    Wu, Taifeng; Orgel, Leslie E.

    1992-01-01

    Nonenzymatic template-directed incorporation of adenosine and uridine residues into template sequences was obtained using nucleoside-5-prime phosphoro (2-methyl)imidazolides as substrates and hairpin oligonucleotides as templates. The reactions are regiospecific, producing mainly 3-prime-5-prime phosphodiester bonds. Limited synthesis of CA and AC sequences was observed along with some synthesis of the AA sequences on templates containing TG and GT sequences, along wilth some synthesis of the AA sequences on templates containing TT sequences.

  13. Direct Oligonucleotide-Photosensitizer Conjugates for Photochemical Delivery of Antisense Oligonucleotides†

    PubMed Central

    Yuan, Ahu; Laing, Brian; Hu, Yiqiao

    2015-01-01

    Activation of photosensitizers in endosomes enables release of therapeutic macromolecules into cytosol of the target cells for pharmacological actions. In this study, we demonstrate that direct conjugation of photosensitizer 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. PMID:25786195

  14. Transcript copy number estimation using a mouse whole-genome oligonucleotide microarray

    PubMed Central

    Carter, Mark G; Sharov, Alexei A; VanBuren, Vincent; Dudekula, Dawood B; Carmack, Condie E; Nelson, Charlie; Ko, Minoru SH

    2005-01-01

    The ability to quantitatively measure the expression of all genes in a given tissue or cell with a single assay is an exciting promise of gene-expression profiling technology. An in situ-synthesized 60-mer oligonucleotide microarray designed to detect transcripts from all mouse genes was validated, as well as a set of exogenous RNA controls derived from the yeast genome (made freely available without restriction), which allow quantitative estimation of absolute endogenous transcript abundance. PMID:15998450

  15. Near-infrared silver cluster optically signaling oligonucleotide hybridization and assembling two DNA hosts.

    PubMed

    Petty, Jeffrey T; Nicholson, David A; Sergev, Orlin O; Graham, Stuart K

    2014-09-16

    Silver clusters with ~10 atoms form within DNA strands, and the conjugates are chemical sensors. The DNA host hybridizes with short oligonucleotides, and the cluster moieties optically respond to these analytes. Our studies focus on how the cluster adducts perturb the structure of their DNA hosts. Our sensor is comprised of an oligonucleotide with two components: a 5'-cluster domain that complexes silver clusters and a 3'-recognition site that hybridizes with a target oligonucleotide. The single-stranded sensor encapsulates an ~11 silver atom cluster with violet absorption at 400 nm and with minimal emission. The recognition site hybridizes with complementary oligonucleotides, and the violet cluster converts to an emissive near-infrared cluster with absorption at 730 nm. Our key finding is that the near-infrared cluster coordinates two of its hybridized hosts. The resulting tertiary structure was investigated using intermolecular and intramolecular variants of the same dimer. The intermolecular dimer assembles in concentrated (~5 μM) DNA solutions. Strand stoichiometries and orientations were chromatographically determined using thymine-modified complements that increase the overall conjugate size. The intramolecular dimer develops within a DNA scaffold that is founded on three linked duplexes. The high local cluster concentrations and relative strand arrangements again favor the antiparallel dimer for the near-infrared cluster. When the two monomeric DNA/violet cluster conjugates transform to one dimeric DNA/near-infrared conjugate, the DNA strands accumulate silver. We propose that these correlated changes in DNA structure and silver stoichiometry underlie the violet to near-infrared cluster transformation.

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

    PubMed

    Shahmuradyan, Anna; Krull, Ulrich J

    2016-03-15

    Quantum dots (QDs) have been widely used in chemical and biosensing due to their unique photoelectrical properties and are well suited as donors in fluorescence resonance energy transfer (FRET). Selective hybridization interactions of oligonucleotides on QDs have been determined by FRET. Typically, the QD-FRET constructs have made use of labeled targets or have implemented labeled sandwich format assays to introduce dyes in proximity to the QDs for the FRET process. The intention of this new work is to explore a method to incorporate the acceptor dye into the probe molecule. Thiazole orange (TO) derivatives are fluorescent intercalating dyes that have been used for detection of double-stranded nucleic acids. One such dye system has been reported in which single-stranded oligonucleotide probes were doubly labeled with adjacent thiazole orange derivatives. In the absence of the fully complementary (FC) oligonucleotide target, the dyes form an H-aggregate, which results in quenching of fluorescence emission due to excitonic interactions between the dyes. The hybridization of the FC target to the probe provides for dissociation of the aggregate as the dyes intercalate into the double stranded duplex, resulting in increased fluorescence. This work reports investigation of the dependence of the ratiometric signal on the type of linkage used to conjugate the dyes to the probe, the location of the dye along the length of the probe, and the distance between adjacent dye molecules. The limit of detection for 34mer and 90mer targets was found to be identical and was 10 nM (2 pmol), similar to analogous QD-FRET using labeled oligonucleotide target. The detection system could discriminate a one base pair mismatch (1BPM) target and was functional without substantial compromise of the signal in 75% serum. The 1BPM was found to reduce background signal, indicating that the structure of the mismatch affected the environment of the intercalating dyes.

  17. Simple and efficient oligonucleotide-directed mutagenesis using one primer and circular plasmid DNA template.

    PubMed

    Marotti, K R; Tomich, C S

    1989-01-01

    A rapid and simple procedure for site-directed mutagenesis is described. This method uses only a single oligonucleotide primer with the double-stranded circular plasmid DNA as the template for mutagenesis. The phage T4 gene 32 product is included during primer extension in vitro to increase efficiency. Single and multiple changes as well as deletions have been obtained at an efficiency of 1-2%.

  18. Targeted Delivery of Therapeutic Oligonucleotides for the Treatment of Prostate Cancer

    DTIC Science & Technology

    2004-05-01

    ODN were entrapped in a lipid vector that was 200 stable in human plasma. This involves the formation in 0. Im= and extraction of cationic lipid/ODN...more pro- (8) Cumin , F., Asselbergs, F., Lartigot, M., and Felder, E. (1993) nounced with increasing amounts of ceramide PEG Modulation of human...oligonucleotides, Mol.Pharmacol. 41 (1992) 1023-1033. treatment. A much lower dose of DOX was re- [4] F. Cumin , F. Asselbergs, M. Lartigot, E. Felder

  19. End-labeling and analysis of Spo11-oligonucleotide complexes in Saccharomyces cerevisiae.

    PubMed

    Neale, Matthew J; Keeney, Scott

    2009-01-01

    During meiosis Spo11 catalyzes the formation of DNA double-strand breaks, becoming covalently attached to the 5' ends on both sides of the break during this process. Spo11 is removed from the DSB by single-stranded endonucleolytic cleavage flanking the DSB, liberating a short-lived species consisting of Spo11 protein covalently linked to a short oligonucleotide. The method presented here details how to detect these Spo11-oligo complexes in extracts made from meiotic yeast cells.

  20. GenoMass software: a tool based on electrospray ionization tandem mass spectrometry for characterization and sequencing of oligonucleotide adducts

    PubMed Central

    Sharma, Vaneet K; Glick, James; Liao, Qing; Shen, Chang; Vouros, Paul

    2012-01-01

    The analysis of DNA adducts is of importance in understanding DNA damage, and in the last few years mass spectrometry (MS) has emerged as the most comprehensive and versatile tool for routine characterization of modified oligonucleotides. The structural analysis of modified oligonucleotides, although routinely analyzed using mass spectrometry, is followed by a large amount of data, and a significant challenge is to locate the exact position of the adduct by computational spectral interpretation, which still is a bottleneck. In this report, we present an additional feature of the in-house developed GenoMass software, which determines the exact location of an adduct in modified oligonucleotides by connecting tandem mass spectrometry (MS/MS) to a combinatorial isomer library generated in silico for nucleic acids. The performance of this MS/MS approach using GenoMass software was evaluated by MS/MS data interpretation for an unadducted and its corresponding N-acetylaminofluorene (AAF) adducted 17-mer (5′OH-CCT ACC CCT TCC TTG TA-3′OH) oligonucleotide. Further computational screening of this AAF adducted 17-mer oligonucleotide (5′OH-CCT ACC CCT TCC TTG TA-3′OH) from a complex oligonucleotide mixture was performed using GenoMass. Finally, GenoMass was also used to identify the positional isomers of the AAF adducted 15-mer oligonucleotide (5′OH-ATGAACCGGAGGCCC-3′OH). GenoMass is a simple, fast, data interpretation software that uses an in silico constructed library to relate the MS/MS sequencing approach to identify the exact location of adduct on oligonucleotides. PMID:22689626

  1. Triple helix formation with purine-rich phosphorothioate-containing oligonucleotides covalently linked to an acridine derivative.

    PubMed Central

    Lacoste, J; François, J C; Hélène, C

    1997-01-01

    Purine-rich (GA)- and (GT)-containing oligophosphorothioates were investigated for their triplex-forming potential on a 23 bp DNA duplex target. In our system, GA-containing oligophosphorothioates (23mer GA-PS) were capable of triplex formation with binding affinities lower than (GA)-containing oligophosphodiesters (23mer GA-PO). The orientation of the third strand 23mers GA-PS and GA-PO was antiparallel to the purine strand of the duplex DNA target. In contrast, (GT)-containing oligophosphorothioates (23mer GT-PS) did not support triplex formation in either orientation, whereas the 23mer GT-PO oligophosphodiester demonstrated triplex formation in the antiparallel orientation. GA-PS oligonucleotides, in contrast to GT-PS oligonucleotides, were capable of self-association, but these self-associated structures exhibited lower stabilities than those formed with GA-PO oligonucleotides, suggesting that homoduplex formation (previously described for the 23mer GA-PO sequence by Noonberg et al.) could not fully account for the decrease in triplex stability when phosphorothioate linkages were used. The 23mer GA-PS oligonucleotide was covalently linked via its 5'-end to an acridine derivative (23mer Acr-GA-PS). In the presence of potassium cations, this conjugate demonstrated triplex formation with higher binding affinity than the unmodified 23mer GA-PS oligonucleotide and even than the 23mer GA-PO oligonucleotide. A (GA)-containing oligophosphodiester with two phosphorothioate linkages at both the 5'- and 3'-ends exhibited similar binding affinity to duplex DNA compared with the unmodified GA-PO oligophosphodiester. This capped oligonucleotide was more resistant to nucleases than the GA-PO oligomer and thus represents a good alternative for ex vivo applications of (GA)-containing, triplex-forming oligonucleotides, allowing a higher binding affinity for its duplex target without rapid cellular degradation. PMID:9115367

  2. Oligonucleotide-based systems: DNA, microRNAs, DNA/RNA aptamers

    PubMed Central

    Jolly, Pawan; Estrela, Pedro

    2016-01-01

    There are an increasing number of applications that have been developed for oligonucleotide-based biosensing systems in genetics and biomedicine. Oligonucleotide-based biosensors are those where the probe to capture the analyte is a strand of deoxyribonucleic acid (DNA), ribonucleic acid (RNA) or a synthetic analogue of naturally occurring nucleic acids. This review will shed light on various types of nucleic acids such as DNA and RNA (particularly microRNAs), their role and their application in biosensing. It will also cover DNA/RNA aptamers, which can be used as bioreceptors for a wide range of targets such as proteins, small molecules, bacteria and even cells. It will also highlight how the invention of synthetic oligonucleotides such as peptide nucleic acid (PNA) or locked nucleic acid (LNA) has pushed the limits of molecular biology and biosensor development to new perspectives. These technologies are very promising albeit still in need of development in order to bridge the gap between the laboratory-based status and the reality of biomedical applications. PMID:27365033

  3. Targeting the r(CGG) Repeats That Cause FXTAS with Modularly Assembled Small Molecules and Oligonucleotides

    PubMed Central

    2015-01-01

    We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)exp) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)exp toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)expin vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)exp’s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2′-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide. PMID:24506227

  4. Antisense oligonucleotide treatment ameliorates alpha-1 antitrypsin–related liver disease in mice

    PubMed Central

    Guo, Shuling; Booten, Sheri L.; Aghajan, Mariam; Hung, Gene; Zhao, Chenguang; Blomenkamp, Keith; Gattis, Danielle; Watt, Andrew; Freier, Susan M.; Teckman, Jeffery H.; McCaleb, Michael L.; Monia, Brett P.

    2013-01-01

    Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease that results from mutations in the alpha-1 antitrypsin (AAT) gene. The mutant AAT protein aggregates and accumulates in the liver leading to AATD liver disease, which is only treatable by liver transplant. The PiZ transgenic mouse strain expresses a human AAT (hAAT) transgene that contains the AATD-associated Glu342Lys mutation. PiZ mice exhibit many AATD symptoms, including AAT protein aggregates, increased hepatocyte death, and liver fibrosis. In the present study, we systemically treated PiZ mice with an antisense oligonucleotide targeted against hAAT (AAT-ASO) and found reductions in circulating levels of AAT and both soluble and aggregated AAT protein in the liver. Furthermore, AAT-ASO administration in these animals stopped liver disease progression after short-term treatment, reversed liver disease after long-term treatment, and prevented liver disease in young animals. Additionally, antisense oligonucleotide treatment markedly decreased liver fibrosis in this mouse model. Administration of AAT-ASO in nonhuman primates led to an approximately 80% reduction in levels of circulating normal AAT, demonstrating potential for this approach in higher species. Antisense oligonucleotides thus represent a promising therapy for AATD liver disease. PMID:24355919

  5. Determination of pKa values for deprotonable nucleobases in short model oligonucleotides.

    PubMed

    González-Olvera, Julio C; Martínez-Reyes, José; González-Jasso, Eva; Pless, Reynaldo C

    2015-11-01

    The deprotonation of ionizable nucleobases centrally placed in short model oligonucleotides was examined under different physical conditions, using UV absorption spectroscopy. The oligonucleotide sequences were designed so that only the central base would be ionized over the pH range examined. pKa values of 9.90±0.01 and 9.34±0.04 were determined for the guanine group in the oligomer d-ACAGCAC and 2'-deoxyguanosine, respectively, both at 25°C and 0.1M NaCl. Lengthening the oligonucleotide up to the tridecamer stage further increases the pKa of the central guanine moiety. Electrolyte concentration, temperature, and mixed water-ethanol solvents affect the acidity of the central base. Changes in the sequence surrounding the central guanine can also have a significant effect, especially in the case of strongly stacking sequences. The pKa values were also determined for the hepta(2'-O-methyl)ribonucleotide and the heptamer PNA of identical sequence, as well as for oligodeoxyribonucleotides with different deprotonable bases, viz. thymine, uracil, or hypoxanthine, in the central position. The results are interpreted in terms of the electric-field effect exerted on the departing proton by the negative electric charges located on the internucleotide phosphate groups, and calculations show this effect to approximately explain the magnitude of the pKa difference observed between the deoxyriboheptanucleotide and its electroneutral PNA analogue.

  6. Identification of enterotoxigenic Escherichia coli with synthetic alkaline phosphatase-conjugated oligonucleotide DNA probes.

    PubMed Central

    Seriwatana, J; Echeverria, P; Taylor, D N; Sakuldaipeara, T; Changchawalit, S; Chivoratanond, O

    1987-01-01

    Alkaline phosphatase-conjugated (AP) 26-base oligonucleotide DNA probes were compared with the same probes labeled with gamma-32P for the identification of heat-labile (LT) and heat-stable (ST) enterotoxigenic Escherichia coli (ETEC). The AP oligonucleotide probes were as sensitive as the radiolabeled (RL) probes in detecting LT and STA-2 target cell DNA, but the AP ST probe, which differed from STA-1 by two bases, was less sensitive than the RL probe in detecting STA-1 DNA (6.25 versus 0.78 ng). Of 94 ETEC that were identified with the RL probe, the AP probes detected 93% (28 of 30) of ST, 73% (25 of 34) of LT, and 67% (20 of 30) of LTST ETEC. When colony lysates of these ETEC were examined, the AP probes identified all 94 ETEC. In examinations of stool blots, the RL and AP probes were shown to have sensitivities of 71 and 59%, specificities of 91 and 86%, positive predictive values of 87 and 73%, and negative predictive values of 86 and 74%, respectively. AP oligonucleotide probes to detect ETEC were less sensitive in detecting ETEC by colony or stool blot hybridization than the RL probes but could be used by laboratories without access to radioisotopes to examine colony lysates. Images PMID:3305559

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

    PubMed

    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.

  8. Pericellular matrix formation alters the efficiency of intracellular uptake of oligonucleotides in osteosarcoma cells.

    PubMed

    Suzuki, Yoshitaka; Nishida, Yoshihiro; Naruse, Takahiro; Gemba, Takefumi; Ishiguro, Naoki

    2009-03-01

    One of the crucial roles of tumor extracellular matrix is to act as a barrier to drug delivery. In this study, we analyzed the relationship between the formation of tumor extracellular matrix and the efficiency of intracellular uptake of oligonucleotides in human osteosarcoma cell lines, HOS, and MG-63. Oligonucleotides used in this study were nuclear factor-kappa B (NF-kappaB) decoy, which might be a therapeutic tool for neoplasms. Pericellular matrix formation was examined by particle exclusion assay. Cellular uptake of fluorescein isothiocyanate-labeled NF-kappaB decoy was evaluated by fluorescent microscopy and flow cytometry. Effects of NF-kappaB decoy on cell viability and cell cycle arrest in MG-63 cells were determined by MTT assay and flow cytometry, respectively. MG-63 cells exhibited abundant pericellular matrix with time compared with HOS cells. Uptake of fluorescein isothiocyanate-labeled NF-kappaB decoy decreased in MG-63 cells with time but not in HOS cells in both monolayer and three-dimensional culture using matrigel. However, after enzymatic removal of pericellular matrix, the uptake markedly recovered in MG-63 cells. NF-kappaB decoy inhibited cell proliferation and induced G0/G1 cell cycle arrest in MG-63 cells. These results suggest that abundant pericellular matrix might disturb the uptake of NF-kappaB decoy, and modification of pericellular matrix composition would increase the efficacy of exogenous oligonucleotides treatment for neoplasms.

  9. Fundamental mechanisms of DNA radiosensitization: damage induced by low-energy electrons in brominated oligonucleotide trimers.

    PubMed

    Park, Yeunsoo; Polska, Katarzyna; Rak, Janusz; Wagner, J Richard; Sanche, Léon

    2012-08-16

    The replacement of nucleobases with brominated analogs enhances DNA radiosensitivity. We examine the chemistry of low-energy electrons (LEEs) in this sensitization process by experiments with thin films of the oligonucleotide trimers TBrXT, where BrX = 5-BrU (5-bromouracil), 5-BrC (5-bromocytosine), 8-BrA (8-bromoadenine), or 8-BrG (8-bromoguanine). The products induced from irradiation of thin (∼ 2.5 nm) oligonucleotide films, with 10 eV electrons, under ultrahigh vacuum (UHV) are analyzed by HPLC-UV. The number of damaged brominated trimers ranges from about 12 to 15 × 10(-3) molecules per incident electron, whereas under the identical conditions, these numbers drop to 4-7 × 10(-3) for the same, but nonbrominated oligonucleotides. The results of HPLC analysis show that the main degradation pathway of trinucleotides containing brominated bases involve debromination (i.e., loss of the bromine atom and its replacement with a hydrogen atom). The electron-induced sum of products upon bromination increases by factors of 2.1 for the pyrimidines and 3.2 for the purines. Thus, substitution of any native nucleobase with a brominated one in simple models of DNA increases LEE-induced damage to DNA and hence its radiosensitivity. Furthermore, besides the brominated pyrimidines that have already been tested in clinical trials, brominated purines not only appear to be promising sensitizers for radiotherapy, but could provide a higher degree of radiosensitization.

  10. Electron stimulated desorption of anions from native and brominated single stranded oligonucleotide trimers.

    PubMed

    Polska, Katarzyna; Rak, Janusz; Bass, Andrew D; Cloutier, Pierre; Sanche, Léon

    2012-02-21

    We measured the low energy electron stimulated desorption (ESD) of anions from thin films of native (TXT) and bromine monosubstituted (TBrXT) oligonucleotide trimers deposited on a gold surface (T = thymidine, X = T, deoxycytidine (C), deoxyadenosine (A) or deoxyguanosine (G), Br = bromine). The desorption of H(-), CH(3)(-)/NH(-), O(-)/NH(2)(-), OH(-), CN(-), and Br(-) was induced by 0 to 20 eV electrons. Dissociative electron attachment, below 12 eV, and dipolar dissociation, above 12 eV, are responsible for the formation of these anions. The comparison of the results obtained for the native and brominated trimers suggests that the main pathways of TBrXT degradation correspond to the release of the hydride and bromide anions. Significantly, the presence of bromine in oligonucleotide trimers blocks the electron-induced degradation of nuclobases as evidenced by a dramatic decrease in CN(-) desorption. An increase in the yields of OH(-) is also observed. The debromination yield of particular oligonucleotides diminishes in the following order: BrdU > BrdA > BrdG > BrdC. Based on these results, 5-bromo-2(')-deoxyuridine appears to be the best radiosensitizer among the studied bromonucleosides.

  11. Treatment of experimental autoimmune encephalomyelitis with antisense oligonucleotides against the low affinity neurotrophin receptor.

    PubMed

    Soilu-Hänninen, M; Epa, R; Shipham, K; Butzkueven, H; Bucci, T; Barrett, G; Bartlett, P F; Kilpatrick, T J

    2000-03-15

    Upregulated expression of the low-affinity neurotrophin receptor (p75) in the central nervous system (CNS) during experimental autoimmune encephalomyelitis (EAE) has recently been demonstrated. To investigate whether p75 plays a role in disease pathogenesis, we adopted a gene therapy approach, utilizing antisense oligonucleotides to downregulate p75 expression during EAE. Phosphorothioate antisense oligonucleotides (AS), nonsense oligonucleotides (NS) or phosphate buffered saline (PBS) were injected daily for 18 days after immunization of SJL/J (H-2s)-mice with myelin proteolipid protein (PLP) peptide 139-151. In the AS group, there was a statistically significant reduction in both the mean maximal disease score (1.85 in the AS, 2.94 in the NS and 2.75 in the PBS-groups, respectively, P < 0.025) and in the cumulative disease incidence ( approximately 60% in the AS group and approximately 90% in the control groups). Histological and immunohistochemical analysis showed reduced inflammation and demyelination, as well as reduced p75 expression at the blood-brain barrier (BBB) in the AS-treated mice in comparison with both control groups. There was no difference, however, in p75 expression on neural cells within the CNS between the three groups of mice. We conclude that p75 could play a proactive role in the pathogenesis of EAE and may exert its effect at the level of the BBB.

  12. Delivery of Splice Switching Oligonucleotides by Amphiphilic Chitosan-Based Nanoparticles.

    PubMed

    Moreno, Pedro M D; Santos, Joyce C; Gomes, Carla P; Varela-Moreira, Aida; Costa, Artur; Leiro, Victoria; Mansur, Herman; Pêgo, Ana P

    2016-02-01

    Splice switching oligonucleotides (SSOs) are a class of single-stranded antisense oligonucleotides (ssONs) being used as gene therapeutics and demonstrating great therapeutic potential. The availability of biodegradable and biocompatible delivery vectors that could improve delivery efficiencies, reduce dosage, and, in parallel, reduce toxicity concerns could be advantageous for clinical translation. In this work we explored the use of quaternized amphiphilic chitosan-based vectors in nanocomplex formation and delivery of splice switching oligonucleotides (SSO) into cells, while providing insights regarding cellular uptake of such complexes. Results show that the chitosan amphiphilic character is important when dealing with SSOs, greatly improving colloidal stability under serum conditions, as analyzed by dynamic light scattering, and enhancing cellular association. Nanocomplexes were found to follow an endolysosomal route with a long lysosome residence time. Conjugation of a hydrophobic moiety, stearic acid, to quaternized chitosan was a necessary condition to achieve transfection, as an unmodified quaternary chitosan was completely ineffective. We thus demonstrate that amphiphilic quaternized chitosan is a biomaterial that holds promise and warrants further development as a platform for SSO delivery strategies.

  13. Image-based detection of oligonucleotides--a low cost alternative to spectrophotometric or fluorometric methods.

    PubMed

    Ahirwar, Rajesh; Tanwar, Swati; Parween, Shahila; Kumar, Ashok; Nahar, Pradip

    2014-05-07

    Herein, we report a sensitive and low cost image-based (photocolorimetric) method for the detection of oligonucleotides on an activated polypropylene microtest plate (APPμTP). The assay was developed on the APPμTP by covalently immobilising 20-mer amino-modified oligonucleotides. Biotin-tagged complementary target sequences were then hybridised with the immobilised oligonucleotides. Colour was developed by streptavidin-HRP conjugate and the image of the coloured assay solution was taken by a desktop scanner and analysed using colour saturation. The developed method was analysed for its detection limit, accuracy, sensitivity and interference. The linearity range was found to be 1.7-170 ng mL(-1) while the lower limit of detection and limit of quantification were 1.7 and 5.6 ng mL(-1) respectively. The method showed comparable sensitivity to fluorometric methods, and was found to be correlated to fluorescence (R(2) = 0.8081, p-value < 0.0001) and absorbance (R(2) = 0.9394, p-value < 0.0001)-based quantification. It discriminates mismatched base sequences from perfectly matched sequences efficiently. Validation of the method was carried out by detecting por A DNA of Neisseria meningitidis in bacterial meningitis samples. The por A-specific probe having a 6-carbon spacer at its 5'-NH2 terminus was immobilised covalently to the APPμTP and hybridised with different samples of biotinylated single-stranded por A DNA.

  14. Antisense oligonucleotide treatment ameliorates alpha-1 antitrypsin-related liver disease in mice.

    PubMed

    Guo, Shuling; Booten, Sheri L; Aghajan, Mariam; Hung, Gene; Zhao, Chenguang; Blomenkamp, Keith; Gattis, Danielle; Watt, Andrew; Freier, Susan M; Teckman, Jeffery H; McCaleb, Michael L; Monia, Brett P

    2014-01-01

    Alpha-1 antitrypsin deficiency (AATD) is a rare genetic disease that results from mutations in the alpha-1 antitrypsin (AAT) gene. The mutant AAT protein aggregates and accumulates in the liver leading to AATD liver disease, which is only treatable by liver transplant. The PiZ transgenic mouse strain expresses a human AAT (hAAT) transgene that contains the AATD-associated Glu342Lys mutation. PiZ mice exhibit many AATD symptoms, including AAT protein aggregates, increased hepatocyte death, and liver fibrosis. In the present study, we systemically treated PiZ mice with an antisense oligonucleotide targeted against hAAT (AAT-ASO) and found reductions in circulating levels of AAT and both soluble and aggregated AAT protein in the liver. Furthermore, AAT-ASO administration in these animals stopped liver disease progression after short-term treatment, reversed liver disease after long-term treatment, and prevented liver disease in young animals. Additionally, antisense oligonucleotide treatment markedly decreased liver fibrosis in this mouse model. Administration of AAT-ASO in nonhuman primates led to an approximately 80% reduction in levels of circulating normal AAT, demonstrating potential for this approach in higher species. Antisense oligonucleotides thus represent a promising therapy for AATD liver disease.

  15. Effect of Molecular Crowding and Ionic Strength on the Isothermal Hybridization of Oligonucleotides

    PubMed Central

    Markarian, Marie Z.; Schlenoff, Joseph B.

    2010-01-01

    The isothermal hybridization of complimentary oligonucleotides, 15-mer, 25-mer, 35-mer, and a molecular beacon, was investigated under varying conditions of molecular crowding and ionic strength, using hypochromicity to follow strand pairing and polyethylene glycol as a crowding agent. Thermodynamic analysis of the results revealed the addition of counterions to the oligonucleotide backbones, Δψ, to be dependent on the strand G-C content and the molecular crowding. A decrease in Δψ was observed with both increasing GC% and solution PEG content. In contrast, the number of bound water molecules depended on the activity of Na+, where two regimes were observed. At aNa+⟨0.05 and increasing molecular crowding, water molecules were released into the DNA solutions and oligonucleotide pairing was favored with both increasing hydrophobic forces, while at aNa+≥0.05, water molecules were bound to the strands and the extent of double strand formation decreased with increasing PEG wt%. PMID:20701389

  16. Electron stimulated desorption of anions from native and brominated single stranded oligonucleotide trimers

    SciTech Connect

    Polska, Katarzyna; Rak, Janusz; Bass, Andrew D.; Cloutier, Pierre; Sanche, Leon

    2012-02-21

    We measured the low energy electron stimulated desorption (ESD) of anions from thin films of native (TXT) and bromine monosubstituted (TBrXT) oligonucleotide trimers deposited on a gold surface (T = thymidine, X = T, deoxycytidine (C), deoxyadenosine (A) or deoxyguanosine (G), Br = bromine). The desorption of H{sup -}, CH{sub 3}{sup -}/NH{sup -}, O{sup -}/NH{sub 2}{sup -}, OH{sup -}, CN{sup -}, and Br{sup -} was induced by 0 to 20 eV electrons. Dissociative electron attachment, below 12 eV, and dipolar dissociation, above 12 eV, are responsible for the formation of these anions. The comparison of the results obtained for the native and brominated trimers suggests that the main pathways of TBrXT degradation correspond to the release of the hydride and bromide anions. Significantly, the presence of bromine in oligonucleotide trimers blocks the electron-induced degradation of nuclobases as evidenced by a dramatic decrease in CN{sup -} desorption. An increase in the yields of OH{sup -} is also observed. The debromination yield of particular oligonucleotides diminishes in the following order: BrdU > BrdA > BrdG > BrdC. Based on these results, 5-bromo-2{sup '}-deoxyuridine appears to be the best radiosensitizer among the studied bromonucleosides.

  17. Attomolar Zika virus oligonucleotide detection based on loop-mediated isothermal amplification and AC susceptometry.

    PubMed

    Tian, Bo; Qiu, Zhen; Ma, Jing; Zardán Gómez de la Torre, Teresa; Johansson, Christer; Svedlindh, Peter; Strömberg, Mattias

    2016-12-15

    Because of the serological cross-reactivity among the flaviviruses, molecular detection methods, such as reverse-transcription polymerase chain reaction (RT-PCR), play an important role in the recent Zika outbreak. However, due to the limited sensitivity, the detection window of RT-PCR for Zika viremia is only about one week after symptom onset. By combining loop-mediated isothermal amplification (LAMP) and AC susceptometry, we demonstrate a rapid and homogeneous detection system for the Zika virus oligonucleotide. Streptavidin-magnetic nanoparticles (streptavidin-MNPs) are premixed with LAMP reagents including the analyte and biotinylated primers, and their hydrodynamic volumes are dramatically increased after a successful LAMP reaction. Analyzed by a portable AC susceptometer, the changes of the hydrodynamic volume are probed as Brownian relaxation frequency shifts, which can be used to quantify the Zika virus oligonucleotide. The proposed detection system can recognize 1 aM synthetic Zika virus oligonucleotide in 20% serum with a total assay time of 27min, which can hopefully widen the detection window for Zika viremia and is therefore promising in worldwide Zika fever control.

  18. Detection of Glucose with Atomic Absorption Spectroscopy by Using Oligonucleotide Functionalized Gold Nanoparticle.

    PubMed

    Zhang, Hong; Yan, Honglian; Ling, Liansheng

    2016-06-01

    A novel method for the detection of glucose was established with atomic absorption spectroscopy by using the label of gold nanoparticle (AuNP). Silver-coated glass assembled with oligonucleotide 5'-SH-T12-AGA CAA GAG AGG-3' (Oligo 1) was acted as separation probe, oligonucleotide 5'-CAA CAG AGA ACG-T12-SH-3' modified gold nanoparticle (AuNP-Oligo 2) was acted as signal-reporting probe. Oligonucleotide 5'-CGT TCT CTG TTG CCT CTC TTG TCT-3' (Oligo 3) could hybridize with Oligo 1 on the surface of silver-coated glass and AuNP-Oligo 2, and free AuNP-Oligo 2 could be removed by rinsing with buffer. Hence the concentration of Oligo 3 was transformed into the concentration of gold element. In addition, Oligo 3 could be cleaved into DNA fragments by glucose, glucose oxidase and Fe(2+)-EDTA through Fenton reaction. Thereby the concentration of glucose could be transformed to the absorbance of gold element. Under the optimum conditions, the integrated absorbance decreased proportionally to the concentration of glucose over the range from 50.0 μM to 1.0 mM with a detection limit of 40.0 μM. Moreover, satisfactory result was obtained when the assay was used to determinate glucose in human serum.

  19. Bisulfite oligonucleotide-capture sequencing for targeted base- and strand-specific absolute 5-methylcytosine quantitation.

    PubMed

    Masser, Dustin R; Stanford, David R; Hadad, Niran; Giles, Cory B; Wren, Jonathan D; Sonntag, William E; Richardson, Arlan; Freeman, Willard M

    2016-06-01

    Epigenetic regulation through DNA methylation (5mC) plays an important role in development, aging, and a variety of diseases. Genome-wide studies of base- and strand-specific 5mC are limited by the extensive sequencing required. Targeting bisulfite sequencing to specific genomic regions through sequence capture with complimentary oligonucleotide probes retains the advantages of bisulfite sequencing while focusing sequencing reads on regions of interest, enables analysis of more samples by decreasing the amount of sequence required per sample, and provides base- and strand-specific absolute quantitation of CG and non-CG methylation levels. As an example, an oligonucleotide capture set to interrogate 5mC levels in all rat RefSeq gene promoter regions (18,814) and CG islands, shores, and shelves (18,411) was generated. Validation using whole-genome methylation standards and biological samples demonstrates enrichment of the targeted regions and accurate base-specific quantitation of CG and non-CG methylation for both forward and reverse genomic strands. A total of 170 Mb of the rat genome is covered including 6.6 million CGs and over 67 million non-CG sites, while reducing the amount of sequencing required by ~85 % as compared to existing whole-genome sequencing methods. This oligonucleotide capture targeting approach and quantitative validation workflow can also be applied to any genome of interest.

  20. Targeting the r(CGG) repeats that cause FXTAS with modularly assembled small molecules and oligonucleotides.

    PubMed

    Tran, Tuan; Childs-Disney, Jessica L; Liu, Biao; Guan, Lirui; Rzuczek, Suzanne; Disney, Matthew D

    2014-04-18

    We designed small molecules that bind the structure of the RNA that causes fragile X-associated tremor ataxia syndrome (FXTAS), an incurable neuromuscular disease. FXTAS is caused by an expanded r(CGG) repeat (r(CGG)(exp)) that inactivates a protein regulator of alternative pre-mRNA splicing. Our designed compounds modulate r(CGG)(exp) toxicity in cellular models of FXTAS, and pull-down experiments confirm that they bind r(CGG)(exp) in vivo. Importantly, compound binding does not affect translation of the downstream open reading frame (ORF). We compared molecular recognition properties of our optimal compound to oligonucleotides. Studies show that r(CGG)(exp)'s self-structure is a significant energetic barrier for oligonucleotide binding. A fully modified 2'-OMethyl phosphorothioate is incapable of completely reversing an FXTAS-associated splicing defect and inhibits translation of the downstream ORF, which could have deleterious effects. Taken together, these studies suggest that a small molecule that recognizes structure may be more well suited for targeting highly structured RNAs that require strand invasion by a complementary oligonucleotide.

  1. Genetic repair of mutations in plant cell-free extracts directed by specific chimeric oligonucleotides.

    PubMed

    Rice, M C; May, G D; Kipp, P B; Parekh, H; Kmiec, E B

    2000-06-01

    Chimeric oligonucleotides are synthetic molecules comprised of RNA and DNA bases assembled in a double hairpin conformation. These molecules have been shown to direct gene conversion events in mammalian cells and animals through a process involving at least one protein from the DNA mismatch repair pathway. The mechanism of action for gene repair in mammalian cells has been partially elucidated through the use of a cell-free extract system. Recent experiments have expanded the utility of chimeric oligonucleotides to plants and have demonstrated genotypic and phenotypic conversion, as well as Mendelian transmission. Although these experiments showed correction of point and frameshift mutations, the biochemical and mechanistic aspects of the process were not addressed. In this paper, we describe the establishment of cell-free extract systems from maize (Zea mays), banana (Musa acuminata cv Rasthali), and tobacco (Nicotiana tabacum). Using a genetic readout system in bacteria and chimeric oligonucleotides designed to direct the conversion of mutations in antibiotic-resistant genes, we demonstrate gene repair of point and frameshift mutations. Whereas extracts from banana and maize catalyzed repair of mutations in a precise fashion, cell-free extracts prepared from tobacco exhibited either partial repair or non-targeted nucleotide conversion. In addition, an all-DNA hairpin molecule also mediated repair albeit in an imprecise fashion in all cell-free extracts tested. This system enables the mechanistic study of gene repair in plants and may facilitate the identification of DNA repair proteins operating in plant cells.

  2. Cationic derivatives of biocompatible hyaluronic acids for delivery of siRNA and antisense oligonucleotides.

    PubMed

    Han, Su-Eun; Kang, Hyungu; Shim, Ga Yong; Kim, Sun Jae; Choi, Han-Gon; Kim, Jiseok; Hahn, Sei Kwang; Oh, Yu-Kyoung

    2009-02-01

    In this study, we tested the use of cationic polymer derivatives of biocompatible hyaluronic acid (HA) as a delivery system of siRNA and antisense oligonucleotides. HA was modified with cationic polymer polyethylenimine (PEI). When compared with PEI alone, cationic PEI derivatives of HA (HA-PEI) provided increased cellular delivery of Small interfering RNA (siRNA) in B16F1, A549, HeLa, and Hep3B tumor cells. Indeed, more than 95% of the cells were positive for siRNA following its delivery with HA-PEI. A survivin-specific siRNA that was delivered using HA-PEI potently reduced the mRNA expression levels of the target gene in all of the cell lines. By contrast, survivin-specific siRNA delivered by PEI alone did not induce a significant reduction in mRNA levels. In green fluorescent protein (GFP)-expressing 293 T cells, a loss of GFP expression was evident in the cells that had been treated with GFP-specific siRNA and HA-PEI complex. The inhibition of target gene expression by antisense oligonucleotide G3139 was also enhanced after delivery with HA-PEI. Moreover, HA-PEI displayed lower cytotoxicity than PEI alone. These results suggest that HA-PEI could be further developed as biocompatible delivery systems of siRNA and antisense oligonucleotides for enhanced cellular uptake and inhibition of target gene expression.

  3. Selection of optimal oligonucleotide probes for microarrays usingmultiple criteria, global alignment and parameter estimation.

    SciTech Connect

    Li, Xingyuan; He, Zhili; Zhou, Jizhong

    2005-10-30

    The oligonucleotide specificity for microarray hybridizationcan be predicted by its sequence identity to non-targets, continuousstretch to non-targets, and/or binding free energy to non-targets. Mostcurrently available programs only use one or two of these criteria, whichmay choose 'false' specific oligonucleotides or miss 'true' optimalprobes in a considerable proportion. We have developed a software tool,called CommOligo using new algorithms and all three criteria forselection of optimal oligonucleotide probes. A series of filters,including sequence identity, free energy, continuous stretch, GC content,self-annealing, distance to the 3'-untranslated region (3'-UTR) andmelting temperature (Tm), are used to check each possibleoligonucleotide. A sequence identity is calculated based on gapped globalalignments. A traversal algorithm is used to generate alignments for freeenergy calculation. The optimal Tm interval is determined based on probecandidates that have passed all other filters. Final probes are pickedusing a combination of user-configurable piece-wise linear functions andan iterative process. The thresholds for identity, stretch and freeenergy filters are automatically determined from experimental data by anaccessory software tool, CommOligo_PE (CommOligo Parameter Estimator).The program was used to design probes for both whole-genome and highlyhomologous sequence data. CommOligo and CommOligo_PE are freely availableto academic users upon request.

  4. De novo sequencing of highly modified therapeutic oligonucleotides by hydrophobic tag sequencing coupled with LC-MS.

    PubMed

    Goto, R; Miyakawa, S; Inomata, E; Takami, T; Yamaura, J; Nakamura, Y

    2017-02-01

    Correct sequences are prerequisite for quality control of therapeutic oligonucleotides. However, there is no definitive method available for determining sequences of highly modified therapeutic RNAs, and thereby, most of the oligonucleotides have been used clinically without direct sequence determination. In this study, we developed a novel sequencing method called 'hydrophobic tag sequencing'. Highly modified oligonucleotides are sequenced by partially digesting oligonucleotides conjugated with a 5'-hydrophobic tag, followed by liquid chromatography-mass spectrometry analysis. 5'-Hydrophobic tag-printed fragments (5'-tag degradates) can be separated in order of their molecular masses from tag-free oligonucleotides by reversed-phase liquid chromatography. As models for the sequencing, the anti-VEGF aptamer (Macugen) and the highly modified 38-mer RNA sequences were analyzed under blind conditions. Most nucleotides were identified from the molecular weight of hydrophobic 5'-tag degradates calculated from monoisotopic mass in simple full mass data. When monoisotopic mass could not be assigned, the nucleotide was estimated using the molecular weight of the most abundant mass. The sequences of Macugen and 38-mer RNA perfectly matched the theoretical sequences. The hydrophobic tag sequencing worked well to obtain simple full mass data, resulting in accurate and clear sequencing. The present study provides for the first time a de novo sequencing technology for highly modified RNAs and contributes to quality control of therapeutic oligonucleotides. Copyright © 2016 John Wiley & Sons, Ltd.

  5. Lipid-modified oligonucleotide conjugates: Insights into gene silencing, interaction with model membranes and cellular uptake mechanisms.

    PubMed

    Ugarte-Uribe, Begoña; Grijalvo, Santiago; Pertíñez, Samuel Núñez; Busto, Jon V; Martín, César; Alagia, Adele; Goñi, Félix M; Eritja, Ramón; Alkorta, Itziar

    2017-01-01

    The ability of oligonucleotides to silence specific genes or inhibit the biological activity of specific proteins has generated great interest in their use as research tools and therapeutic agents. Unfortunately, their biological applications meet the limitation of their poor cellular accessibility. Developing an appropriate delivery system for oligonucleotides is essential to achieve their efficient cellular uptake. In the present work a series of phosphorothioate lipid-oligonucleotide hybrids were synthesized introducing covalently single or double lipid tails at both 3'- and 5'-termini of an antisense oligonucleotide. Gene transfections in cultured cells showed antisense luciferase inhibition without the use of a transfecting agent for conjugates modified with the double-lipid tail at 5'-termini. The effect of the double lipid-tailed modification was further studied in detail in several model membrane systems as well as in cellular uptake experiments. During these studies the spontaneous formation of self-assembled microstructures is clearly observed. Lipidation allowed the efficient incorporation of the oligonucleotide in HeLa cells by a macropinocytosis mechanism without causing cytotoxicity in cells or altering the binding properties of the oligonucleotide conjugates. In addition, both single- and double-tailed compounds showed a similar behavior in lipid model membranes, making them useful in nucleotide-based technologies.

  6. Detection of Ligand-Induced Conformational Changes in Oligonucleotides by Second-Harmonic Generation at a Supported Lipid Bilayer Interface.

    PubMed

    Butko, Margaret T; Moree, Ben; Mortensen, Richard B; Salafsky, Joshua

    2016-11-01

    There is a high demand for characterizing oligonucleotide structural changes associated with binding interactions as well as identifying novel binders that modulate their structure and function. In this study, second-harmonic generation (SHG) was used to study RNA and DNA oligonucleotide conformational changes associated with ligand binding. For this purpose, we developed an avidin-based biotin capture surface based on a supported lipid bilayer membrane. The technique was applied to two well-characterized aptamers, both of which undergo conformational changes upon binding either a protein or a small molecule ligand. In both cases, SHG was able to resolve conformational changes in these oligonucleotides sensitively and specifically, in solution and in real time, using nanogram amounts of material. In addition, we developed a competition assay for the oligonucleotides between the specific ligands and known, nonspecific binders, and we demonstrated that intercalators and minor groove binders affect the conformation of the DNA and RNA oligonucleotides in different ways upon binding and subsequently block specific ligand binding in all cases. Our work demonstrates the broad potential of SHG for studying oligonucleotides and their conformational changes upon interaction with ligands. As SHG offers a powerful, high-throughput screening approach, our results here also open an important new avenue for identifying novel chemical probes or sequence-targeted drugs that disrupt or modulate DNA or RNA structure and function.

  7. Oligonucleotide and Parylene Surface Coating of Polystyrene and ePTFE for Improved Endothelial Cell Attachment and Hemocompatibility

    PubMed Central

    Schleicher, Martina; Hansmann, Jan; Elkin, Bentsian; Kluger, Petra J.; Liebscher, Simone; Huber, Agnes J. T.; Fritze, Olaf; Schille, Christine; Müller, Michaela; Schenke-Layland, Katja; Seifert, Martina; Walles, Heike; Wendel, Hans-Peter; Stock, Ulrich A.

    2012-01-01

    In vivo self-endothelialization by endothelial cell adhesion on cardiovascular implants is highly desirable. DNA-oligonucleotides are an intriguing coating material with nonimmunogenic characteristics and the feasibility of easy and rapid chemical fabrication. The objective of this study was the creation of cell adhesive DNA-oligonucleotide coatings on vascular implant surfaces. DNA-oligonucleotides immobilized by adsorption on parylene (poly(monoaminomethyl-para-xylene)) coated polystyrene and ePTFE were resistant to high shear stress (9.5 N/m2) and human blood serum for up to 96 h. Adhesion of murine endothelial progenitor cells, HUVECs and endothelial cells from human adult saphenous veins as well as viability over a period of 14 days of HUVECs on oligonucleotide coated samples under dynamic culture conditions was significantly enhanced (P < 0.05). Oligonucleotide-coated surfaces revealed low thrombogenicity and excellent hemocompatibility after incubation with human blood. These properties suggest the suitability of immobilization of DNA-oligonucleotides for biofunctionalization of blood vessel substitutes for improved in vivo endothelialization. PMID:22481939

  8. High-quality genotyping data from formalin-fixed, paraffin-embedded tissue on the drug metabolizing enzymes and transporters plus array.

    PubMed

    Vos, Hanneke I; van der Straaten, Tahar; Coenen, Marieke J H; Flucke, Uta; te Loo, D Maroeska W M; Guchelaar, Henk-Jan

    2015-01-01

    The Affymetrix Drug Metabolizing Enzymes and Transporters (DMET) Plus array covers 1936 markers in 231 genes involved in drug metabolism and transport. Blood- and saliva-derived DNA works well on the DMET array, but the utility of DNA from FFPE tissue has not been reported for this array. As the ability to use DNA from FFPE tissue on the array could open the potential for large retrospective sample collections, we examined the performance and reliability of FFPE-derived DNA on the DMET Plus array. Germline DNA isolated from archived normal FFPE tissue blocks stored for 3 to 19 years and matched blood or saliva from 16 patients with osteosarcoma were genotyped on the DMET Plus array. Concordance was assessed by calculating agreement and the κ-statistic. We observed high call rates for both the blood- or saliva-derived DNA samples (99.4%) and the FFPE-derived DNA samples (98.9%). Moreover, the concordance among the 16 blood- or saliva-derived DNA and FFPE DNA pairs was high (97.4%, κ = 0.915). This is the first study showing that DNA from normal FFPE tissue provides accurate and reliable genotypes on the DMET Plus array compared with blood- or saliva-derived DNA. This finding provides an opportunity for pharmacogenetic studies in diseases with high mortality rates and prevents a bias in studies where otherwise only alive patients can be included.

  9. Superconducting Bolometer Array Architectures

    NASA Technical Reports Server (NTRS)

    Benford, Dominic; Chervenak, Jay; Irwin, Kent; Moseley, S. Harvey; Shafer, Rick; Staguhn, Johannes; Wollack, Ed; Oegerle, William (Technical Monitor)

    2002-01-01

    The next generation of far-infrared and submillimeter instruments require large arrays of detectors containing thousands of elements. These arrays will necessarily be multiplexed, and superconducting bolometer arrays are the most promising present prospect for these detectors. We discuss our current research into superconducting bolometer array technologies, which has recently resulted in the first multiplexed detections of submillimeter light and the first multiplexed astronomical observations. Prototype arrays containing 512 pixels are in production using the Pop-Up Detector (PUD) architecture, which can be extended easily to 1000 pixel arrays. Planar arrays of close-packed bolometers are being developed for the GBT (Green Bank Telescope) and for future space missions. For certain applications, such as a slewed far-infrared sky survey, feedhorncoupling of a large sparsely-filled array of bolometers is desirable, and is being developed using photolithographic feedhorn arrays. Individual detectors have achieved a Noise Equivalent Power (NEP) of -10(exp 17) W/square root of Hz at 300mK, but several orders of magnitude improvement are required and can be reached with existing technology. The testing of such ultralow-background detectors will prove difficult, as this requires optical loading of below IfW. Antenna-coupled bolometer designs have advantages for large format array designs at low powers due to their mode selectivity.

  10. Electronic Switch Arrays for Managing Microbattery Arrays

    NASA Technical Reports Server (NTRS)

    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.

  11. Carbon nanotube nanoelectrode arrays

    DOEpatents

    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.

  12. Pacific Array (Transportable Broadband Ocean Floor Array)

    NASA Astrophysics Data System (ADS)

    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.

  13. Active aperture phased arrays

    NASA Astrophysics Data System (ADS)

    Shenoy, R. P.

    1989-04-01

    Developments towards the realization of active aperture phased arrays are reviewed. The technology and cost aspects of the power amplifier and phase shifter subsystems are discussed. Consideration is given to research concerning T/R modules, MESFETs, side lobe control, beam steering, optical control techniques, and printed circuit antennas. Methods for configuring the array are examined, focusing on the tile and brick configurations. It is found that there is no technological impediment for introducing active aperture phased arrays.

  14. A photovoltaic array simulator

    NASA Astrophysics Data System (ADS)

    Vachtsevanos, G. J.; Grimbas, E. J.

    A system simulating the output voltage-current characteristics of a photovoltaic array is described. The simulator may be used to test the performance of PV arrays and associated power conditioning equipment necessary for the autonomous or interconnected operation of photovoltaic energy sources. The simulator's main features include simplicity of construction, wide parametric variability and low cost. It is capable of reproducing the output characteristics of commercially available arrays at varying solar irradiation levels with sufficient accuracy. The design ensures the lowest possible power dissipation and minimal thermal drift. It is estimated that the cost of the simulator is an insignificant fraction of the actual array cost in the kilowatt power range.

  15. Interferometric array generation

    NASA Astrophysics Data System (ADS)

    Patra, A. S.; Khare, Alika

    2006-02-01

    We report the formation of square, rectangular as well as hexagonal arrays of small light spots in one single setup using Michelson and Mach-Zehnder interferometers in tandem. The geometry of arrays can be altered easily online, by changing the relative orientations of the mirrors. The arrays could be scanned over large longitudinal distances and could be compressed to give large spot density. The expression for the resultant intensity distribution for the arrays has been worked out and the computed pattern is compared with the experimental data.

  16. Integrated avalanche photodiode arrays

    DOEpatents

    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.

  17. DETECTING LOW-LEVEL SYNTHESIS IMPURITIES IN MODIFIED PHOSPHOROTHIOATE OLIGONUCLEOTIDES USING LIQUID CHROMATOGRAPHY – HIGH RESOLUTION MASS SPECTROMETRY

    PubMed Central

    Nikcevic, Irena; Wyrzykiewicz, Tadeusz K.; Limbach, Patrick A.

    2010-01-01

    Summary An LC-MS method based on the use of high resolution Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS) for profiling oligonucleotides synthesis impurities is described. Oligonucleotide phosphorothioatediesters (phosphorothioate oligonucleotides), in which one of the non-bridging oxygen atoms at each phosphorus center is replaced by a sulfur atom, are now one of the most popular oligonucleotide modifications due to their ease of chemical synthesis and advantageous pharmacokinetic properties. Despite significant progress in the solid-phase oligomerization chemistry used in the manufacturing of these oligonucleotides, multiple classes of low-level impurities always accompany synthetic oligonucleotides. Liquid chromatography-mass spectrometry has emerged as a powerful technique for the identification of these synthesis impurities. However, impurity profiling, where the entire complement of low-level synthetic impurities is identified in a single analysis, is more challenging. Here we present an LC-MS method based the use of high resolution-mass spectrometry, specifically Fourier transform ion cyclotron resonance mass spectrometry (FTIRCMS or FTMS). The optimal LC-FTMS conditions, including the stationary phase and mobile phases for the separation and identification of phosphorothioate oligonucleotides, were found. The characteristics of FTMS enable charge state determination from single m/z values of low-level impurities. Charge state information then enables more accurate modeling of the detected isotopic distribution for identification of the chemical composition of the detected impurity. Using this approach, a number of phosphorothioate impurities can be detected by LC-FTMS including failure sequences carrying 3′-terminal phosphate monoester and 3′-terminal phosphorothioate monoester, incomplete backbone sulfurization and desulfurization products, high molecular weight impurities, and chloral, isobutyryl, and N3 (2-cyanoethyl) adducts

  18. Focal plane array with modular pixel array components for scalability

    SciTech Connect

    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.

  19. Applications of DNA tiling arrays for whole-genome analysis.

    PubMed

    Mockler, Todd C; Chan, Simon; Sundaresan, Ambika; Chen, Huaming; Jacobsen, Steven E; Ecker, Joseph R

    2005-01-01

    DNA microarrays are a well-established technology for measuring gene expression levels. Microarrays designed for this purpose use relatively few probes for each gene and are biased toward known and predicted gene structures. Recently, high-density oligonucleotide-based whole-genome microarrays have emerged as a preferred platform for genomic analysis beyond simple gene expression profiling. Potential uses for such whole-genome arrays include empirical annotation of the transcriptome, chromatin-immunoprecipitation-chip studies, analysis of alternative splicing, characterization of the methylome (the methylation state of the genome), polymorphism discovery and genotyping, comparative genome hybridization, and genome resequencing. Here we review different whole-genome microarray designs and applications of this technology to obtain a wide variety of genomic scale information.

  20. Solar array stepping to minimize array excitation

    NASA Technical Reports Server (NTRS)

    Bhat, Mahabaleshwar K. P. (Inventor); Liu, Tung Y. (Inventor); Plescia, Carl T. (Inventor)

    1989-01-01

    Mechanical oscillations of a mechanism containing a stepper motor, such as a solar-array powered spacecraft, are reduced and minimized by the execution of step movements in pairs of steps, the period between steps being equal to one-half of the period of torsional oscillation of the mechanism. Each pair of steps is repeated at needed intervals to maintain desired continuous movement of the portion of elements to be moved, such as the solar array of a spacecraft. In order to account for uncertainty as well as slow change in the period of torsional oscillation, a command unit may be provided for varying the interval between steps in a pair.

  1. Large scale explorative oligonucleotide probe selection for thousands of genetic groups on a computing grid: application to phylogenetic probe design using a curated small subunit ribosomal RNA gene database.

    PubMed

    Jaziri, Faouzi; Peyretaillade, Eric; Missaoui, Mohieddine; Parisot, Nicolas; Cipière, Sébastien; Denonfoux, Jérémie; Mahul, Antoine; Peyret, Pierre; Hill, David R C

    2014-01-01

    Phylogenetic Oligonucleotide Arrays (POAs) were recently adapted for studying the huge microbial communities in a flexible and easy-to-use way. POA coupled with the use of explorative probes to detect the unknown part is now one of the most powerful approaches for a better understanding of microbial community functioning. However, the selection of probes remains a very difficult task. The rapid growth of environmental databases has led to an exponential increase of data to be managed for an efficient design. Consequently, the use of high performance computing facilities is mandatory. In this paper, we present an efficient parallelization method to select known and explorative oligonucleotide probes at large scale using computing grids. We implemented a software that generates and monitors thousands of jobs over the European Computing Grid Infrastructure (EGI). We also developed a new algorithm for the construction of a high-quality curated phylogenetic database to avoid erroneous design due to bad sequence affiliation. We present here the performance and statistics of our method on real biological datasets based on a phylogenetic prokaryotic database at the genus level and a complete design of about 20,000 probes for 2,069 genera of prokaryotes.

  2. Triplex-forming ability of oligonucleotides containing 1-aryl-1,2,3-triazole nucleobases linked via a two atom-length spacer.

    PubMed

    Hari, Yoshiyuki; Nakahara, Motoi; Obika, Satoshi

    2013-09-01

    Phosphoramidites containing 2-propynyloxy or 1-butyn-4-yl as nucleobase precursors were synthesized and introduced into oligonucleotides using an automated DNA synthesizer. Copper-catalyzed alkyne-azide 1,3-dipolar cycloaddition of the oligonucleotides with various azides gave the corresponding triazolylated oligonucleotides, triplex-forming ability of these synthetic oligonucleotides with double-stranded DNA targets was evaluated by UV melting experiments. It was found that nucleobases containing 2-(1-m-carbonylaminophenyl-1,2,3-triazol-4-yl)ethyl units likely interacted with A of a TA base pair in a parallel triplex DNA.

  3. Cell and tissue distribution of synthetic oligonucleotides in healthy and tumor-bearing nude mice. An autoradiographic, immunohistological, and direct fluorescence microscopy study.

    PubMed Central

    Plenat, F.; Klein-Monhoven, N.; Marie, B.; Vignaud, J. M.; Duprez, A.

    1995-01-01

    Antisense oligonucleotides have the ability to inhibit individual gene expression in the potential treatment of cancer and viral diseases. However, the way parenterally administered oligonucleotides distribute themselves into healthy tissues or tumors is poorly understood. In this study, the cell and tissue distribution of two modified or unmodified phosphodiester pentadeca-beta-oligonucleotides intravenously administered to healthy or tumor-bearing nude mice was assessed by autoradiography as well as by direct fluorescence and immunoenzymatic histological methods. Resistance of oligonucleotides to degradation by nuclease activity was previously studied in vitro. Using these methods we were able to show the following: 1) within minutes, oligonucleotides permeate all cells and tissues with the exceptions of erythrocytes and intervertebral discs; 2) cell and tissue distribution does not depend on the sequence of the given oligonucleotide; 3) concentration of oligonucleotides is higher within the connective tissue cells than in the interstitial matrix; 4) after uptake, oligomers partition throughout all of the cellular compartments, including at the highest intracellular concentrations in the nuclei; 5) oligonucleotides penetrate easily the tumor cell compartments, oligonucleotide diffusion being unimpeded by the extracellular matrix. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 PMID:7604874

  4. Synthetic oligonucleotides with particular base sequences from the cDNA encoding proteins of Mycobacterium bovis BCG induce interferons and activate natural killer cells.

    PubMed

    Tokunaga, T; Yano, O; Kuramoto, E; Kimura, Y; Yamamoto, T; Kataoka, T; Yamamoto, S

    1992-01-01

    Thirteen kinds of 45-mer single-stranded oligonucleotide, having sequence randomly selected from the known cDNA encoding BCG proteins, were tested for their capability to augment natural killer (NK) cell activity of mouse spleen cells in vitro. Six out of the 13 oligonucleotides showed the activity, while the others did not. In order to know the minimal and essential sequence(s) responsible for the biological activity, 2 kinds of 30-mer and 5 kinds of 15-mer oligonucleotide fragments of an active 45-mer nucleotide were tested for their activity. One of the 30-mer oligonucleotides, designated BCG-A4a, was active, but the other 30-mer was inactive. All of the 15-mer oligonucleotide fragments were inactive. The BCG-A4a also stimulated the spleen cells to produce interferon (IFN)-alpha and -gamma. An experiment using anti-IFN antisera showed that the NK cell activation by the oligonucleotide was ascribed to the IFN-alpha produced. It was noticed that all of the biologically active oligonucleotides possessed one or more palindrome sequence(s), and the inactive ones did not, with an exception of a 45-mer inactive oligonucleotide containing overlapping palindrome sequences (GGGCCCGGG). These findings strongly suggest that certain palindrome sequences, like GACGTC, GGCGCC and TGCGCA, are essential for 30-mer oligonucleotides, like BCG-A4a, to induce IFNs.

  5. The ICAM-1 antisense oligonucleotide ISIS-3082 prevents the development of postoperative ileus in mice.

    PubMed

    The, Frans O; de Jonge, Wouter J; Bennink, Roel J; van den Wijngaard, Rene M; Boeckxstaens, Guy E

    2005-09-01

    Intestinal manipulation (IM) during abdominal surgery triggers the influx of inflammatory cells, leading to postoperative ileus. Prevention of this local muscle inflammation, using intercellular adhesion molecule-1 (ICAM-1) and leukocyte function-associated antigen-1-specific antibodies, has been shown to shorten postoperative ileus. However, the therapeutic use of antibodies has considerable disadvantages. The aim of the current study was to evaluate the effect of ISIS-3082, a mouse-specific ICAM-1 antisense oligonucleotide, on postoperative ileus in mice. Mice underwent a laparotomy or a laparotomy combined with IM after treatment with ICAM-1 antibodies, 0.1-10 mg kg(-1) ISIS-3082, saline or ISIS-8997 (scrambled control antisense oligonucleotides, 1 and 3 mg kg(-1)). At 24 h after surgery, gastric emptying of a 99mTC labelled semi-liquid meal was determined using scintigraphy. Intestinal inflammation was assessed by myeloperoxidase (MPO) activity in ileal muscle whole mounts. IM significantly reduced gastric emptying compared to laparotomy. Pretreatment with ISIS-3082 (0.1-1 mg kg(-1)) as well as ICAM-1 antibodies (10 mg kg(-1)), but not ISIS-8997 or saline, improved gastric emptying in a dose-dependent manner. This effect diminished with higher doses of ISIS-3082 (3-10 mg kg(-1)). Similarly, ISIS-3082 (0.1-1 mg kg(-1)) and ICAM-1 antibodies, but not ISIS-8997 or higher doses of ISIS-3082 (3-10 mg kg(-1)), reduced manipulation-induced inflammation. Immunohistochemistry showed reduction of ICAM-1 expression with ISIS-3082 only. ISIS-3082 pretreatment prevents postoperative ileus in mice by reduction of manipulation-induced local intestinal muscle inflammation. Our data suggest that targeting ICAM-1 using antisense oligonucleotides may represent a new therapeutic approach to the prevention of postoperative ileus.

  6. Detection of cyclin D1 mRNA by hybridization sensitive NIC-oligonucleotide probe.

    PubMed

    Kovaliov, Marina; Segal, Meirav; Kafri, Pinhas; Yavin, Eylon; Shav-Tal, Yaron; Fischer, Bilha

    2014-05-01

    A large group of fluorescent hybridization probes, includes intercalating dyes for example thiazole orange (TO). Usually TO is coupled to nucleic acids post-synthetically which severely limits its use. Here, we have developed a phosphoramidite monomer, 10, and prepared a 2'-OMe-RNA probe, labeled with 5-(trans-N-hexen-1-yl-)-TO-2'-deoxy-uridine nucleoside, dU(TO), (Nucleoside bearing an Inter-Calating moiety, NIC), for selective mRNA detection. We investigated a series of 15-mer 2'-OMe-RNA probes, targeting the cyclin D1 mRNA, containing one or several dU(TO) at various positions. dU(TO)-2'-OMe-RNA exhibited up to 7-fold enhancement of TO emission intensity upon hybridization with the complementary RNA versus that of the oligomer alone. This NIC-probe was applied for the specific detection of a very small amount of a breast cancer marker, cyclin D1 mRNA, in total RNA extract from cancerous cells (250 ng/μl). Furthermore, this NIC-probe was found to be superior to our related NIF (Nucleoside with Intrinsic Fluorescence)-probe which could detect cyclin D1 mRNA target only at high concentrations (1840 ng/μl). Additionally, dU(T) can be used as a monomer in solid-phase oligonucleotide synthesis, thus avoiding the need for post-synthetic modification of oligonucleotide probes. Hence, we propose dU(TO) oligonucleotides, as hybridization probes for the detection of specific RNA in homogeneous solutions and for the diagnosis of breast cancer.

  7. Evaluation of "at risk" alpha 1-antitrypsin genotype SZ with synthetic oligonucleotide gene probes.

    PubMed Central

    Nukiwa, T; Brantly, M; Garver, R; Paul, L; Courtney, M; LeCocq, J P; Crystal, R G

    1986-01-01

    Alpha 1-antitrypsin (alpha 1AT), a 52,000-mol-wt serum glycoprotein produced by hepatocytes and mononuclear phagocytes, functions as the major inhibitor of neutrophil elastase. The alpha 1AT haplotype S is associated with childhood liver disease and/or adult emphysema when inherited with the Z haplotype to give the phenotype SZ. To accurately identify the SZ phenotype at the level of genomic DNA, four 32P-labeled 19-mer synthetic oligonucleotide probes were prepared; two to identify the M and S difference in exon III, and two to identify the M and Z difference in exon V. These probes were hybridized with various cloned DNAs and genomic DNAs cut with the restriction endonucleases BgII and EcoRI; the genomic DNAs represented all six possible phenotype combinations of the M, S, and Z haplotypes (MM, MS, MZ, SS, ZZ, and SZ). Using the four probes to evaluate 42 samples of genomic DNA, the "at risk" SZ and ZZ phenotypes were correctly identified in all cases, as were the "not at risk" phenotypes SS, MS, MM, and MZ, demonstrating that both exon III and exon V directed probes are necessary to properly identify all of the major "at risk" alpha 1AT genes. However, when used to evaluate a very rare family carrying a null allele, these four oligonucleotide probes misidentified the "at risk" null-null and S null phenotypes as "not at risk" MM and SM combinations. These observations indicate that oligonucleotide gene probes yielded reliable and accurate assessment of "at risk" alpha 1AT genotypes in almost all situations, but in the context of prenatal diagnosis and genetic counseling this approach must be used with caution and in combination with family studies so as not to misidentify rare genotypes that may be associated with a risk for disease. Images PMID:3484754

  8. Sequence-dependent structural changes in a self-assembling DNA oligonucleotide.

    PubMed

    Saoji, Maithili; Paukstelis, Paul J

    2015-12-01

    DNA has proved to be a remarkable molecule for the construction of sophisticated two-dimensional and three-dimensional architectures because of its programmability and structural predictability provided by complementary Watson-Crick base pairing. DNA oligonucleotides can, however, exhibit a great deal of local structural diversity. DNA conformation is strongly linked to both environmental conditions and the nucleobase identities inherent in the oligonucleotide sequence, but the exact relationship between sequence and local structure is not completely understood. This study examines how a single-nucleotide addition to a class of self-assembling DNA 13-mers leads to a significantly different overall structure under identical crystallization conditions. The DNA 13-mers self-assemble in the presence of Mg(2+) through a combination of Watson-Crick and noncanonical base-pairing interactions. The crystal structures described here show that all of the predicted Watson-Crick base pairs are present, with the major difference being a significant rearrangement of noncanonical base pairs. This includes the formation of a sheared A-G base pair, a junction of strands formed from base-triple interactions, and tertiary interactions that generate structural features similar to tandem sheared G-A base pairs. The adoption of this alternate noncanonical structure is dependent in part on the sequence in the Watson-Crick duplex region. These results provide important new insights into the sequence-structure relationship of short DNA oligonucleotides and demonstrate a unique interplay between Watson-Crick and noncanonical base pairs that is responsible for crystallization fate.

  9. Effect of Terminal Groups of Dendrimers in the Complexation with Antisense Oligonucleotides and Cell Uptake.

    PubMed

    Márquez-Miranda, Valeria; Peñaloza, Juan Pablo; Araya-Durán, Ingrid; Reyes, Rodrigo; Vidaurre, Soledad; Romero, Valentina; Fuentes, Juan; Céric, Francisco; Velásquez, Luis; González-Nilo, Fernando D; Otero, Carolina

    2016-12-01

    Poly(amidoamine) dendrimers are the most recognized class of dendrimer. Amino-terminated (PAMAM-NH2) and hydroxyl-terminated (PAMAM-OH) dendrimers of generation 4 are widely used, since they are commercially available. Both have different properties, mainly based on their different overall charges at physiological pH. Currently, an important function of dendrimers as carriers of short single-stranded DNA has been applied. These molecules, known as antisense oligonucleotides (asODNs), are able to inhibit the expression of a target mRNA. Whereas PAMAM-NH2 dendrimers have shown to be able to transfect plasmid DNA, PAMAM-OH dendrimers have not shown the same successful results. However, little is known about their interaction with shorter and more flexible molecules such as asODNs. Due to several initiatives, the use of these neutral dendrimers as a scaffold to introduce other functional groups has been proposed. Because of its low cytotoxicity, it is relevant to understand the molecular phenomena involving these types of dendrimers. In this work, we studied the behavior of an antisense oligonucleotide in presence of both types of dendrimers using molecular dynamics simulations, in order to elucidate if they are able to form stable complexes. In this manner, we demonstrated at atomic level that PAMAM-NH2, unlike PAMAM-OH, could form a well-compacted complex with asODN, albeit PAMAM-OH can also establish stable interactions with the oligonucleotide. The biological activity of asODN in complex with PAMAM-NH2 dendrimer was also shown. Finally, we revealed that in contact with PAMAM-OH, asODN remains outside the cells as TIRF microscopy results showed, due to its poor interaction with this dendrimer and cell membranes.

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

    NASA Astrophysics Data System (ADS)

    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

  11. Therapeutic potentialities of EWS-Fli-1 mRNA-targeted vectorized antisense oligonucleotides.

    PubMed

    Maksimenko, A; Lambert, G; Bertrand, J R; Fattal, E; Couvreur, P; Malvy, C

    2003-12-01

    We have used structured antisense oligonucleotides (AON), which are protected against extra and intracellular degradation by their internal structure. We have shown that if correctly designed this structure does not prevent them from hybridizing to the mRNA target. This concept allows reducing the number of thioate groups in the oligonucleotide and therefore the potential toxicity. Junction oncogenes are found in cancers such as certain leukemias, Ewing sarcoma, and thyroid papillary carcinomas. Ewing sarcoma is a cancer of children and young adults with bone metastasis. It is caused by a chromosomic translocation t(11;22) (q24;q12) creating a fusion gene between the genes EWS and Fli-1 giving rise to a chimeric protein which is an unnatural transcription factor. Immortalized NIH/3T3 cells transfected by the EWS-Fli-1 cDNA under the control of the LTR retroviral promoter--which do not undergo apoptosis and which became tumoral--were used for this study. As a model of Ewing sarcoma in nude mice, we have used permanently expressing human EWS-Fli-1 cells grafted to nude mice. The nanospheres or nanocapsules have been used to deliver two different AON: a phosphorothioate, and a structured chimeric AON, both targeted toward the junction area of EWS-Fli-1. Both types of AON-loaded nanoparticles inhibited the growth of the xenografted tumor after intratumoral injections into nude mice, whereas similar nanoparticles with control oligonucleotides had no effect. With AON in nanospheres, we have shown after 24 hours that the mRNA of EWS-Fli-1 was specifically down-regulated, confirming the antisense activity of the targeted AON.

  12. Characterization of chemically modified oligonucleotides targeting a pathogenic mutation in human mitochondrial DNA.

    PubMed

    Tonin, Yann; Heckel, Anne-Marie; Dovydenko, Ilya; Meschaninova, Mariya; Comte, Caroline; Venyaminova, Alya; Pyshnyi, Dmitrii; Tarassov, Ivan; Entelis, Nina

    2014-05-01

    Defects in mitochondrial genome can cause a wide range of clinical disorders, mainly neuromuscular diseases. Most of the deleterious mitochondrial mutations are heteroplasmic, meaning that wild type and mutated forms of mtDNA coexist in the same cell. Therefore, a shift in the proportion between mutant and wild type molecules could restore mitochondrial functions. The anti-replicative strategy aims to induce such a shift in heteroplasmy by mitochondrial targeting specifically designed molecules in order to inhibit replication of mutant mtDNA. Recently, we developed mitochondrial RNA vectors that can be used to address anti-replicative oligoribonucleotides into human mitochondria and impact heteroplasmy level, however, the effect was mainly transient, probably due to a rapid degradation of RNA molecules. In the present study, we introduced various chemically modified oligonucleotides in anti-replicative RNAs. We show that the most important increase of anti-replicative molecules' lifetime can be achieved by using synthetic RNA-DNA chimerical molecules or by ribose 2'-O-methylation in nuclease-sensitive sites. The presence of inverted thymidine at 3' terminus and modifications of 2'-OH ribose group did not prevent the mitochondrial uptake of the recombinant molecules. All the modified oligonucleotides were able to anneal specifically with the mutant mtDNA fragment, but not with the wild-type one. Nevertheless, the modified oligonucleotides did not cause a significant effect on the heteroplasmy level in transfected transmitochondrial cybrid cells bearing a pathogenic mtDNA deletion, proving to be less efficient than non-modified RNA molecules.

  13. Genotypic resistance testing in HIV by arrayed primer extension

    PubMed Central

    Bodem, Jochen; Gerhold-Ay, Aslihan; Jacob, Anette; Fellenberg, Kurt; Kräusslich, Hans-Georg; Hoheisel, Jörg D.

    2008-01-01

    The analysis of mutations that are associated with the occurrence of drug resistance is important for monitoring the antiretroviral therapy of patients infected with human immunodeficiency virus (HIV). Here, we describe the establishment and successful application of Arrayed Primer Extension (APEX) for genotypic resistance testing in HIV as a rapid and economical alternative to standard sequencing. The assay is based on an array of oligonucleotide primers that are immobilised via their 5′-ends. Upon hybridisation of template DNA, a primer extension reaction is performed in the presence of the four dideoxynucleotides, each labelled with a distinct fluorophore. The inserted label immediately indicates the sequence at the respective position. Any mutation changes the colour pattern. We designed a microarray for the analysis of 26 and 33 codons in the HIV protease and reverse transcriptase, respectively, which are of special interest with respect to drug resistance. The enormous genome variability of HIV represents a big challenge for genotypic resistance tests, which include a hybridisation step, both in terms of specificity and probe numbers. The use of degenerated oligonucleotides resulted in a significant reduction in the number of primers needed. For validation, DNA of 94 and 48 patients that exhibited resistance to inhibitors of HIV protease and reverse transcriptase, respectively, were analysed. The validation included HIV subtype B, prevalent in industrialised countries, as well as non-subtype B samples that are more common elsewhere. Electronic supplementary material The online version of this article (doi:10.1007/s00216-007-1775-0) contains supplementary material, which is available to authorized users. PMID:18202840

  14. Gene editing activity on extrachromosomal arrays in C. elegans transgenics.

    PubMed

    Falgowski, Kerry A; Kmiec, Eric B

    2011-04-15

    Gene editing by modified single-stranded oligonucleotides is a strategy aimed at inducing single base changes into the genome, generating a permanent genetic change. The work presented here explores gene editing capabilities in the model organism Caenorhabditis elegans. Current approaches to gene mutagenesis in C. elegans have been plagued by non-specificity and thus the ability to induce precise, directed alterations within the genome of C. elegans would offer a platform upon which structure/function analyses can be carried out. As such, several in vivo assay systems were developed to evaluate gene editing capabilities in C. elegans. Fluorescence was chosen as the selectable endpoint as fluorescence can be easily detected through the transparent worm body even from minimal expression. Two tissue specific fluorescent expression vectors containing either a GFP or mCherry transgene were mutagenized to create a single nonsense mutation within the open reading frame of each respective fluorescent gene. These served as the target site to evaluate the frequency of gene editing on extrachromosomal array transgenic lines. Extrachromosomal arrays can carry hundreds of copies of the transgene, therefore low frequency events (like those in the gene editing reaction) may be detected. Delivery of the oligonucleotide was accomplished by microinjection into the gonads of young adult worms in an effort to induce repair of the mutated fluorescent gene in the F1 progeny. Despite many microinjections on the transgenic strains with varying concentrations of ODNs, no gene editing events were detected. This result is consistent with the previous research, demonstrating the difficulties encountered in targeting embryonic stem cells and the pronuclei of single-celled embryos.

  15. Recursive construction of perfect DNA molecules and libraries from imperfect oligonucleotides.

    PubMed

    Linshiz, Gregory; Yehezkel, Tuval Ben; Shapiro, Ehud

    2012-01-01

    Making faultless complex objects from potentially faulty building blocks is a fundamental challenge in computer engineering, nanotechnology, and synthetic biology. We developed an error-correcting recursive construction procedure that attempts to address this challenge. Making DNA molecules from synthetic oligonucleotides using the procedure described here surpasses existing methods for de novo DNA synthesis in speed, precision, and amenability to automation. It provides for the first time a unified DNA construction platform for combining synthetic and natural DNA fragments, for constructing designer DNA libraries, and for making the faultless long synthetic DNA building blocks needed for de novo genome construction.

  16. Oligonucleotides as probes for studying polymerization reactions in dilute aqueous solution. 2: Polycondensations

    NASA Technical Reports Server (NTRS)

    Kolb, Vera; Orgel, Leslie E.

    1995-01-01

    We have prepared a (P-32)-labeled oligonucleotide probe carrying a ureido (-NH-CO-NH2) function at its 3'-terminus. This labeled oligomer was used to study polycondensations of urea and formaldehyde and of various phenols and formaldehyde in aqueous solution. The formation of formaldehyde copolymers attached to the amido-function of the probe was monitored by gel electrophoresis. Our results are generally in agreement with those obtained using conventional techniques. Our method is suitable for monitoring potentially prebiotic polycondensation reactions involving formaldehyde.

  17. Development and validation of an oligonucleotide ligation assay to detect lamivudine resistance in hepatitis B virus.

    PubMed

    Beck, Ingrid A; Payant, Rachel; Ngo-Giang-Huong, Nicole; Khamduang, Woottichai; Laomanit, Laddawan; Jourdain, Gonzague; Frenkel, Lisa M

    2016-07-01

    Treatment of chronic hepatitis B virus (HBV) infection with lamivudine-monotherapy rapidly selects mutant variants in a high proportion of individuals. Monitoring lamivudine resistance by consensus sequencing is costly and insensitive for detection of minority variants. An oligonucleotide ligation assay (OLA) for HBV lamivudine-resistance was developed and compared to consensus sequencing. Both assays detected drug resistance mutations in 35/64 (54.7%) specimens evaluated, and OLA detected minority mutants in an additional six (9.4%). OLA may offer a sensitive and inexpensive alternative to consensus sequencing for detection of HBV drug resistance in resource-limited settings.

  18. Oligonucleotides as probes for studying polymerization reactions in dilute aqueous solution: II. Polycondensations

    NASA Technical Reports Server (NTRS)

    Kolb, V.; Orgel, L. E.

    1995-01-01

    We have prepared a [32P]-labeled oligonucleotide probe carrying a ureido (-NH-CO-NH2) function at its 3'-terminus. This labeled oligomer was used to study polycondensations of urea and formaldehyde and of various phenols and formaldehyde in aqueous solution. The formation of formaldehyde copolymers attached to the amido-function of the probe was monitored by gel electrophoresis. Our results are generally in agreement with those obtained using conventional techniques. Our method is suitable for monitoring potentially prebiotic polycondensation reactions involving formaldehyde.

  19. An expedient biocatalytic procedure for abasic site precursors useful in oligonucleotide synthesis.

    PubMed

    Martínez-Montero, Saúl; Fernández, Susana; Sanghvi, Yogesh S; Gotor, Vicente; Ferrero, Miguel

    2011-09-07

    Preparation of abasic site precursors through a divergent chemoenzymatic synthesis has been accomplished. Several biocatalysts and acylating agents were studied furnishing a practical and scalable green method useful for industrial applications. Highly regioselective acylation and deacylation reactions with 1,2-dideoxy-D-ribose are described resulting in excellent yield. A fast, atom-efficient and convenient synthesis of 3-, and 5-O-DMTr-1,2-dideoxyribose 17 and 19 has been achieved. These compounds are useful precursors for the preparation of phosphoramidites required for the assembly of oligonucleotides containing the tetrahydrofuran abasic lesions.

  20. Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer.

    PubMed

    Scheiermann, Julia; Klinman, Dennis M

    2014-11-12

    Synthetic oligonucleotides (ODN) that express unmethylated "CpG motifs" trigger cells that express Toll-like receptor 9. In humans this includes plasmacytoid dendritic cells and B cells. CpG ODN induce an innate immune response characterized by the production of Th1 and pro-inflammatory cytokines. Their utility as vaccine adjuvants was evaluated in a number of clinical trials. Results indicate that CpG ODN improve antigen presentation and the generation of vaccine-specific cellular and humoral responses. This work provides an up-to-date overview of the utility of CpG ODN as adjuvants for vaccines targeting infectious agents and cancer.

  1. Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer

    PubMed Central

    Scheiermann, Julia; Klinman, Dennis M.

    2014-01-01

    Synthetic oligonucleotides (ODN) that express unmethylated “CpG motifs” trigger cells that express Toll-like receptor 9. In humans this includes plasmacytoid dendritic cells and B cells. CpG ODN induce an innate immune response characterized by the production of Th1 and pro-inflammatory cytokines. Their utility as vaccine adjuvants was evaluated in a number of clinical trials. Results indicate that CpG ODN improve antigen presentation and the generation of vaccine-specific cellular and humoral responses. This work provides an up-to-date overview of the utility of CpG ODN as adjuvants for vaccines targeting infectious agents and cancer. PMID:24975812

  2. Synthesis of antisense oligonucleotides containing acyclic alkynyl nucleoside analogs and their biophysical and biological properties.

    PubMed

    Ogata, Aya; Maeda, Yusuke; Ueno, Yoshihito

    2017-04-01

    The synthesis of oligonucleotide (ON) analogs, which can be used as antisense molecules, has recently gained much attention. Here, we report the synthesis and properties of an ON analog containing acyclic thymidine and cytidine analogs with a 4-pentyl-1,2-diol instead of the d-ribofuranose moiety. The incorporation of these analogs into the ON improved its nuclease resistance to 3'-exonucleases. Furthermore, it was found that the incorporation of the acyclic thymidine analog into a DNA/RNA duplex accelerates the RNA cleavage of a DNA/RNA duplex by Escherichia coli RNase H.

  3. Pyrimidine phosphorothioate oligonucleotides form triple-stranded helices and promote transcription inhibition.

    PubMed Central

    Xodo, L; Alunni-Fabbroni, M; Manzini, G; Quadrifoglio, F

    1994-01-01

    The ability of phosphorothioate (POS) oligonucleotides to recognise and bind to homopurine-homopyrimidine DNA double-stranded sites via triple helix formation has been investigated. It has been found that the homologous pyrimidine POS sequences Y11-Si (i = 0, 1,2,3,4,10), which have been obtained by an increasing sulphur substitution in the sugar-phosphate backbone of d(CTTCCTCCTCT) (Y11), and the target hairpin duplex d(GAAGGAGGAGA-T4-TCTCCTCCTTC) (h26) can form stable triple helices, as indicated by PAGE, CD and UV melting experiments. The thermal stability of the triple helices depends on the number of POS linkages in the third Y11 strand, varying from 48 degrees C (Y11, with only phosphate groups, PO2) to 31 degrees C (Y11-S10 containing exclusively thioate groups). On average, a Tm depression of about 2 degrees C per POS linkage introduced in Y11 was observed. CD data indicate that the sulphurization of the third strand results in minimal changes of triple-stranded structures. The energetics of the triplex-to-hairpin plus single-strand transition has been determined by van't Hoff analyses of the melting curves. In free energy terms, the POS triplexes h26.Y11-Si are less stable than the normal PO2 h26.Y11 triplex by values between 2.7 and 5.4 kcal/mol, depending on the number of POS linkages contained in the third strand. Phosphorothioate oligonucleotides being resistant towards several nucleases offer an interesting choice as gene blockers in antisense strategy. Thus, their ability to inhibit transcription via triple helix formation has been examined in vitro. We found that triplex-forming POS oligonucleotides of 20 bases in length (with a cytosine contents of 45%), containing either 10% or 26% thioate groups, strongly repress the transcription activity of the bacteriophage T7 RNA polymerase at pH 6.9, when used in excess compared to the target (mol oligo/mol template = 125). The here reported data are useful for designing phosphorothioate oligonucleotides

  4. ISS Solar Array Management

    NASA Technical Reports Server (NTRS)

    Williams, James P.; Martin, Keith D.; Thomas, Justin R.; Caro, Samuel

    2010-01-01

    The International Space Station (ISS) Solar Array Management (SAM) software toolset provides the capabilities necessary to operate a spacecraft with complex solar array constraints. It monitors spacecraft telemetry and provides interpretations of solar array constraint data in an intuitive manner. The toolset provides extensive situational awareness to ensure mission success by analyzing power generation needs, array motion constraints, and structural loading situations. The software suite consists of several components including samCS (constraint set selector), samShadyTimers (array shadowing timers), samWin (visualization GUI), samLock (array motion constraint computation), and samJet (attitude control system configuration selector). It provides high availability and uptime for extended and continuous mission support. It is able to support two-degrees-of-freedom (DOF) array positioning and supports up to ten simultaneous constraints with intuitive 1D and 2D decision support visualizations of constraint data. Display synchronization is enabled across a networked control center and multiple methods for constraint data interpolation are supported. Use of this software toolset increases flight safety, reduces mission support effort, optimizes solar array operation for achieving mission goals, and has run for weeks at a time without issues. The SAM toolset is currently used in ISS real-time mission operations.

  5. Array for detecting microbes

    DOEpatents

    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.

  6. Solar array deployment mechanism

    NASA Technical Reports Server (NTRS)

    Calassa, Mark C.; Kackley, Russell

    1995-01-01

    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.

  7. Antiviral activity of a phosphorothioate oligonucleotide complementary to RNA of the human cytomegalovirus major immediate-early region.

    PubMed Central

    Azad, R F; Driver, V B; Tanaka, K; Crooke, R M; Anderson, K P

    1993-01-01

    Phosphorothioate oligonucleotides complementary to mRNA of the human cytomegalovirus (HCMV) DNA polymerase gene or to RNA transcripts of the major immediate-early regions 1 and 2 (IE1 and IE2) of HCMV were evaluated for antiviral activity in a 96-well immunoassay with primary human dermal fibroblasts as host cells. Oligonucleotides complementary to RNA of the IE2 region exhibited the most potent antiviral activity. One of these oligonucleotides, ISIS 2922, was at least 30-fold more potent than the nucleoside analog, ganciclovir, with a 50% effective concentration of 0.37 microM in the 96-well immunoassay. In an infectious virus yield reduction assay, ISIS 2922 and ganciclovir reduced production of infectious virus by 2 log units at concentrations of 2.2 and 36 microM, respectively. A control oligonucleotide showed no inhibition of virus production at concentrations as high as 3 microM. ISIS 2922 reduced IE protein synthesis in HCMV-infected cells in a dose-dependent manner which correlated with antiviral activity. The antiviral activity of ISIS 2922 was not due to oligonucleotide-induced cytotoxicity since effects on cell viability or proliferation were observed only at concentrations well in excess of effective antiviral concentrations. The specificity and potency of ISIS 2922 suggest that it may be useful for the treatment of cytomegalovirus disease in humans. Images PMID:8239610

  8. Automated SNP genotype clustering algorithm to improve data completeness in high-throughput SNP genotyping datasets from custom arrays.

    PubMed

    Smith, Edward M; Littrell, Jack; Olivier, Michael

    2007-12-01

    High-throughput SNP genotyping platforms use automated genotype calling algorithms to assign genotypes. While these algorithms work efficiently for individual platforms, they are not compatible with other platforms, and have individual biases that result in missed genotype calls. Here we present data on the use of a second complementary SNP genotype clustering algorithm. The algorithm was originally designed for individual fluorescent SNP genotyping assays, and has been optimized to permit the clustering of large datasets generated from custom-designed Affymetrix SNP panels. In an analysis of data from a 3K array genotyped on 1,560 samples, the additional analysis increased the overall number of genotypes by over 45,000, significantly improving the completeness of the experimental data. This analysis suggests that the use of multiple genotype calling algorithms may be advisable in high-throughput SNP genotyping experiments. The software is written in Perl and is available from the corresponding author.

  9. High density pixel array

    NASA Technical Reports Server (NTRS)

    Wiener-Avnear, Eliezer (Inventor); McFall, James Earl (Inventor)

    2004-01-01

    A pixel array device is fabricated by a laser micro-milling method under strict process control conditions. The device has an array of pixels bonded together with an adhesive filling the grooves between adjacent pixels. The array is fabricated by moving a substrate relative to a laser beam of predetermined intensity at a controlled, constant velocity along a predetermined path defining a set of grooves between adjacent pixels so that a predetermined laser flux per unit area is applied to the material, and repeating the movement for a plurality of passes of the laser beam until the grooves are ablated to a desired depth. The substrate is of an ultrasonic transducer material in one example for fabrication of a 2D ultrasonic phase array transducer. A substrate of phosphor material is used to fabricate an X-ray focal plane array detector.

  10. Micromachined electrode array

    DOEpatents

    Okandan, Murat; Wessendorf, Kurt O.

    2007-12-11

    An electrode array is disclosed which has applications for neural stimulation and sensing. The electrode array, in certain embodiments, can include a plurality of electrodes each of which is flexibly attached to a common substrate using a plurality of springs to allow the electrodes to move independently. In other embodiments of the electrode array, the electrodes can be fixed to the substrate. The electrode array can be formed from a combination of bulk and surface micromachining, and can include electrode tips having an electroplated metal (e.g. platinum, iridium, gold or titanium) or a metal oxide (e.g. iridium oxide) for biocompatibility. The electrode array can be used to form a part of a neural prosthesis, and is particularly well adapted for use in an implantable retinal prosthesis.

  11. Photovoltaic array loss mechanisms

    NASA Astrophysics Data System (ADS)

    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.

  12. Multibeam Phased Array Antennas

    NASA Technical Reports Server (NTRS)

    Popovic, Zoya; Romisch, Stefania; Rondineau, Sebastien

    2004-01-01

    In this study, a new architecture for Ka-band multi-beam arrays was developed and demonstrated experimentally. The goal of the investigation was to demonstrate a new architecture that has the potential of reducing the cost as compared to standard expensive phased array technology. The goals of this specific part of the project, as stated in the yearly statement of work in the original proposal are: 1. Investigate bounds on performance of multi-beam lens arrays in terms of beamwidths, volume (size), isolation between beams, number of simultaneous beams, etc. 2. Design a small-scale array to demonstrate the principle. The array will be designed for operation around 3OGHz (Ka-band), with two 10-degree beamwidth beams. 3. Investigate most appropriate way to accomplish fine-tuning of the beam pointing within 5 degrees around the main beam pointing angle.

  13. Microfabricated ion trap array

    DOEpatents

    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.

  14. A new diagnostic workflow for patients with mental retardation and/or multiple congenital abnormalities: test arrays first.

    PubMed

    Gijsbers, Antoinet C J; Lew, Janet Y K; Bosch, Cathy A J; Schuurs-Hoeijmakers, Janneke H M; van Haeringen, Arie; den Hollander, Nicolette S; Kant, Sarina G; Bijlsma, Emilia K; Breuning, Martijn H; Bakker, Egbert; Ruivenkamp, Claudia A L

    2009-11-01

    High-density single-nucleotide polymorphism (SNP) genotyping technology enables extensive genotyping as well as the detection of increasingly smaller chromosomal aberrations. In this study, we assess molecular karyotyping as first-round analysis of patients with mental retardation and/or multiple congenital abnormalities (MR/MCA). We used different commercially available SNP array platforms, the Affymetrix GeneChip 262K NspI, the Genechip 238K StyI, the Illumina HumanHap 300 and HumanCNV 370 BeadChip, to detect copy number variants (CNVs) in 318 patients with unexplained MR/MCA. We found abnormalities in 22.6% of the patients, including six CNVs that overlap known microdeletion/duplication syndromes, eight CNVs that overlap recently described syndromes, 63 potentially pathogenic CNVs (in 52 patients), four large segments of homozygosity and two mosaic trisomies for an entire chromosome. This study shows that high-density SNP array analysis reveals a much higher diagnostic yield as that of conventional karyotyping. SNP arrays have the potential to detect CNVs, mosaics, uniparental disomies and loss of heterozygosity in one experiment. We, therefore, propose a novel diagnostic approach to all MR/MCA patients by first analyzing every patient with an SNP array instead of conventional karyotyping.

  15. Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in FaciosScapuloHumeral Muscular Dystrophy (FSHD)

    PubMed Central

    Ansseau, Eugénie; Vanderplanck, Céline; Wauters, Armelle; Harper, Scott Q.; Coppée, Frédérique; Belayew, Alexandra

    2017-01-01

    FacioScapuloHumeral muscular Dystrophy (FSHD) is one of the most prevalent hereditary myopathies and is generally characterized by progressive muscle atrophy affecting the face, scapular fixators; upper arms and distal lower legs. The FSHD locus maps to a macrosatellite D4Z4 repeat array on chromosome 4q35. Each D4Z4 unit contains a DUX4 gene; the most distal of which is flanked by a polyadenylation site on FSHD-permissive alleles, which allows for production of stable DUX4 mRNAs. In addition, an open chromatin structure is required for DUX4 gene transcription. FSHD thus results from a gain of function of the toxic DUX4 protein that normally is only expressed in germ line and stem cells. Therapeutic strategies are emerging that aim to decrease DUX4 expression or toxicity in FSHD muscle cells. We review here the heterogeneity of DUX4 mRNAs observed in muscle and stem cells; and the use of antisense oligonucleotides (AOs) targeting the DUX4 mRNA to interfere either with transcript cleavage/polyadenylation or intron splicing. We show in primary cultures that DUX4-targeted AOs suppress the atrophic FSHD myotube phenotype; but do not improve the disorganized FSHD myotube phenotype which could be caused by DUX4c over-expression. Thus; DUX4c might constitute another therapeutic target in FSHD. PMID:28273791

  16. Multiplex detection of bacteria associated with normal microbiota and with bacterial vaginosis in vaginal swabs by use of oligonucleotide-coupled fluorescent microspheres.

    PubMed

    Dumonceaux, Tim J; Schellenberg, John; Goleski, Vanessa; Hill, Janet E; Jaoko, Walter; Kimani, Joshua; Money, Deborah; Ball, T Blake; Plummer, Francis A; Severini, Alberto

    2009-12-01

    Bacterial vaginosis (BV) is a recurrent condition that is associated with a range of negative outcomes, including the acquisition of human immunodeficiency virus and other sexually transmitted diseases, preterm births, and pelvic inflammatory disease. In contrast to the Lactobacillus-dominated normal vaginal microbiota, BV is characterized by a lack of lactobacilli and an abundance of anaerobic and gram-negative organisms, including Gardnerella vaginalis and Atopobium vaginae. To date, the laboratory diagnosis of BV has relied upon the fulfillment of criteria determined by microscopic observation of Gram-stained vaginal swabs. We describe a molecular-based method for the easy determination of the species profile within the vaginal microbiota based on the amplification of the chaperonin-60 genes of all bacteria present in the swab and hybridization of the amplicon to species-specific oligonucleotide-coupled fluorescent beads that are identified by flow cytometry with a Luminex instrument. We designed a nineplex Luminex array for characterization of the vaginal microbiota and applied it to the analysis of vaginal swabs from individuals from Africa and North America. Using the presence of A. vaginae or G. vaginalis, or both, as the defining criterion for BV, we found that the method was highly specific and sensitive for the diagnosis of BV using microscopy as a gold standard.

  17. Antisense Oligonucleotides Used to Target the DUX4 mRNA as Therapeutic Approaches in FaciosScapuloHumeral Muscular Dystrophy (FSHD).

    PubMed

    Ansseau, Eugénie; Vanderplanck, Céline; Wauters, Armelle; Harper, Scott Q; Coppée, Frédérique; Belayew, Alexandra

    2017-03-03

    FacioScapuloHumeral muscular Dystrophy (FSHD) is one of the most prevalent hereditary myopathies and is generally characterized by progressive muscle atrophy affecting the face, scapular fixators; upper arms and distal lower legs. The FSHD locus maps to a macrosatellite D4Z4 repeat array on chromosome 4q35. Each D4Z4 unit contains a DUX4 gene; the most distal of which is flanked by a polyadenylation site on FSHD-permissive alleles, which allows for production of stable DUX4 mRNAs. In addition, an open chromatin structure is required for DUX4 gene transcription. FSHD thus results from a gain of function of the toxic DUX4 protein that normally is only expressed in germ line and stem cells. Therapeutic strategies are emerging that aim to decrease DUX4 expression or toxicity in FSHD muscle cells. We review here the heterogeneity of DUX4 mRNAs observed in muscle and stem cells; and the use of antisense oligonucleotides (AOs) targeting the DUX4 mRNA to interfere either with transcript cleavage/polyadenylation or intron splicing. We show in primary cultures that DUX4-targeted AOs suppress the atrophic FSHD myotube phenotype; but do not improve the disorganized FSHD myotube phenotype which could be caused by DUX4c over-expression. Thus; DUX4c might constitute another therapeutic target in FSHD.

  18. Improved Genome Editing Efficiency and Flexibility Using Modified Oligonucleotides with TALEN and CRISPR-Cas9 Nucleases.

    PubMed

    Renaud, Jean-Baptiste; Boix, Charlotte; Charpentier, Marine; De Cian, Anne; Cochennec, Julien; Duvernois-Berthet, Evelyne; Perrouault, Loïc; Tesson, Laurent; Edouard, Joanne; Thinard, Reynald; Cherifi, Yacine; Menoret, Séverine; Fontanière, Sandra; de Crozé, Noémie; Fraichard, Alexandre; Sohm, Frédéric; Anegon, Ignacio; Concordet, Jean-Paul; Giovannangeli, Carine

    2016-03-08

    Genome editing has now been reported in many systems using TALEN and CRISPR-Cas9 nucleases. Precise mutations can be introduced during homology-directed repair with donor DNA carrying the wanted sequence edit, but efficiency is usually lower than for gene knockout and optimal strategies have not been extensively investigated. Here, we show that using phosphorothioate-modified oligonucleotides strongly enhances genome editing efficiency of single-stranded oligonucleotide donors in cultured cells. In addition, it provides better design flexibility, allowing insertions more than 100 bp long. Despite previous reports of phosphorothioate-modified oligonucleotide toxicity, clones of edited cells are readily isolated and targeted sequence insertions are achieved in rats and mice with very high frequency, allowing for homozygous loxP site insertion at the mouse ROSA locus in particular. Finally, when detected, imprecise knockin events exhibit indels that are asymmetrically positioned, consistent with genome editing taking place by two steps of single-strand annealing.

  19. Spectroscopic (UV/VIS, Raman) and Electrophoresis Study of Cytosine-Guanine Oligonucleotide DNA Influenced by Magnetic Field

    PubMed Central

    Banihashemian, Seyedeh Maryam; Periasamy, Vengadesh; Boon Tong, Goh; Abdul Rahman, Saadah

    2016-01-01

    Studying the effect of a magnetic field on oligonucleotide DNA can provide a novel DNA manipulation technique for potential application in bioengineering and medicine. In this work, the optical and electrochemical response of a 100 bases oligonucleotides DNA, cytosine-guanine (CG100), is investigated via exposure to different magnetic fields (250, 500, 750, and 1000 mT). As a result of the optical response of CG100 to the magnetic field, the ultra-violet-visible spectrum indicated a slight variation in the band gap of CG100 of about 0.3 eV. Raman spectroscopy showed a significant deviation in hydrogen and phosphate bonds’ vibration after exposure to the magnetic field. Oligonucleotide DNA mobility was investigated in the external electric field using the gel electrophoresis technique, which revealed a small decrease in the migration of CG100 after exposure to the magnetic field. PMID:26999445

  20. Developing an Inflatable Solar Array

    NASA Technical Reports Server (NTRS)

    Malone, Patrick K.; Jankowski, Francis J.; Williams, Geoffery T.; Vendura, George J., Jr.

    1992-01-01

    Viewgraphs describing the development of an inflatable solar array as part of the Inflatable Torus Solar Array Technology (ITSAT) program are presented. Program phases, overall and subsystem designs, and array deployment are addressed.