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PubMedDate not supplied

Stabilization of DNA triple-helix formation by appended cationic peptides.

We have investigated the impact of appended cationic peptides on the triplex-forming and thermal-stabilizing abilities of an oligonucleotide "third strand" using a homopurine-homopyrimidine 37-mer as the host duplex target. The family of appended cationic peptides studied here contains arginine, lysine, ornithine, and diaminobutyric acid, with the residues being linked through either their alpha-amino or side chain amino groups. On the basis of optical melting profiles, we find that peptides with different amino acid compositions, but with four positive charges in common, are able to enhance, in a similar manner, the triplex-forming capability of an oligonucleotide. We note the implications of these results for the rational design of third-strand probes.

Aminobutyrates↗
PubMed2006

Dynamic assembly of primers on nucleic acid templates.

A strategy is presented that uses dynamic equlibria to assemble in situ composite DNA polymerase primers, having lengths of 14 or 16 nt, from DNA fragments that are 6 or 8 nt in length. In this implementation, the fragments are transiently joined under conditions of dynamic equilibrium by an imine linker, which has a dissociation constant of approximately 1 muM. If a polymerase is able to extend the composite, but not the fragments, it is possible to prime the synthesis of a target DNA molecule under conditions where two useful specificities are combined: (i) single nucleotide discrimination that is characteristic of short oligonucleotide duplexes (four to six nucleobase pairs in length), which effectively excludes single mismatches, and (ii) an overall specificity of priming that is characteristic of long (14 to 16mers) oligonucleotides, potentially unique within a genome. We report here the screening of a series of polymerases that combine an ability not to accept short primer fragments with an ability to accept the long composite primer held together by an unnatural imine linkage. Several polymerases were found that achieve this combination, permitting the implementation of the dynamic combinatorial chemical strategy.

Base Pair Mismatch↗
PubMed1980

Characterization of the viral RNA species of prototype dengue viruses.

Dengue virus isolates have been grown in the Igarashi clone C6/36 Aedes albopictus cells in amounts suitable for biochemical studies. Oligonucleotide fingerprint analyses have been used to characterize the virion 40 S RNA species of the four prototype dengue (DEN) serotype viruses: DEN 1, Hawaiian strain; DEN 2, New Guinea C strain (both insect and mouse brain passaged derivatives); DEN 3, H-87 strain; and DEN 4, H-241 strain. Comparisons of the largest oligonucleotides derived by ribonuclease T1 digestion of these 40 S DEN virion RNA species indicate that ther are few, if any, large oligonucleotides that are homologous between any two fo the four dengue prototype strains. Preliminary analyses of the 40 S RNA 5' terminal sequence of DEN 3 virion RNA indicate that it has the composition of m7GpppAmpXp.

Aedes↗
PubMed1999

Synthesis and characterization of oligonucleotides containing 5',8-cyclopurine 2'-deoxyribonucleosides: (5'R)-5',8-cyclo-2'-deoxyadenosine, (5'S)-5',8-cyclo-2'-deoxyguanosine, and (5'R)-5',8-cyclo-2'-deoxyguanosine.

Radiation-induced degradation of purine and pyrimidine nucleosides gave rise to carbon-bridged cyclocompounds. Such cyclonucleosides represent a class of tandem lesions in which modification of both the base and 2-deoxyribose has occurred. A solid-phase synthetic method was designed for the incorporation of both 5'R and 5'S diastereoisomers of 5',8-cyclopurine 2'-deoxyribonucleosides into oligodeoxynucleotides to facilitate the assessment of the biochemical and biophysical features of such lesions. We report the preparation of the phosphoramidite synthons of (5'R)-5', 8-cyclo-2'-deoxyadenosine (2), (5'S)-5',8-cyclo-2'-deoxyguanosine (3), and (5'R)-5',8-cyclo-2'-deoxyguanosine (4). Fully protected compounds 10, 18, and 25 were then inserted into several oligonucleotides by automated procedures. Analysis of modified DNA oligomers 26-31 by electrospray mass spectrometry and enzymatic digestions with exo- and endonucleases confirmed the base compositions and the integrity of free radical-induced tandem lesions 2-4 that were chemically inserted.

Alkaline Phosphatase↗
PubMed2002

Extensive set of 16S rRNA-based probes for detection of bacteria in human feces.

For the detection of six groups of anaerobic bacteria in human feces, we designed seven new 16S rRNA-based oligonucleotide probes. This set of probes extends the current set of probes and gives more data on the composition of the human gut flora. Probes were designed for Phascolarctobacterium and relatives (Phasco741), Veillonella (Veil223), Eubacterium hallii and relatives (Ehal1469), Lachnospira and relatives (Lach571), and Eubacterium cylindroides and relatives (Ecyl387), and two probes were designed for Ruminococcus and relatives (Rbro730 and Rfla729). The hybridization conditions for the new probes were optimized for fluorescent in situ hybridization, and the probes were validated against a set of reference organisms. The probes were applied to fecal samples of 11 volunteers to enumerate their target bacterial groups. The Phasco741 and Veil223 probes both detected average numbers below 1% of the total number of bacteria as determined with the bacterial kingdom-specific Bact338 probe. The Ecyl387 probe detected about 1.4%, the Lach571 and Ehal1469 probes detected 3.8 and 3.6%, respectively, and a combination of the Rbro730 and Rfla729 probes detected 10.3%. A set of 15 probes consisting of probes previously described and those presented here were evaluated in hybridization with the fecal samples of the same volunteers. Together, the group-specific probes detected 90% of the total bacterial cells.

Bacteria↗
PubMedDate not supplied

Computational systems analysis of developmental toxicity: design, development and implementation of a Birth Defects Systems Manager (BDSM).

Birth defects and developmental disabilities remain an important public health issue worldwide. With the availability of genomic sequences from a growing number of human and model organisms and the rapid expansion of the public repositories holding large-scale gene expression datasets, a computational systems analysis of developmental toxicology can incorporate this vast digital information toward the realization of predictive models for complex disease. Here we describe the initial design, development and implementation of a Birth Defects Systems Manager (BDSM). The project was motivated by the need for a computational-bioinformatics infrastructure to manage vast digital information from functional genomics and for a new knowledge environment specifically engineered for the analysis of developmental processes and toxicities. Proof-of-concept tested BDSM using meta-analysis of gene expression data collected from different laboratories, technology platforms, and study models. The composite dataset incorporated 232 microarray comparisons of RNA samples by single or dual microarray platforms, cDNA or oligonucleotide based probes, and human or mouse sequence information. Preliminary results identified system-level features in the embryonic transcriptome as it reacted to various developmental-teratological stimuli. BDSM is open access through the worldwide web (http://systemsanalysis.louisville.edu/) and can be integrated with other bioinformatics tools and resources to advance the pace of discovery in birth defects research.

Animals↗
PubMed2006

Homogeneous versus heterogeneous probes for microbial ecological microarrays.

Microbial ecological microarrays have been developed for investigating the composition and functions of microorganism communities in environmental niches. These arrays include microbial identification microarrays, which use oligonucleotides, gene fragments or microbial genomes as probes. In this article, the advantages and disadvantages of each type of probe are reviewed. Oligonucleotide probes are currently useful for probing uncultivated bacteria that are not amenable to gene fragment probing, whereas the functional gene fragments amplified randomly from microbial genomes require phylogenetic and hierarchical categorization before use as microbial identification probes, despite their high resolution for both specificity and sensitivity. Until more bacteria are sequenced and gene fragment probes are thoroughly validated, heterogeneous bacterial genome probes will provide a simple, sensitive and quantitative tool for exploring the ecosystem structure.

Bacteria↗
PubMed2006

Sequential injection analysis system for the sandwich hybridization-based detection of nucleic acids.

A sequential injection analysis lab-on-valve (SIA-LOV) system was developed for the specific detection of single-stranded nucleic acid sequences via sandwich hybridization of specific DNA probes to the target sequence. One DNA probe was tagged with fluorescein; the other was biotinylated and immobilized to streptavidin-coated porous beads. The system was optimized with respect to buffer composition, length of hybridization and wash steps, and volumes and concentrations of components used. On-bead oligonucleotide hybridization was studied using UV detection at 260 nm, while a final dose response curve was quantified using fluorescence detection. A dynamic range of 1-1000 pmol was obtained for a synthetic DNA sequence that was homologous to a segment in the B. anthracis atxA mRNA. A within-day variation of 7.2% and a day-to-day variation of 9.9% was observed. Each analysis was completed within 20 min. Subsequently, the system was applied to the detection of atxA mRNA expressed in a surrogate organism and amplified using NASBA. The SIA-LOV will find its application in routine laboratory-based analysis of specific single-stranded DNA/RNA sequences. Future improvements will include the integration of dye-encapsulating liposomes for signal enhancement used in lieu of the single fluorophore-labeled probe in order to lower the limit of detection.

Biosensing Techniques↗
PubMed2006

Nucleobase orientation and ordering in films of single-stranded DNA on gold.

We demonstrate how the orientation and ordering of DNA bases in ultrahigh vacuum (UHV) and ambient environments can be determined using complementary spectroscopic methods. Near-edge X-ray absorption fine structure (NEXAFS) with fluorescence detection, X-ray photoelectron (XPS), and Fourier transform infrared (FTIR) spectroscopies are used to quantify the coverage, chemical composition, orientation, and ordering of thymine bases in model self-assembled monolayers of thymine homo-oligonucleotides [oligo(dT)] on gold. We find that, in monolayers of thiol-modified oligo(dT), thymine bases tend to orient parallel to the Au substrate, and this preferential orientation is significantly more pronounced in monolayers of thiolated 5-mers compared to 25-mers. We interpret this preferential orientation as a signature of significant correlations (local ordering) between individual nuleobases, which offers a way to quantify and compare nucleobase interactions in films under both ambient and UHV conditions.

DNA, Single-Stranded↗
PubMed2004

Matching strategies for genetic association studies in structured populations.

Association studies in populations that are genetically heterogeneous can yield large numbers of spurious associations if population subgroups are unequally represented among cases and controls. This problem is particularly acute for studies involving pooled genotyping of very large numbers of single-nucleotide-polymorphism (SNP) markers, because most methods for analysis of association in structured populations require individual genotyping data. In this study, we present several strategies for matching case and control pools to have similar genetic compositions, based on ancestry information inferred from genotype data for approximately 300 SNPs tiled on an oligonucleotide-based genotyping array. We also discuss methods for measuring the impact of population stratification on an association study. Results for an admixed population and a phenotype strongly confounded with ancestry show that these simple matching strategies can effectively mitigate the impact of population stratification.

Case-Control Studies↗
PubMed2003

Effect of CaCl2 concentration on the rate of foreign gene transfer and expression by in vivo electroporation in the mouse ovary.

We tested the effect of electroporation (EP) medium composition on the rate of gene transfer and expression in the mouse ovary in vivo. FITC labeled oligonucleotides were dissolved in a medium with varying levels of CaCl2 concentration from 0 to 250 mM, and transferred by in vivo EP. Gene transfer efficiency was assessed by examining fluorescence signal intensity with a fluorescent microscope at 3 h after in vivo EP. The results indicated that CaCl2 concentration at 50 mM gave the highest transfer efficiency of the oligonucleotides only in the presence of phosphate buffered saline (PBS). Without PBS or CaCl2, the oligonucleotide transfer was negligible. A further increase in CaCl2 from 50 to 250 mM lowered the transfer efficiency. Little fluorescence signal was attained by substituting CaCl2 for MgCl2, NaCl or KCl. Addition of glycerol to the EP medium with 50 mM CaCl2 did not improve the transfer efficiency in the presence of PBS, although a marginal increase was observed in the absence of PBS. The stimulating effect of increased CaCl2 concentration from 0 to 50 mM was further evaluated by examining the intensity of reporter protein expression after transferring the bacterial lacZ gene. The results of X-gal staining demonstrated that CaCl2 with a range of 20 to 100 mM, showed enhanced gene expression in comparison with 0 mM. However, no remarkable difference was observed between the different CaCl2 concentrations, suggesting that the stimulating effect of CaCl2 on gene transfer and expression in the mouse ovary in vivo may not necessarily parallel in terms of the optimal concentration.

Animals↗
PubMed1994

Further studies on the use of oligonucleotide facilitators to increase ribozyme turnover.

We showed previously that the turnover of a hammerhead ribozyme cleaving a substrate RNA could be increased by an oligonucleotide (facilitator) that bound to the substrate continguously with the ribozyme. This phenomenon has been investigated further by varying the temperature and the number of base pairs formed between the ribozyme and substrate. The results support the hypothesis that facilitators act by cooperative binding and are beneficial when the rate of cleavage is limited by the stability of the ribozyme/substrate complex. The facilitator also promotes cleavage at lower concentrations of magnesium.

Base Composition↗
PubMed1999

Population structure and phylogenetic characterization of marine benthic Archaea in deep-sea sediments.

During the past few years Archaea have been recognized as a widespread and significant component of marine picoplankton assemblages and, more recently, the presence of novel archaeal phylogenetic lineages has been reported in coastal marine benthic environments. We investigated the relative abundance, vertical distribution, phylogenetic composition, and spatial variability of Archaea in deep-sea sediments collected from several stations in the Atlantic Ocean. Quantitative oligonucleotide hybridization experiments indicated that the relative abundance of archaeal 16S rRNA in deep-sea sediments (1500 m deep) ranged from about 2.5 to 8% of the total prokaryotic rRNA. Clone libraries of PCR-amplified archaeal rRNA genes (rDNA) were constructed from 10 depth intervals obtained from sediment cores collected at depths of 1,500, 2,600, and 4,500 m. Phylogenetic analysis of rDNA sequences revealed the presence of a complex archaeal population structure, whose members could be grouped into discrete phylogenetic lineages within the two kingdoms, Crenarchaeota and Euryarchaeota. Comparative denaturing gradient gel electrophoresis profile analysis of archaeal 16S rDNA V3 fragments revealed a significant depth-related variability in the composition of the archaeal population.

Archaea↗
PubMedDate not supplied

Oligonucleotide probes for the identification of three algal groups by dot blot and fluorescent whole-cell hybridization.

Photosynthetic pico- and nanoplankton dominate phytoplankton biomass and primary production in the oligotrophic open ocean. Species composition, community structure, and dynamics of the eukaryotic components of these size classes are poorly known primarily because of the difficulties associated with their preservation and identification. Molecular techniques utilizing 18S rRNA sequences offer a number of new and rapid means of identifying the picoplankton. From the available 18S rRNA sequence data for the algae, we designed new group-specific oligonucleotide probes for the division Chlorophyta, the division Haptophyta, and the class Pelagophyceae (division Heterokonta). Dot blot hybridization with polymerase chain reaction amplified target rDNA and whole-cell hybridization assays with fluorescence microscopy and flow cytometry were used to demonstrate probe specificity. Hybridization results with representatives from seven algal classes supported the phylogenetic affinities of the cells. Such group- or taxon-specific probes will be useful in examining community structure, for identifying new algal isolates, and for in situ detection of these three groups, which are thought to be the dominant algal taxa in the oligotrophic regions of the ocean.

Chlorophyta↗
PubMed2002

The microbial community composition of a nitrifying-denitrifying activated sludge from an industrial sewage treatment plant analyzed by the full-cycle rRNA approach.

The composition of the microbial community present in the nitrifying-denitrifying activated sludge of an industrial wastewater treatment plant connected to a rendering facility was investigated by the full-cycle rRNA approach. After DNA extraction using three different methods, 94 almost full-length 16S rRNA gene clones were retrieved and analyzed phylogenetically. 59% of the clones were affiliated with the Proteobacteria and clustered with the beta- (29 clones), alpha- (24), and delta-class (2 clones), respectively. 15 clones grouped within the green nonsulfur (GNS) bacteria and 11 clones belonged to the Planctomycetes. The Verrucomicrobia, Acidobacteria, Nitrospira, Bacteroidetes, Firmicutes and Actinobacteria were each represented by one to five clones. Interestingly, the highest 'species richness' [measured as number of operational taxonomic units (OTUs)] was found within the alpha-class of Proteobacteria, followed by the Planctomycetes, the beta-class of Proteobacteria, and the GNS-bacteria. The microbial community composition of the activated sludge was determined quantitatively by using 36 group-, subgroup-, and OTU-specific rRNA-targeted oligonucleotide probes for fluorescence in situ hybridization (FISH), confocal laser scanning microscopy and digital image analysis. 89% of all bacteria detectable by FISH with a bacterial probe set could be assigned to specific divisions. Consistent with the 16S rRNA gene library data, members of the beta-class of Proteobacteria dominated the microbial community and represented almost half of the biovolume of all bacteria detectable by FISH. Within the beta-class, 98% of the cells could be identified by the application of genus- or OTU-specific probes demonstrating a high in situ abundance of bacteria related to Zoogloea and Azoarcus sensu lato. Taken together, this study provides the first encompassing, high-resolution insight into the in situ composition of the microbial community present in a full-scale, industrial wastewater treatment plant.

Bacteria↗
PubMed1985

Activation of oestrogen receptor complexes: evidence for the distinct regulation of ligand and oligonucleotide binding sites.

The activation of the rat uterine oestrogen receptor has been measured in vitro by its binding to oligodeoxythymidylate cellulose (oligo(dT] and was found to be sensitive to the time and temperature of prior incubation of cytosol with oestradiol. The presence of 20 mM dithiothreitol promoted receptor activation and was partially inhibited by 10 mM molybdate; molybdate also inhibited the time- and temperature-dependent activation of receptor. The nucleotides GTP, ATP, ADP, CTP and UTP all promoted receptor activation; the effect of GTP was significantly greater than that of ATP. It is unlikely that phosphate donation is involved in receptor activation as the effects of GTP could be reproduced by p[NH]ppG (guanosine 5'-[beta, gamma-imido]triphosphate), while PPi was also effective in activating receptor. The results provide evidence for the distinct regulation of the oligonucleotide- and ligand-binding domains, since manipulations which promoted binding to oligo(dT) did not affect either ligand binding capacity or the rate constant and composition the biphasic dissociation of the ligand receptor complex.

Animals↗