Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “oligonucleotide composition”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 739 records · Page 41Linked to original sources
PubMed1996

Actinomycin D binding to single-stranded DNA: sequence specificity and hemi-intercalation model from fluorescence and 1H NMR spectroscopy.

We have studied the sequence specificity in the binding of the potent antitumor drug actinomycin D (AMD) to single-stranded DNA (ssDNA) by fluorescence and NMR spectroscopy and by molecular modeling. The significant absorption and emission changes accompanying the interaction of the fluorescent derivative 7-amino-AMD with DNAs varying in length and base composition were used to calculate affinity constants for the drug-DNA complexes. The guanine-containing trinucleotide sequences AGT, AGA, and TGT embedded within 25-base oligonucleotides, constituted favorable binding sites. In contrast, the sequence TGA did not bind the drug appreciably. Among the DNAs studied, the highest affinity was for the tetranucleotide sequence TAGT. The binding was length dependent, an oligonucleotide of at least 14 bases being required for effective complex formation (Ka > 10(4) M1=). AMD also bound to poly(d(AGT)). Gel electrophoresis confirmed that the complex was formed between the drug and individual unstructured DNA strands. The 1H NMR spectra of oligonucleotides containing the TAGT site and their complexes with AMD provided further insight into the mode(s) of interaction. A comparison of the measured chemical shifts with those estimated from ring-current calculations provided strong evidence for a hemi-intercalation of AMD between the A and G purine bases with a preference for one of two possible relative orientations. The latter were modeled as complexes with the sequence T3AGT3 and refined by force field calculations with the AMBER program. The biological implications for this novel form of interaction of AMD with single-stranded DNA are discussed.

Base Sequence↗
PubMed1996

Through-bond correlation of imino and aromatic resonances in 13C-, 15N-labeled RNA via heteronuclear TOCSY.

Novel HCCNH TOCSY NMR experiments are presented that provide unambiguous assignment of the exchangeable imino proton resonances by intranucleotide through-bond connectivities to the (assigned) nonexchangeable purine H8 and pyrimidine H6 protons in uniformly 15N-, 13C-labeled RNA oligonucleotides. The HCCNH TOCSY experiments can be arranged as a two-dimensional experiment, correlating solely GH8/UH6 and GH1/UH3 proton resonances (HCCNH), 51 as three-dimensional experiments, in which additional chemical shift labeling either by GN1/UN3 (HCCNH) or by GC8/UC6 (HCCNH) chemical shifts is introduced. The utility of these experiments for the assignment of relatively large RNA oligonucleotides is demonstrated for two different RNA molecules.

Base Composition↗
PubMed1993

The development of chromosome-specific composite DNA probes for the mouse and their application to chromosome painting.

The speed and ease of human cytogenetic analysis has been greatly enhanced by the technique of fluorescence in situ hybridization (FISH). Non-radioactive fluorescently tagged complex DNA probes specific for individual chromosomes can be hybridized to conventionally obtained metaphase chromosome spreads. Several chromosomes may be "painted" concurrently by using combinations of different labeled probes. Surveys of chromosome breakage and rearrangement may be performed very quickly by avoiding the time consuming process of GTG-banding. The application of FISH to mouse cytogenetics would allow large scale molecular toxicology studies to be conducted on the effects of such environmental insults as potential carcinogens, mutagens and radiation. Progress has been hampered, however, as the Mus musculus karyotype consists of 40 acrocentric chromosomes of approximately the same size, making the recognition and separation of individual chromosomes very difficult. We now describe the successful production and application of chromosome-specific composite DNA probes for M. musculus chromosomes 2 and 8. Stable Robertsonian translocated chromosomes were isolated on a flow sorter and their DNA subsequently amplified by degenerate oligonucleotide primer (DOP) PCR. Small pools (300 copies) of each chromosome were denatured at 94 degrees C then annealed with the primer at 30 degrees C for 15 cycles. This was followed by 20 cycles at an annealing temperature of 62 degrees C. Additional amplification was performed at an annealing temperature of 62 degrees C. The chromosome-specific DNA was labeled with biotin 11-dUTP by nick translation and used for FISH. The usefulness of the technique for translocation detection is demonstrated by analyzing chromosome exchanges induced in mice irradiated with 137Cs gamma rays.

Animals↗
PubMed2000

Parallel-stranded DNA with Hoogsteen base pairing stabilized by a trans-[Pt(NH3)2]2+ cross-link: characterization and conversion into a homodimer and a triplex.

The oligonucleotides 5'-d(TTTTCTTTTG) and 5'-d(AAAAGAAAAG) were cross-linked with a trans-[Pt(NH3)2]2+ entity via the N7 positions of the 3'-end guanine bases to give parallel-stranded (ps) DNA. At pH 4.2, CD and NMR spectroscopy indicate the presence of Hoogsteen base pairing. In addition, temperature-dependent UV spectroscopy shows an increase in melting temperature for the platinated duplex (35 degrees C) as compared to the non-platinated, antiparallel-stranded duplex formed from the same oligonucleotides (21 degrees C). A monomer-dimer equilibrium for the platinated 20mer is revealed by gel electrophoresis. At pH 4.2, addition of a third strand of composition 5'-d(AGCTTTTCTTTTAG) to the ps duplex leads to the formation of a triple helix with two distinct melting points at 38 degrees C (platinum cross-linked Hoogsteen part) and 21 degrees C (Watson-Crick part), respectively.

Base Pairing↗
PubMed1985

In vitro reconstitution of messenger ribonucleoprotein particles from globin messenger RNA and cytosol proteins.

Deproteinized globin poly(A) + mRNAs reassociate readily in vitro with soluble RNA-binding proteins of the cytosol; reconstituted messenger ribonucleoprotein complexes are obtained which are very similar to native globin polyribosomal-mRNP as far as bouyant density in Cs2SO4 and the composition of proteins which can be crosslinked to the mRNA are concerned. Proteins thus identified bind specifically to mRNA and not to ribosomal RNA or any synthetic oligonucleotides, with one exception: a 78-kDa protein could be cross-linked to poly(A).

Animals↗
PubMed2006

Single-stranded conformational polymorphism for separation of mixed rRNAS (rRNA-SSCP): a new method for profiling microbial communities.

We show that non-denaturing gel electrophoresis, or single-stranded conformational polymorphism (SSCP), can be used to separate mixtures of full-length rRNAs. Individual bands can then be excised for identification by RT-PCR and sequencing. This has the advantage over profiling methods such as DGGE and T-RFLP that no PCR amplification is involved prior to sequencing; thus, extraction biases aside, it should yield a quantitative picture of community composition in terms of ribosome content. To simplify banding patterns, RNA subsamples (e.g. bacterial 16S rRNA) can first be isolated by magnetic bead capture hybridization. Alternatively, oligonucleotide-directed ribonuclease H (RNase H) digestion can be used to identify bands of interest by running digested samples in parallel to undigested ones. We illustrate the use of this technique to identify a potentially predominant species in a hypersaline microbial mat. We anticipate that rRNA-SSCP will be useful for community profiling; for clone library construction by directed cloning of individual rRNAs; and for following incorporation of radiolabeled substrates at the species level, by gel autoradiography, without advance information or guesswork about which species might be active and abundant.

Bacteria↗
PubMed1987

Cloning of cDNAs coding for the heavy chain region and connecting region of human factor V, a blood coagulation factor with four types of internal repeats.

Human factor V is a high molecular weight plasma glycoprotein that participates as a cofactor in the conversion of prothrombin to thrombin by factor Xa. Prior to its participation in the coagulation cascade, factor V is converted to factor Va by thrombin generating a heavy chain and a light chain, and these two chains are held together by calcium ions. A connecting region originally located between the heavy and light chains is liberated during the activation reaction. In a previous study, a cDNA of 2970 nucleotides that codes for the carboxyl-terminal 938 amino acids of factor V was isolated and characterized from a Hep G2 cDNA library [Kane, W. H., & Davie, E. W. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 6800-6804]. This cDNA has been used to obtain additional clones from Hep G2 and human liver cDNA libraries. Furthermore, a Hep G2 cDNA library prepared with an oligonucleotide from the 5' end of these cDNAs was screened to obtain overlapping cDNA clones that code for the amino-terminal region of the molecule. The composite sequence of these clones spans 6911 nucleotides and is consistent with the size of the factor V message present in Hep G2 cells (approximately 7 kilobases). The cDNA codes for a leader sequence of 28 amino acids and a mature protein of 2196 amino acids. The amino acid sequence predicted from the cDNA was in complete agreement with 139 amino acid residues that were identified by Edman degradation of cyanogen bromide peptides isolated from the heavy chain region and connecting region of plasma factor V.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2006

Controlling DNA orientation on mixed ssDNA/OEG SAMs.

We present and characterize a mixed self-assembled monolayer (SAM) consisting of single-stranded oligonucleotide (ssDNA)- and oligo(ethylene glycol) (OEG)-terminated thiols. The ssDNA/OEG SAMs are prepared by simultaneous coadsorption from a common thiol solution over a broad range of compositions. Electron spectroscopy for chemical analysis (ESCA) is used to measure the surface coverage of ssDNA, whereas surface plasmon resonance (SPR) sensor is used to measure the hybridization of complementary ssDNA and protein resistance. Through the complementary use of these techniques, we find that the composition of OEG in the assembly solution controls a key parameter: the surface coverage of ssDNA on the surface. There is evidence that it influences the orientation of the immobilized ssDNA probes. Lower OEG concentrations yield a surface with higher ssDNA coverage and less favorable orientation, whereas higher OEG concentrations produce a surface with lower DNA coverage and more favorable orientation. Competition between these two effects controls the hybridization efficiency of the ssDNA surface. Compared to ssDNA surfaces prepared with other diluent thiols, the use of OEG improves the protein resistance of the surface, making it more broadly applicable.

Adsorption↗
PubMed2001

Microbial community dynamics and evaluation of bioremediation strategies in oil-impacted salt marsh sediment microcosms.

Microbial community dynamics in wetlands microcosms emended with commercial products (surfactant, a biological agent, and nutrients) designed to enhance bioremediation was followed for 3 months. The effectiveness of enhanced degradation was assessed by determining residual concentrations of individual petroleum hydrocarbons by GC/MS. The size and composition of the sediment microbial community was assessed using a variety of indices, including bacterial plate counts, MPNs, and DNA hybridizations with domain- and group-specific oligonucleotide probes. The addition of inorganic nutrients was the most effective treatment for the enhancement of oil degradation, resulting in marked degradation of petroleum alkanes and a lesser extent of degradation of aromatic oil constituents. The enhanced degradation was associated with increases in the amount of extractable microbial DNA and Streptomyces in the sediment, although not with increased viable counts (plate counts, MPN). Bacteria introduced with one of the proprietary products were still detected in the microcosms after 3 months, but were not a major quantitative constituent of the community. The biological product enhanced oil degradation relative to the control, but to a lesser extent than the nutrient additions alone. In contrast, application of the surfactant to the oil-impacted sediment decreased oil degradation.

Biodegradation, Environmental↗
PubMed1983

Biotin and fluorescent labeling of RNA using T4 RNA ligase.

Biotin, fluorescein, and tetramethylrhodamine derivatives of P1-(6-aminohex-1-yl)-P2-(5'-adenosine) pyrophosphate were synthesized and used as substrates with T4 RNA ligase. In the absence of ATP, the non-adenylyl portion of these substrates is transferred to the 3'-hydroxyl of an RNA acceptor to form a phosphodiester bond and the AMP portion is released. E. coli and D. melanogaster 5S RNA, yeast tRNAPhe, (Ap)3C, and (Ap)3A serve as acceptors with yields of products varying from 50 to 100%. Biotin-labeled oligonucleotides are bound selectively and quantitatively to avidin-agarose and may be eluted with 6 M guanidine hydrochloride, pH 2.5. Fluorescein and tetramethylrhodamine-labeled oligonucleotides are highly fluorescent and show no quenching due to attachment to the acceptor. The diverse structures of the appended groups and of the chain lengths and compositions of the acceptor RNAs show that T4 RNA ligase will be a useful modification reagent for the addition of various functional groups to the 3'-terminus of RNA molecules.

Animals↗
PubMed1995

Solution structures of unimolecular quadruplexes formed by oligonucleotides containing Oxytricha telomere repeats.

BACKGROUND: Oligonucleotides containing the guanine-rich telomeric sequence of Oxytricha chromosomes (dT4G4) have previously been shown to form DNA quadruplexes comprising guanine quartets stabilized by cations. Two different structures have been reported for both d(G4T4G4) (Oxy1.5) and d(G4T4G4T4G4T4G4) (Oxy3.5). RESULTS: Here we present the solution structure of a uracil- and inosine-containing derivative of Oxy3.5, d(G4TUTUG4T4G4UUTTG3I) (Oxy3.5-U4128), determined using two-dimensional 1H and 31P NMR techniques. This oligonucleotide forms a unimolecular quadruplex that is very similar to the dimeric Oxy1.5 solution structure, in that it contains a loop spanning the diagonal of an end quartet. The groove widths, strand polarities, and positions of the syn bases along the G4 tracts and within the quartets are all as reported for Oxy1.5. The first and third pyrimidine tracts form parallel loops spanning a wide groove and a narrow groove respectively. CONCLUSIONS: Both Oxy3.5 and Oxy3.5-U(4)128 form unimolecular quadruplexes in solution with a diagonal central T4 loop. These results conflict with those reported for d(G4TUTUG4TTUUG4UUTTG4) in solution, in which the central loop spans a wide groove.

Animals↗
PubMed1994

Replacement of the conserved G.U with a G-C pair at the cleavage site of the Tetrahymena ribozyme decreases binding, reactivity, and fidelity.

There is a phylogenetically conserved G.U pair at the 5'-splice site of group I introns. When this is mutagenized to a G-C pair, splicing of these introns is greatly reduced. We have used a ribozyme derived from the Tetrahymena group I intron to compare the binding and reactivity of oligonucleotides that form either a G.U or a G-C pair at this position. Ribozyme binding of oligonucleotides at 42 degrees C was measured by native gel electrophoresis and equilibrium dialysis. Binding of GGCCCUCC (C(-1)P), which base-pairs with the ribozyme guide sequence to form a G-C at the cleavage site, was 10-fold weaker than the binding of GGCCCUCU (U(-1)P), which maintains the conserved G.U pair at the cleavage site. This is surprising since a terminal G-C enhances the binding between oligonucleotides by 20-fold relative to a terminal G.U. Thermal denaturation studies indicate that C(-1)P and several analogs with deoxy substitutions bind the guide-sequence oligonucleotide, GGAGGGAAA, as strongly as they bind the ribozyme. In contrast, U(-1)P binds 240-fold more strongly to the ribozyme than to GGAGGGAAA, a difference that is decreased by deoxy substitutions. Thus, while U(-1)P binds the ribozyme through a combination of base-pairing and specific 2-OH and other tertiary interactions, C(-1)P may bind by base-pairing alone. The substrate GGCCCUCCAAAAA (C(-1)S) is cleaved 100-fold more slowly than GGCCCUCUAAAAA (U(-1)S) and also has a higher propensity to be cleaved at the wrong nucleotide position.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed2003

[Characterization of several synthetic and environmental microbial samples by quantitative hybridization].

Limitations of traditional techniques based on selective enrichment and pure-culture isolation make it difficult to precisely characterize the natural microbial ecosystems. Molecular techniques are now being developed and used to address these limitations. Group-specific 16S rRNAs-targeted oligonucleotide probes of different phylogenetic levels are increasingly used to identify and quantify the microbial members in complex environmental samples. Two domain-specific probes were used in this study to elementarily characterize the defined mixtures of RNAs extracted from pure culture (synthetic samples) and a RNA sample obtained from landfill leachate (environmental sample). The results demonstrated that 16S rRNA quantitative hybridization provided an excellent estimation of domain level community composition of these samples, and thus, had a huge potential of usefulness in microbial ecology studies.

Ecology↗
PubMed1996

Fate of cationic liposomes and their complex with oligonucleotide in vivo.

The present studies describe the biodistribution of cationic liposomes and cationic liposome/oligonucleotide complex following intravenous injection into mice via the tail vein. (111)In-diethylenetriaminepentaacetic acid stearylamide ((111)In-DTPA-SA) was used as a lipid-phase radiolabel. Inclusion of up to 5 mol% DTPA-SA in liposomes composed of 3beta-(N-(N',N'-dimethylaminoethane)carbamoyl)cholesterol (DC-Chol) and dioleoylphosphatidylethanolamine (DOPE) did not influence liposome formation or size, nor the binding/uptake or fusion of the cationic liposomes with CHO cells in vitro. Moreover, nuclear delivery of oligonucleotide to CHO cells was unaffected by the probe. The biodistribution of liposomes with increasing concentration of DC-Chol (1:4-4:1, DC-Chol/DOPE, mol/mol) at 24 h post-injection revealed no dependence on lipid composition. Uptake was primarily by liver, and accumulation in spleen and skin was also observed. Comparatively little accumulation occurred in lung. Clearance of injected liposomes by liver was very rapid (approximately 84.5% of the injected dose by 7.5 h post-injection). Liposome uptake by liver and spleen were equally efficient in the dose range of 3.33 to 33.33 mg/kg body weight, yet possible saturation of liver uptake at a dose of 66.80 mg/kg may have allowed for increased spleen accumulation. Preincubation of cationic liposomes with phosphorothioate oligonucleotide induced a dramatic yet transient accumulation of the lipid in lung which gradually redistributed to liver. Similar results were observed when monitoring iodinated oligonucleotide in the complex. Immuno-histochemical studies revealed large aggregates of oligonucleotide within pulmonary capillaries at 15 min post-injection, suggesting the early accumulation in lung was due to embolism. Immuno-histochemical studies further revealed labeled oligonucleotide to be localized primarily to Kupffer cells at 24 h post-injection. Immuno-electron microscopy revealed localization of oligonucleotide primarily to the lumen of pulmonary capillaries at 15 min post-injection. Immuno-electron microscopy revealed localization of oligonucleotide primarily to the lumen of pulmonary capillaries at 15 min post-injection, and to phagocytic vacuoles of Kupffer cells at 24 h post-injection. By these methods, nuclear delivery of oligonucleotide in vivo was not observed. Increasing concentration of mouse serum inhibited cellular binding/uptake of cationic liposomes in vitro, without or with complexed oligonucleotide. We therefore postulate that interaction with plasma components, including opsonin(s), inhibits cellular uptake of the injected liposomes as well as the liposome/oligonucleotide complex, and mediates rapid uptake by Kupffer cells of the liver. These results are relevant to the design of cationic liposomes for efficient delivery of nucleic acid in vivo.

Animals↗
PubMed1993

Conformational changes in oligo-nucleotides upon binding to a peptide representing the N-terminal region of a viral coat protein. An optical spectroscopy study.

Conformational changes of the oligonucleotides r(A)12, d(GC)5, and d(AT)5 upon binding to a pentacosapeptide representing the RNA-binding N-terminus of the coat protein of cowpea chlorotic mottle virus (CCMV) were studied by using absorption and circular dichroism spectroscopy. The peptide destabilizes the single-stranded structure of r(A)12 at pH 7.2, but disrupts the double-stranded structure of r(A)12 at pH 5.0. However, at pH 4.0, the peptide is not able to disrupt this double-stranded structure. The double-stranded structures of d(GC)5 and d(AT)5 with Watson-Crick base-pairing are stabilized upon binding to the peptide.

Adenine Nucleotides↗
PubMed2004

Global features of sequences of bacterial chromosomes, plasmids and phages revealed by analysis of oligonucleotide usage patterns.

BACKGROUND: Oligonucleotide frequencies were shown to be conserved signatures for bacterial genomes, however, the underlying constraints have yet not been resolved in detail. In this paper we analyzed oligonucleotide usage (OU) biases in a comprehensive collection of 155 completely sequenced bacterial chromosomes, 316 plasmids and 104 phages. RESULTS: Two global features were analyzed: pattern skew (PS) and variance of OU deviations normalized by mononucleotide content of the sequence (OUV). OUV reflects the strength of OU biases and taxonomic signals. PS denotes asymmetry of OU in direct and reverse DNA strands. A trend towards minimal PS was observed for almost all complete sequences of bacterial chromosomes and plasmids, however, PS was substantially higher in separate genomic loci and several types of plasmids and phages characterized by long stretches of non-coding DNA and/or asymmetric gene distribution on the two DNA strands. Five of the 155 bacterial chromosomes have anomalously high PS, of which the chromosomes of Xylella fastidiosa 9a5c and Prochlorococcus marinus MIT9313 exhibit extreme PS values suggesting an intermediate unstable state of these two genomes. CONCLUSIONS: Strand symmetry as indicated by minimal PS is a universally conserved feature of complete bacterial genomes that results from the matching mutual compensation of local OU biases on both replichors while OUV is more a taxon specific feature. Local events such as inversions or the incorporation of genome islands are balanced by global changes in genome organization to minimize PS that may represent one of the leading evolutionary forces driving bacterial genome diversification.

Bacteriophages↗