SEARCH · PubMed Health

Results for “duplex sequencing”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

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

At least 1,099 records · Page 61Linked to original sources

PubMed2005

Self-organisation of an oligodeoxynucleotide containing the G- and C-rich stretches of the direct repeats of the human mitochondrial DNA.

Stretches of cytosines and guanosines have been shown in vitro to adopt non-canonical structures known as i-motifs and G-quartets, respectively. When combined, such sequences are expected to either retain their structure or form duplexes or triple helices. All these structures may occur in vivo whenever the sequence criteria are met. Such stretches are present in the circular genome of human mitochondria, as two 10 nucleotide-long perfect tandem direct repeats (DR1 and DR2). The DR1 and DR2 repeats are G-rich on the heavy strand and C-rich on the light strand. Previous results suggested that during replication, transient formation of a parallel GGC triple helix between the neo-synthesised G-rich DR1 and the double-stranded homologous DR2 could be involved in a rearrangement process leading to genome instability. In order to get structural insights into the interaction between the two repeats, we have studied by nuclear magnetic resonance (NMR) the assembly properties of a 24-mer oligodeoxyribonucleotide in which the C- and G-rich segments of the DRs are covalently tethered by a TTTT linker. We show here that this 24-mer self-associates into a triplex-containing symmetrical tetramer. The core of the structure is composed of anti-parallel Watson-Crick (WC) base pairs. Two additional strands are hydrogen-bonded to the Hoogsteen side of the Gs, thus forming CGC(+) triple helices, with G-rich ends folding into G-quartets. These results suggest that such structures could occur when the two DRs are put to close proximity in a biological context.

Base Sequence↗

PubMed1990

The origin binding protein of herpes simplex virus 1 binds cooperatively to the viral origin of replication oris.

The origin binding protein (OBP) or herpes simplex virus 1 has been expressed in Escherichia coli and used to study the role of multiple OBP binding sites in the herpes simplex virus #1 origin of replication, oris. Our results showed that the sequence CGTTCGCACTT was required for the binding of OBP to duplex DNA with high affinity. The minimal oris contains three repeats of this sequence or close derivatives thereof. Filter binding experiments have demonstrated that specific binding occurs to two of these repeats, box I and box II. An investigation using the DNase I footprinting technique revealed that the binding of OBP to box I and box II was cooperative and led to the formation of a highly organized complex in which the entire oris sequence was induced. We observed furthermore that the AT-rich sequence of the oris dyad was readily accessible to macromolecules even in the OBP.oris complex. The DNase I cleavage pattern of this sequence was, however, altered radically, indicating that a significant conformational change had occurred. A tentative structural model for the OBP-oris interaction is discussed on the basis of these observations.

Base Sequence↗

PubMed1997

Resolution of an early RecA-recombination intermediate by a junction-specific endonuclease.

The nucleoprotein filament formed on a circular single strand by Escherichia coli RecA protein in vitro can pair with homologous duplex DNA even when the latter lacks a free homologous end, but subsequent progression of the reaction through strand exchange requires an end in at least one strand of the duplex DNA. We purified from E. coli an endonuclease activity that cleaves the outgoing strand of duplex DNA at the junction of homologous and heterologous sequences in three-stranded RecA-recombination intermediates. This endonuclease activity also cleaves specifically at the junctions of duplex and single-stranded regions in synthetic double-stranded oligonucleotides whose central portion consists of unpaired heterologous sequences. These activities are consistent with a role in recombination and repair of DNA.

Base Sequence↗

PubMed1989

Conformational transitions in cytidine bulge-containing deoxytridecanucleotide duplexes: extra cytidine equilibrates between looped out (low temperature) and stacked (elevated temperature) conformations in solution.

High-resolution homonuclear and heteronuclear two-dimensional NMR studies have been carried out on the self-complementary d(C-C-G-C-G-A-A-T-T-C-C-G-G) duplex (designated GCG 13-mer) in aqueous solution. This sequence contains an extra cytidine located between residues G3 and G4 on each strand of the duplex. The exchangeable and nonexchangeable proton resonances have been assigned from an analysis of two-dimensional nuclear Overhauser enhancement (NOESY) and correlated (COSY and relay COSY) spectra for the GCG 13-mer duplex in H2O and D2O solution. The extra cytidine at the bulge site (designated CX) results in more pronounced changes in the NOE distance connectivities for the G3-CX-G4 segment centered about the CX residue compared to the C9-C10 segment on the partner strand opposite the CX residue for the GCG 13-mer duplex at 25 degrees C. The cross-peak intensities in the short mixing time NOESY spectrum also establish that all glycosidic torsion angles including that of CX are anti in the GCG 13-mer duplex at 25 degrees C. The observed chemical shift changes for the CX base protons and the G3pCX phosphorus resonance with temperature between 0 and 40 degrees C demonstrate a temperature-dependent conformational equilibrium in the premelting transition region. The NOE and chemical shift parameters establish that the predominant conformation at low temperature (0 degree C) has the extra cytidine looped out of the helix with the flanking G3.C10 and G4.C9 base pairs stacked on each other. These results support conclusions based on earlier one-dimensional NMR studies of extra cytidine containing complementary duplexes in aqueous solution [Morden, K. M., Chu, Y. G., Martin, F. H., & Tinoco, I., Jr. (1983) Biochemistry 22, 5557-5563. Woodson, S. A., & Crothers, D. M. (1987) Biochemistry 26, 904-912]. By contrast, the chemical shift and NOE parameters demonstrate that the conformational equilibrium shifts toward a structure with a stacked extra cytidine on raising the temperature to 40 degrees C prior to the helix-coil melting transition. The most downfield shifted phosphorus resonance in the GCG 13-mer duplex has been assigned to the phosphate in the C2-G3 step, and this observation demonstrates that the perturbation in the phosphodiester backbone extends to regions removed from the (G3-CX-G4).(C9-C10) bulge site.

Base Composition↗

PubMed2004

Specific versus nonspecific binding of cationic PNAs to duplex DNA.

Although peptide nucleic acids (PNAs) are neutral by themselves, they are usually appended with positively charged lysine residues to increase their solubility and binding affinity for nucleic acid targets. Thus obtained cationic PNAs very effectively interact with the designated duplex DNA targets in a sequence-specific manner forming strand-invasion complexes. We report on the study of the nonspecific effects in the kinetics of formation of sequence-specific PNA-DNA complexes. We find that in a typical range of salt concentrations used when working with strand-invading PNAs (10-20 mM NaCl) the PNA binding rates essentially do not depend on the presence of nontarget DNA in the reaction mixture. However, at lower salt concentrations (<10 mM NaCl), the rates of PNA binding to DNA targets are significantly slowed down by the excess of unrelated DNA. This effect of nontarget DNA arises from depleting the concentration of free PNA capable of interacting with DNA target due to adhesion of positively charged PNA molecules on the negatively charged DNA duplex. As expected, the nonspecific electrostatic effects are more pronounced for more charged PNAs. We propose a simple model quantitatively describing all major features of the observed phenomenon. This understanding is important for design of and manipulation with the DNA-binding polycationic ligands in general and PNA-based drugs in particular.

Adsorption↗

PubMed2003

Cell cycle modulation of gene targeting by a triple helix-forming oligonucleotide.

Successful gene-targeting reagents must be functional under physiological conditions and must bind chromosomal target sequences embedded in chromatin. Triple helix-forming oligonucleotides (TFOs) recognize and bind specific sequences via the major groove of duplex DNA and may have potential for gene targeting in vivo. We have constructed chemically modified, psoralen-linked TFOs that mediate site-specific mutagenesis of a chromosomal gene in living cells. Here we show that targeting efficiency is sensitive to the biology of the cell, specifically, cell cycle status. Targeted mutagenesis was variable across the cycle with the greatest activity in S phase. This was the result of differential TFO binding as measured by cross-link formation. Targeted cross-linking was low in quiescent cells but substantially enhanced in S phase cells with adducts in approximately 20-30% of target sequences. 75-80% of adducts were repaired faithfully, whereas the remaining adducts were converted into mutations (>5% mutation frequency). Clones with mutations could be recovered by direct screening of colonies chosen at random. These results demonstrate high frequency target binding and target mutagenesis by TFOs in living cells. Successful protocols for TFO-mediated manipulation of chromosomal sequences are likely to reflect a combination of appropriate oligonucleotide chemistry and manipulation of the cell biology.

Animals↗

PubMed1994

DNA substrate requirements for different activities of the human immunodeficiency virus type 1 integrase protein.

The integrase protein (IN) of human immunodeficiency virus type 1 removes two nucleotides from both 3' ends of the viral DNA (donor cleavage) and subsequently couples the newly generated 3' OH groups to phosphates in the target DNA (integration). The sequence requirements of IN for cleavage as well as for integration of viral DNA substrates have previously been studied by mutational analyses and by adduct interference assays. We extended these studies by analysis of heteroduplex oligonucleotide substrates and by missing-base analysis. We found for some base pairs that mutation of only one of the two bases and not the other affected IN activity. These base pairs center around the cleavage site. Besides donor cleavage and integration, IN can also perform "intermolecular disintegration," which has been described as the reversal of the integration reaction. We found that this reaction is independent of viral DNA sequences. In addition, the optimum spacing between the integration sites in intermolecular disintegration does not reflect the spacing found in vivo. These results indicate that this reaction is not the exact reversal of integration but rather is a sequence-independent phosphoryl transfer reaction between gapped DNA duplex molecules.

Base Sequence↗

PubMed1997

Effect of radionuclide linker structure on DNA cleavage by 125I-labeled oligonucleotides.

We studied the yield and distribution of DNA strand breaks produced by decay of 125I introduced into triplex-forming and duplex-forming oligodeoxyribonucleotide (ODNs) through linkers of various lengths. ODNs were prepared with 125I attached at the 5'-end with a long linker or to an internal nucleotide position with a short linker. The 125I-ODNs were hybridized to either a single-stranded target to form duplexes or to a double-stranded target to form triplexes. After decay accumulation, the duplex and triplex samples were assayed for strand breaks in a sequencing gel. The yield of strand breaks per decay was 0.34 for duplex with the 5'-modified ODN and 0.66 for duplex with internally modified ODN. The triplex samples with internal 125I have different yields of DNA breaks in the pyrimidine and purine strands, 0.16 and 0.37, respectively. The yield of DNA breaks in the pyrimidine strand of the triplex with the 5'-modified ODN is 0.46. The majority of breaks are located within 5 nucleotides from the decay site. The yield of strand cleavage per decay of 125I was nearly two-fold lower with the described linkers in comparison with the results obtained when 125I is directly attached to the C-5 position of cytosine. Nevertheless, the rapid iodination procedure reported here combined with the possibility of multiple incorporations of 125I on the linkers makes such 125I-ODNs promising agents for sequence-specific cleavage of DNA.

Base Sequence↗

PubMed1989

Sequence recognition protein for the 17-base-pair A + T-rich tract in the replication origin of simian virus 40 DNA.

A DNA-binding protein has been identified that recognizes runs of deoxyadenines and/or deoxythymines (dA/dT sequences) and purified from a chromatographic fraction containing the multiprotein DNA polymerase alpha-primase complex of HeLa cells by successive steps of chromatography on oligo(dT)-cellulose and Q-Sepharose. Polyacrylamide gel electrophoresis of the purified dA/dT sequence-binding protein in the presence of NaDodSO4 showed a single protein band of 62 kDa. Nitrocellulose filter binding assays using homopolydeoxynucleotides indicated that the purified protein preferentially binds to dA/dT sequences in single-stranded or duplex DNAs. Gel mobility shift assays with a variety of DNAs showed that the purified protein specifically binds to a fragment of simian virus 40 DNA containing the minimal (core) origin for replication. The binding occurred in a protein-dependent manner and in the presence of a vast excess of competing DNAs lacking the simian virus replication origin. The origin binding was reduced, however, when DNA fragments from simian virus 40 deletion mutants containing deletions within the 17-base-pair A + T-rich tract in the core DNA replication origin were used in the assays. These results indicate that the dA/dT sequence-binding protein preferentially binds to the 17-base-pair A + T-rich tract and suggest a possible role for the protein in the initiation of DNA replication.

Adenine↗

PubMed2001

Methylation of the nucleobases in RNA oligonucleotides mediates duplex-hairpin conversion.

We have systematically investigated the duplex to hairpin conversion of oligoribonucleotides under the aspect of nucleobase methylation. The first part of our study refers to the self-complementary sequence rCGCGAAUUCGCGA, which forms a stable Watson-Crick base paired duplex under various buffer conditions. It is shown that this sequence is forced to adopt a hairpin conformation if one of the central 6 nt is replaced by the corresponding methylated nucleotide, such as 1-methylguanosine N(2),N(2)-dimethylguanosine, N(6),N(6)-dimethyladenosine (m(6)(2)A) or 3-methyluridine. On the other hand, the duplex structure is retained and even stabilized by replacement of a central nucleotide with N(2)-methylguanosine (m(2)G) or N(4)-methylcytidine. A borderline case is represented by N(6)-methyladenosine (m(6)A). Although generally a duplex-preserving modification, our data indicate that m(6)A in specific strand positions and at low strand concentrations is able to effectuate duplex-hairpin conversion. Our studies also include the ssu ribosomal helix 45 sequence motif, rGACCm(2)GGm(6)(2)Am(6)(2)AGGUC. In analogy, it is demonstrated that the tandem m(6)(2)A nucleobases of this oligoribonucleotide prevent duplex formation with complementary strands. Therefore, it can be concluded that nucleobase methylations at the Watson-Crick base pairing site provide the potential not only to modulate but to substantially affect RNA structure by formation of different secondary structure motifs.

Adenosine↗

PubMed1998

T. brucei RNA editing: adenosine nucleotides inversely affect U-deletion and U-insertion reactions at mRNA cleavage.

In the currently envisioned mechanism of trypanosome mitochondrial RNA editing, U-insertion and U-deletion cycles begin with a common kind of gRNA-directed cleavage. However, natural, altered, and mutationally interconverted editing sites reveal that U-deletional cleavage is inefficient without and activated by ATP and ADP, while U-insertional cleavage shows completely reverse nucleotide effects. The adenosine nucleotides' effects appear to be allosteric and determined solely by sequences immediately adjacent to the anchor duplex. Both U-deletional and U-insertional cleavages are reasonably active at physiological mitochondrial ATP concentration. Notably, ATP and ADP markedly stimulate complete U-deletion and inhibit U-insertion reactions, reflecting their effects on cleavage. These plus previous results suggest that U deletion and U insertion are remarkably distinct.

Adenosine↗

PubMed1997

Comparative studies on parallel and antiparallel duplex and triplex DNA.

Parallel strand models for base sequences d(A)10.d(T)10,d(AT)5.d(TA)5, d(G5C5).d(C5G5), d(GC)5.d(CG)5 and d(CTATAGGGAT).d(GATATCCCTA), where reverse Watson-Crick A-T pairing with two H-bonds and reverse Watson-Crick G-C pairing with one H-bond or with two H-bonds were adopted, and three models of d(T)14.d(A)14.d(T)14 triple helix with different strand orientations were built up by molecular architecture and energy minimization. Comparisons of parallel duplex models with their corresponding B-DNA models and comparisons among the three triple helices showed: (i) conformational energies of parallel AT duplex models were a little lower, while for GC duplex models they were about 8% higher than that of their corresponding B-DNA models; (ii) the energy differences between parallel and B-type duplex models and among the three triple helices arose mainly from base stacking energies, especially for GC base pairing; (iii) the parallel duplexes with one H-bond G-C pairs were less stable than those with two H-bonds G-C pairs. The present paper includes a brief discussion about the effect of base stacking and base sequences on DNA conformations.

Animals↗

PubMedDate not supplied

Double-stranded cleavage by cell extracts near recombinational signal sequences of immunoglobulin genes.

The genes encoding the variable regions of murine immunoglobulin light chains are present in the germ line in two separate segments, V and J. During B lymphocyte differentiation these segments are brought together to form a single unit (for review see ref. 1). Although much is known about the structures of V and J segments, both in germ-line configuration and after rearrangement, essentially nothing is known about the biochemical mechanism of V-J recombination. One possible step in proposed mechanisms of immunoglobulin gene rearrangement is endonucleolytic cleavage of the participating DNA segments before joining. In an attempt to detect such an activity, we have developed an assay for the detection of site-specific double- or single-strand endonucleolytic activity in crude soluble extracts. Using this assay we have detected an activity in extracts of nuclei from mouse B-lymphoid lines and from mouse L cells that is capable of introducing duplex breaks near the recombinational signal sequences of immunoglobulin JK segments. We report the activity here because of its intrinsic interest although we lack any direct evidence that it has a role in V-J recombination.

Abelson murine leukemia virus↗

PubMed1998

Thermodynamics of internal C.T mismatches in DNA.

Thermodynamics of 23 oligonucleotides with internal single C.T mismatches were obtained by measuring UV absorbance as a function of temperature. Results from these 23 duplexes were combined with three measurements from the literature to derive nearest-neighbor thermodynamic parameters for seven linearly independent trimer sequences with internal C.T mismatches. The data show that the nearest-neighbor model is adequate for predicting thermodynamics of oligonucleotides with internal C.T with average deviations for Delta G degrees37, Delta H degrees, Delta S degrees and T m of 6.4%, 9.9%, 10.6%, and 1.9 degreesC respectively. C.T mismatches destabilize the duplex in all sequence contexts. The thermodynamic contribution of C. T mismatches to duplex stability varies weakly depending on the orientation of the mismatch and its context and ranges from +1.02 kcal/mol for GCG/CTC and CCG/GTC to +1.95 kcal/mol for TCC/ATG.

Cytidine↗

PubMed1999

Melting studies of short DNA hairpins: influence of loop sequence and adjoining base pair identity on hairpin thermodynamic stability.

Spectroscopic and calorimetric melting studies of 28 DNA hairpins were performed. These hairpins form by intramolecular folding of 16 base self-complementary DNA oligomer sequences. Sequence design dictated that the hairpin structures have a six base pair duplex linked by a four base loop and that the first five base pairs in the stem are the same in every molecule. Only loop sequence and identity of the duplex base pair closing the loop vary for the set of hairpins. For these DNA samples, melting studies were carried out to investigate effects of the variables on hairpin stability. Stability of the 28 oligomers was ascertained from their temperature-induced melting transitions in buffered 115 mM Na(+) solvent, monitored by ultraviolet absorbance and differential scanning calorimetry (DSC). Experiments revealed the melting temperatures of these molecules range from 32.4 to 60.5 degrees C and are concentration independent over strand concentrations of 0.5 to 260 microM; thus, as expected for hairpins, the melting transitions are apparently unimolecular. Model independent thermodynamic transition parameters, DeltaH(cal), DeltaS(cal), and DeltaG(cal), were determined from DSC measurements. Model dependent transition parameters, DeltaH(vH), DeltaS(vH), and DeltaG(vH) were estimated from a van't Hoff (two-state) analysis of optical melting transitions. Results of these studies reveal a significant sequence dependence to DNA hairpin stability. Thermodynamic parameters evaluated by either procedure reveal the transition enthalpy, DeltaH(cal) (DeltaH(vH)) can differ by as much as 20 kcal/mol depending on sequence. Similarly, values of the transition entropy DeltaS(cal) (DeltaS(vH)) can differ by as much as 60 cal/Kmol (eu) for different molecules. Differences in free energies DeltaG(cal) (DeltaG(vH)) are as large as 4 kcal/mol for hairpins with different sequences. Comparisons between the model independent calorimetric values and the thermodynamic parameters evaluated assuming a two-state model reveal that 10 of the 28 hairpins display non-two-state melting behavior. The database of sequence-dependent melting free energies obtained for the hairpins was employed to extract a set of n-n (nearest-neighbor) sequence dependent loop parameters that were able to reproduce the input data within error (with only two exceptions). Surprisingly, this suggests that the thermodynamic stability of the DNA hairpins can in large part be reasonably represented in terms of sums of appropriate nearest-neighbor loop sequence parameters.

Base Sequence↗

PubMed2001

Thermodynamic and computational studies of DNA triple helices containing a nucleotide or a non-nucleotide linker in the third strand.

In this paper we report a thermodynamic characterisation of stability and melting behaviour of four different triple helices at pH 6.0. The target duplex consists of 16 base pairs in alternate sequence of the type 5'-(purine)(m)(pyrimidine)(m)-3'. The four triplexes are formed by targeting the 16-mer duplex with an all pyrimidine 16-mer or 15-mer or 14-mer third strand. The 16-mer oligonucleotide contains a 3'-3' phosphodiester junction and corresponding triplex was named 16-mer P. The 14-mer oligonucleotide contains a non-nucleotide linker, the 1,2,3 propanetriol residue and the corresponding triplex was named 14-mer PT. For the 15-mer oligonucleotide both junctions were alternatively used and the relative triplexes were named 15-mer P and 15-mer PT, respectively. These linkers introduce the appropriate polarity inversion and let the third strand switch from one oligopurine strand of the duplex to the other. Thermal denaturation profiles indicate the initial loss of the third strand followed by the dissociation of the target duplex. Transition enthalpies, entropies and free energies were derived from differential scanning calorimetric measurements. The comparison of Gibbs energies reveals that a more stable triplex is obtained when in the third strand there is the lack of one nucleotide in the junction region and a propanetriol residue as linker was used. The thermodynamic data were discussed in light of molecular mechanics and dynamics calculations.

Calorimetry, Differential Scanning↗

PubMed2003

The base-intercalated duplexes of d(GCGAXAGC) with mutation at X (X=G, T or C).

DNA fragments containing the sequence d(GCGAAAGC) adopt a base-intercalated (zipper-like) duplex in crystalline state at high ionic strength. To investigate effects of point mutation at the 5th residue on the structure, two crystal structures of d(GCGAGAGC) and d(GCGATAGC) have been determined by X-ray crystallography. In both crystals, the two octamers related by a crystallographic two-fold symmetry are aligned in an anti-parallel fashion and associated to each other to form a duplex, suggesting that the base-intercalated duplex is stable even when the 5th residue is mutated with other bases. The sheared G3:A6 pair formation makes the two phosphate backbones closer and facilitates formation of the A-X*-X-A* base-intercalated motif. The three duplexes are assembled around the two hexamine cobalt chlorides. The central X and X* residues are bound to a different cation depending on the kinds of bases.

Base Pairing↗

PubMed1991

Alpha-oligonucleotides: a unique class of modified chimeric nucleic acids.

alpha-Anomeric oligonucleotides are resistant to nucleases and display parallel hybridization with complementary DNA and RNA sequences. Although alpha-DNA/beta-RNA duplexes are not substrates for RNases H, alpha-oligos are able to inhibit translation through a RNase H independent mechanism. alpha-Oligos and their alpha-phosphorothioate analogs (12 mer) targeted against the splice acceptor site of the HIV-TAT gene were potent inhibitors of de novo HIV infection. Furthermore alpha-phosphorothioate and dithioate homo-oligomers exhibit an in vitro, nonsequence-specific, inhibitory effect on HIV reverse transcriptase.

Base Sequence↗