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Results for “duplex sequencing”

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At least 433 records · Page 24Linked to original sources

PubMed1993

Complement-stabilized D-loop. RecA-catalyzed stable pairing of linear DNA molecules at internal sites.

The RecA protein (RecA) of Escherichia coli has the ability to pair a single-stranded DNA to a homologous sequence in a duplex DNA without requiring denaturation of the duplex. This ability has stimulated interest in the use of RecA for targeting probes to genomic DNA. However, because pairing generally requires that the double-stranded DNA either have a homologous end or be negatively supercoiled, the application of RecA to targeting has been very limited. Here, we show that if the sequence complementary to the probe is also included in the reaction, RecA can pair the two single strands to sites distant from any ends on linear DNA. The resulting structure, termed a complement-stabilized D-loop (csD-loop), is cleavable by restriction endonucleases and, upon removal of RecA, remains stable to temperatures up to the tm of the double-stranded probe. These results indicate that the csD-loop probably consists of two side-by-side Watson-Crick duplexes, much like a replication bubble. This novel reaction of RecA may be useful in gene mapping and isolation, as well as in sequence-specific cleavage of genomic DNA, and might have functions in vivo.

DNA Probes↗

PubMed1986

Mechanism of DNA polymerase I: exonuclease/polymerase activity switch and DNA sequence dependence of pyrophosphorolysis and misincorporation reactions.

Mechanistic features of several processes involved in the idling-turnover reaction catalyzed by the large (Klenow) fragment of Escherichia coli DNA polymerase I have been established. The exonuclease----polymerase activity switch involved in the excision/incorporation mode of idling-turnover occurs without an intervening dissociation of the enzyme from its DNA substrate. Comparative studies on the pyrophosphorolysis kinetics of related DNA substrates indicate a significant dependence of the reaction rate upon the DNA sequence within the duplex region upstream of the primer-template junction. Finally, a gel electrophoretic analysis of the products of the idling-turnover reaction has provided direct evidence for an alternative DNA sequence-dependent misincorporation/excision pathway.

Base Sequence↗

PubMed1991

The specific binding, bending, and unwinding of DNA by RsrI endonuclease, an isoschizomer of EcoRI endonuclease.

To determine whether RsrI endonuclease recognizes and cleaves the sequence GAATTC in duplex DNA similarly to its isoschizomer EcoRI we initiated a functional comparison of the two enzymes. Equilibrium binding experiments showed that at 20 degrees C RsrI endonuclease binds to specific and nonspecific sequences in DNA with affinities similar to those of EcoRI. At 0 degrees C the affinity of RsrI for its specific recognition sequence is reduced 7-fold whereas the affinity for noncanonical sequences remains relatively unchanged. Unlike EcoRI, incubation of RsrI endonuclease with N-ethylmaleimide inactivates the enzyme; however, preincubation with DNA prevents the inactivation. The N-ethylmaleimide-treated enzyme fails to bind DNA as assayed by gel mobility shift assays. Comparison of the deduced amino acid sequences of RsrI and EcoRI endonucleases suggests that modification of Cys245 is responsible for the inactivation. Fe(II). EDTA and methidiumpropyl-EDTA.Fe(II) footprinting results indicate that RsrI, like EcoRI, protects 12 base pairs from cleavage when bound to its specific recognition sequence in the absence of Mg2+. RsrI bends DNA by approximately 50 degrees, as determined by measuring the relative electrophoretic mobilities of specific RsrI-DNA complexes with the binding site in the center or near the end of the DNA fragment. This value is similar to that reported for EcoRI. RsrI also unwinds the DNA helix by 25 degrees +/- 5 degrees, a value close to that reported for EcoRI endonuclease. Collectively, these results indicate that the overall structural changes induced in the DNA by the binding of RsrI and EcoRI endonucleases to DNA in the absence of Mg2+ are similar. In the accompanying paper (Aiken, C. R., McLaughlin, L. W., and Gumport, R. I. (1991) J. Biol. Chem. 266, 19070-19078) we present results of studies of RsrI endonuclease using oligonucleotide substrates containing base analogues which suggest differences in the ways the two enzymes cleave DNA.

Base Sequence↗

PubMed2001

The hybridization-stabilization assay: a solution-based isothermal method for rapid screening and determination of sequence preference of ligands that bind to duplexed nucleic acids.

The gene-to-drug quest will be most directly served by the discovery and development of small molecules that bind to nucleic acids and modulate gene expression at the level of transcription and/or inhibit replication of infectious agents. Full realization of this potential will require implementation of a complete suite of modern drug discovery technologies. Towards this end, here we describe our initial results with a new assay for identification and characterization of novel nucleic acid binding ligands. It is based on the well recognized property of stabilization of hybridization of complementary oligonucleotides by groove and/or intercalation binding ligands. Unlike traditional thermal melt methodologies, this assay is isothermal and, unlike gel-based footprinting techniques, the assay also is performed in solution and detection can be by any number of highly sensitive, non-radioisotopic modalities, such as fluorescence resonance energy transfer, described herein. Thus, the assay is simple to perform, versatile in design and amenable to miniaturization and high throughput automation. Assay validation was performed using various permutations of direct and competitive binding formats and previously well studied ligands, including pyrrole polyamide and intercalator natural products, designed hairpin pyrrole-imidazole polyamides and furan-based non-polyamide dications. DNA specific ligands were identified and their DNA binding site size and sequence preference profiles were determined. A systematic approach to studying the relationship of binding sequence specificity with variation in ligand structure was demonstrated, and preferred binding sites in longer DNA sequences were found by pseudo-footprinting, with results that are in accord with established findings. This assay methodology should promote a more rapid discovery of novel nucleic acid ligands and potential drug candidates.

Base Pairing↗

PubMed1997

Factors influencing the extent and selectivity of alkylation within triplexes by reactive G/A motif oligonucleotides.

G/A motif triplex-forming oligonucleotides (TFOs) complementary to a 21 base pair homopurine/homopyrimidine run were conjugated at one or both ends to chlorambucil. These TFOs were incubated with several synthetic duplexes containing the targeted homopurine run flanked by different sequences. The extent of mono and interstrand cross-linking was compared with the level of binding at equilibrium. Covalent modification took place within a triple-stranded complex and usually occurred at guanine residues in the flanking double-stranded DNA. The efficiency of alkylation was dependent upon the sequence of the flanking duplex, the solution conditions, and the rate of triplex formation relative to the rate of chlorambucil reaction. Self-association of the TFOs as parallel duplexes was demonstrated and this did not interfere with triple strand formation. With an optimal target, cross-linking of the triplex was very efficient when incubation was carried in a physiological buffer supplemented with the triplex selective intercalator coralyne.

Adenosine↗

PubMed1993

DNA triple-helix specific intercalators as antigene enhancers: unfused aromatic cations.

Triple-helical structures involving the interaction of an oligonucleotide third strand with a duplex nucleic acid sequence have recently gained attention as a therapeutic strategy in the "antigene" approach [cf. Helene, C. (1991) Eur. J. Cancer 27, 1466-1471]. This method utilizes the triple helix formed from the cellular duplex and an added third strand to directly regulate the activity of a selected gene. The limited stability of nucleic acid triple-helical interactions, particularly if the third strand has backbone modifications such as methylphosphonate or phosphorothioate substitutions, is a limiting condition for the use of this approach. We have designed and synthesized compounds, on the basis of the following three criteria, that we feel should provide selective interactions and significant stabilization of triplexes: appropriate aromatic surface area for stacking with triplex bases in an intercalation complex, positive charge, and limited torsional freedom in the aromatic system to match the propeller twist of the triple-base interactions in the triplex. A series of quinoline derivatives with an alkylamine side chain at the 4-position and with different aryl substituents at the 2-position has been synthesized as our first compounds. A 2-naphthyl derivative provides significant and selective stabilization of the triplex. In a 0.2 M NaCl buffer, the naphthyl derivative increased the Tm for the triplex (triplex to duplex and third strand transition) by approximately 30 degrees C more than the Tm increase for the duplex (duplex to single strands transition). Spectral changes and energy-transfer results indicate that the naphthyl compound and related derivatives bind to the triplex by intercalation.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA↗

PubMed2004

Influence of DNA structure on DNA polymerase beta active site function: extension of mutagenic DNA intermediates.

In the ternary substrate complex of DNA polymerase (pol) beta, the nascent base pair (templating and incoming nucleotides) is sandwiched between the duplex DNA terminus and polymerase. To probe molecular interactions in the dNTP-binding pocket, we analyzed the kinetic behavior of wild-type pol beta on modified DNA substrates that alter the structure of the DNA terminus and represent mutagenic intermediates. The DNA substrates were modified to 1) alter the sequence of the duplex terminus (matched and mismatched), 2) introduce abasic sites near the nascent base pair, and 3) insert extra bases in the primer or template strands to mimic frameshift intermediates. The results indicate that the nucleotide insertion efficiency (k(cat)/K(m), dGTP-dC) is highly dependent on the sequence identity of the matched (i.e. Watson-Crick base pair) DNA terminus (template/primer, G/C approximately A/T > T/A approximately C/G). Mismatches at the primer terminus strongly diminish correct nucleotide insertion efficiency but do not affect DNA binding affinity. Transition intermediates are generally extended more easily than transversions. Most mismatched primer termini decrease the rate of insertion and binding affinity of the incoming nucleotide. In contrast, the loss of catalytic efficiency with homopurine mismatches at the duplex DNA terminus is entirely due to the inability to insert the incoming nucleotide, since K(d)((dGTP)) is not affected. Abasic sites and extra nucleotides in and around the duplex terminus decrease catalytic efficiency and are more detrimental to the nascent base pair binding pocket when situated in the primer strand than the equivalent position in the template strand.

Base Pair Mismatch↗

PubMed1976

Synchronous digestion of SV40 DNA by exonuclease III.

We have established an optimal condition for the synchronous digestion of SV40 DNA with Escherichia coli exonuclease III. Electron microscopy and polyacrylamide gel electrophoresis were used to obtain accurate measurements on the lengths of DNA before and after exonuclease III digestion. Based on this finding, a new method for determining the sequence of long duplex DNA can be realized. It involves (a) the synchronous digestion of the DNA from the 3' ends with exonuclease III, followed by (b) repair synthesis with labeled nucleotides and DNA polymerase, and (c) sequence analysis of the repaired DNA.

Base Sequence↗

PubMed1976

Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of (C leads to) CO+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fols higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3' -ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.

Cobalt↗

PubMed1976

Terminal labeling and addition of homopolymer tracts to duplex DNA fragments by terminal deoxynucleotidyl transferase.

Terminal deoxynucleotidyl transferase, which requires a single-stranded DNA primer under the usual assay conditions, can be made to accept double-stranded DNA as primer for the addition of either rNMP or dNMP, if Mg+2 ion is replaced by Co+2 ion. The priming efficiency in the presence of Co+2 ion with respect to initial rate tested with 2 single-stranded primer, is 5-6 fold higher than that observed with Mg+2 ion. In the presence of Co+2 ion, the primer specificity is altered so that all forms of duplex DNA molecules can be labeled at their unique 3'-ends regardless of whether such ends are staggered or even. Thus, using ribonucleotide incorporation, we have for the first time employed this reaction for sequence analysis of duplex DNA fragments generated by restriction endonuclease cleavages. Furthermore, by using Co+2 ion, it is possible to add a long homopolymer tract of deoxyribonucleotides to the 3'-terminus of double-stranded DNA. Therefore, without prior treatment with lambda exonuclease to expose the 3' terminus as single-stranded primer, this reaction now permits insertion of homopolymer tails at the 3'-ends of all types of DNA molecules for the purpose of in vitro construction of recombinant DNA.

Cobalt↗

PubMed1976

Ethidium bromide-(dC-dG-dC-dG)2 complex in solution: intercalation and sequence specificity of drug binding at the tetranucleotide duplex level.

The binding of ethidium bromide (EtdBr) to the dC-dG-dC-dG self-complementary duplex has been monitored at the resolvable drug and nucleic acid protons and backbone phosphates at high nucleotide/drug (N/D) ratios by nuclear magnetic resonance (NMR) spectroscopy in aqueous solution. We observe averaged resonances (25 degrees-95 degrees) for the nucleic acid and drug nonexchangeable protons in the presence of excess tetranucleotide (N/D = 24), indicative of rapid exchange relative to the chemical shifts in the free and complexed states. Complex formation results in upfield shifts for the base protons at the terminal and internal base pairs and an increase in the transition midpoint for the duplex-to-strand conversion. We observe upfield chemical shift changes of 1.2 ppm at the Watson-Crick guanine N-1 proton(s) on complex formation (N/D = 24), with slow exchange between (dC-dG-dC-dG)2 and EtdBr-(dC-dG-dC-dG)2 relative to this chemical shift difference at-5 degrees. The EtdBr phenanthridine ring protons shift upfield by about 0.9 ppm (H-2, H-4, H-7, H-9) and greater than 0.5 ppm (H-1, H-10) on complex formation, with the chemical shifts versus temperature plots (25 degrees-95 degrees) monitoring the dissociation of the EtdBr-(dC-dG-dC-dG)2 structure. These upfield shifts at the exchangeable and nonexchangeable base protons and phenanthridine ring (but not side chain) protons demonstrate intercalation of the phenanthridine ring of EtdBr into the dC-dG-dC-dG duplex in solution. The intercalation model may be supported by the observation of downfield shifts (up to 1ppm) at the internucleotide phosphate(s) of the tetranucleotide duplex on addition of EtdBr at low temperatures. We observe stronger binding of EtdBr to the self-complementary dC-dG-dC-dG (2 dC-dG intercalation sites) and dC-dC-dG-dG (1 dC-dG site) duplexes compared to the dG-dG-dC-dC (no dC-dG sites) as monitored by UV absorbance changes at 480 nm. These studies extend to the tetranucleotide duplex level earlier observations that EtdBr exhibits a selectivity for formation of complexes to dinucleoside monophosphates with a pyrimidine (3'-5') purine sequence in the crystal and in solution. The experimental proton NMR upfield shifts at the phenanthridine protons on formation of the EtdBr-(dC-dG-dC-dG)2 complex compare favorably with calculated values (atomic diamagnetic anisotropy and ring current contributions) based on the overlap geometry for EtdBr intercalated into the pyrimidine (3'-5') purine dinucleoside monophosphate duplex in the crystal.

Ethidium↗

PubMed2003

Base sequence effects in radical cation migration in duplex DNA: support for the polaron-like hopping model.

A series of anthraquinone-linked (AQ) duplex DNA oligomers were prepared and investigated. Irradiation of the AQ injects a radical cation into the DNA. The radical cation migrates through the DNA and reacts selectively at GG steps, which leads to strand cleavage after treatment with piperidine. The oligomers investigated in this work were selected to assess the effect on long-distance charge transport of placing a T base (or bases) in a strand of repeating purine bases. With notable exceptions, the amount of strand scission decreases with the distance between the AQ and the GG step. The results are consistent only with models for long-distance transport, such as thermally activated polaron-like hopping, that incorporate radical cation delocalization over two or more adjacent bases.

Anthraquinones↗

PubMed1996

Triplex-mediated cleavage of DNA by 1,10-phenanthroline-linked 2'-O-methyl RNA.

We have previously reported that 2'-O-methyl RNAs are efficient probes for duplex DNA. Here we describe the design, synthesis, and DNA cleaving activity of 1,10-phenanthroline (OP)-linked 2'-O-methyl RNA (OP-m). Although a local triple helix was formed, both with OP-m and a control OP-linked DNA at the target sequence of the duplex DNA, the promoter region of the human thrombomodulin gene, the cleavage efficiencies on both strands were not proportional when OP-M was used as a cleavage agent. These results may reflect the structural differences of the respective triple helices and the duplex-triplex junction, formed from the two types of triplex-forming oligonucleotides, 2'-O-methyl RNA and DNA. Since the OP-ms were found to work as preferential purine-strand cutters for duplex DNA, they would be useful as unique tools for genome analysis.

Base Sequence↗

PubMed1986

Construction and characterization of a site-directed CC-1065-N3-adenine adduct within a 117 base pair DNA restriction fragment.

The design, construction, and characterization of a site-directed CC-1065-N3-adenine adduct in a 117 base pair segment of M13mpI DNA are described. CC-1065 is an extremely potent antitumor antibiotic produced by Streptomyces zelensis. Previous studies have demonstrated that the cyclopropyl ring of CC-1065 reacts quite specifically with N3 of adenine in double-stranded DNA to form a CC-1065-DNA adduct. Following alkylation, the drug molecule lies snugly within the minor groove of DNA, overlapping with five base pairs for which a marked sequence preference exists [Hurley, L. H., Reynolds, V. R., Swenson, D. H., Petzold, G. L., & Scahill, T. A. (1984) Science (Washington, D.C.) 226, 843-844]. On the basis of the unique characteristics of the reaction of CC-1065 with DNA and the structure of the resulting DNA adduct, we have designed a general strategy to construct a site-directed CC-1065-DNA adduct in a restriction fragment. The presence of unique AluI and HaeIII restriction enzymes sites on each side of a high-affinity CC-1065 binding sequence (5'-GATTA) permitted the preparation of a partial duplex DNA molecule containing the CC-1065 binding sequence in the duplex DNA region. Since CC-1065 only binds to duplex DNA, potential CC-1065 binding sequences in the long single-stranded regions were protected from drug binding during the construction process.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine↗

PubMed1996

Sequence-specific alkylation and cleavage of DNA mediated by purine motif triple helix formation.

An N-bromoacetyl electrophile attached to the 5'-phosphate group of a purine-rich oligonucleotide affords sequence-specific alkylation of duplex DNA (at 37 degrees C, pH 7.4) through the formation of a specific purine.purine.pyrimidine triple-helical complex. In a 645 bp restriction fragment containing three consecutive guanine bases adjacent to the 3'-end of an oligonucleotide binding site, the yield of single-strand cleavage after piperidine treatment is 80% at the guanine base directly adjacent to the binding site and 88% overall. In an 837 bp restriction fragment containing two adjacent inverted repeats of the third strand binding site and a single 3'-guanine base, yields of single-strand cleavage are 87% on each strand at the 3'-guanine base. Double-strand cleavage was obtained in 61% yield at a single site in a 6.6 kbp plasmid containing the 837 bp fragment. Extension of triple helix mediated DNA alkylation from the pyrimidine to purine motif formally extends the number of sites in duplex DNA that can be cleaved in a sequence-specific and nucleotide-specific manner in good yields.

Acetates↗

PubMed2006

Selection of primer-template sequences that bind human immunodeficiency virus reverse transcriptase with high affinity.

A SELEX (systematic evolution of ligands by exponential enrichment)-based approach was developed to determine whether HIV-RT showed preference for particular primer-template sequences. A 70 nt duplex DNA was designed with 20 nt fixed flanking sequences at the 3' and 5' ends and a randomized 30 nt internal sequence. The fixed sequence at the 5' end contained a BbsI site six bases removed from the randomized region. BbsI cuts downstream of its recognition site generating four base 5' overhangs with recessed 3' termini. Cleavage produced a 50 nt template and 46 nt primer with the 3' terminus within the randomized region. HIV-RT was incubated with this substrate and material that bound RT was isolated by gel-shift. The recovered material was treated to regenerate the BbsI site, amplified by PCR, cleaved with BbsI and selected with HIV-RT again. This was repeated for 12 rounds. Material from round 12 bound approximately 10-fold more tightly than starting material. All selected round 12 primer-templates had similar sequence configuration with a 6-8 base G run at the 3' primer terminus, similar to the HIV polypurine tract. Further modifications indicate that the Gs were necessary and sufficient for strong binding.

Aptamers, Nucleotide↗

PubMed1995

Azole substituted oligonucleotides promote antiparallel triplex formation at non-homopurine duplex targets.

The ability of certain azole substituted oligodeoxy-ribonucleotides to promote antiparallel triple helix formation with duplex targets having CG or TA interruptions in the otherwise homopurine sequence was examined. 2'-Deoxyribonucleosides of the azoles, which include pyrazole, imidazole, 1,2,4-triazole and 1,2,3,4-tetrazole were synthesized using the stereo-specific sodium salt glycosylation procedure. These nucleosides were successfully incorporated using solid-support, phosphoramidite chemistry, into oligonucleotides designed to interact with the non-homopurine duplex targets. The interaction of these modified oligonucleotides with all four possible base pairs was evaluated and compared to similar data for a series of natural oligonucleotides. The oligonucleotides containing simple azoles enhanced the triplex forming ability considerably at non-homopurine targets. Binding of these modified oligonucleotides to duplex targets containing TA inversion sites was particularly noteworthy, and compare favorably to unmodified oligonucleotides for binding to duplex targets containing CG as well as TA base pairs. The selectivity exhibited by certain azoles is suggestive of base pair specific interactions. Thus, the azoles evaluated during this study show considerable promise for efforts to develop generalized triplex formation at non-homopurine duplex sequences.

Azoles↗

PubMed2003

DNA binding properties of the adenovirus DNA replication priming protein pTP.

The precursor terminal protein pTP is the primer for the initiation of adenovirus (Ad) DNA replication and forms a heterodimer with Ad DNA polymerase (pol). Pol can couple dCTP to pTP directed by the fourth nucleotide of the viral genome template strand in the absence of other replication proteins, which suggests that pTP/pol binding destabilizes the origin or stabilizes an unwound state. We analyzed the contribution of pTP to pTP/pol origin binding using various DNA oligonucleotides. We show that two pTP molecules bind cooperatively to short DNA duplexes, while longer DNA fragments are bound by single pTP molecules as well. Cooperative binding to short duplexes is DNA sequence independent and most likely mediated by protein/protein contacts. Furthermore, we observed that pTP binds single-stranded (ss)DNA with a minimal length of approximately 35 nt and that random ssDNA competed 25-fold more efficiently than random duplex DNA for origin binding by pTP. Remarkably, short DNA fragments with two opposing single strands supported monomeric pTP binding. pTP did not stimulate, but inhibited strand displacement by the Ad DNA binding and unwinding protein DBP. These observations suggest a mechanism in which the ssDNA affinity of pTP stabilizes Ad pol on partially unwound origin DNA.

Binding Sites↗