Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Nucleic Acid Denaturation”

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 19 recordsLinked to original sources
PubMed2004

Locked nucleic acids and intercalating nucleic acids in the design of easily denaturing nucleic acids: thermal stability studies.

Intercalating nucleic acids (INA(R)s) with insertions of (R)-1-O-(1-pyrenylmethyl)glycerol were hybridized with locked nucleic acids (LNAs). INA/LNA duplexes were found to be less stable than the corresponding DNA/LNA duplexes when the INA monomer was inserted as a bulge close to the LNA monomers in the opposite strand. This property was used to make "quenched" complements that possess LNA in hairpins and in duplexes and are consequently more accessible for targeting native DNA. The duplex between a fully modified 13-mer LNA sequence and a complementary INA with six pyrene residues inserted after every second base as a bulge was found to be very unstable (Tm=30.1 degrees C) in comparison with the unmodified double-stranded DNA (Tm=48.7 degrees C) and the corresponding duplexes of LNA/DNA (Tm=81.6 degrees C) and INA/DNA (Tm=66.4 degrees C). A thermal melting experiment of a mixture of an LNA hairpin, with five LNA nucleotides in the stem, and its complementary DNA sequence gave a transition with an extremely low increase in optical density (hyperchromicity). When two INA monomers were inserted into the stem of the LNA hairpin, the same experiment resulted in a significant hyperchromicity comparable with the one obtained for the corresponding DNA/DNA duplex.

DNA↗
PubMed1978

Numerical analysis of thermal denaturation of nucleic acids.

Denaturation of DNA molecules by stepwise incrementation of the temperature leads to melting profiles showing a fine structure, composed of individual melting modes. A method is described by which quantitative physical information brought by the modes can be extracted from the melting profile. Related problems such as data editing and smoothing are also discussed.

DNA↗
PubMed1990

Improved detection of rotavirus RNA in dot-blot hybridization assay by chromatographic extraction and acid denaturation of double-stranded RNA.

One of the commonest methods of denaturing nucleic acids, denaturation by heat, was found to be ineffective for double-stranded (ds)RNA when RNA preparations contain 200 mM or higher concentrations of NaCl. We report acid denaturation of dsRNA (incubation for 10 min in the presence of 80 mM HCl) was particularly useful for such preparations. When genomic dsRNAs of rotavirus were extracted by ion-exchange chromatography columns (Extractor) and then denatured by acid, the detection of rotavirus RNA in stool specimens by the previously reported dot-blot hybridization assay (Yamakawa, K. et al. (1989). Molecular and Cellular Probes 3, 397-401.) was significantly improved.

Chromatography, Ion Exchange↗
PubMed2005

Easily denaturing nucleic acids derived from intercalating nucleic acids: thermal stability studies, dual duplex invasion and inhibition of transcription start.

The bulged insertions of (R)-1-O-(pyren-1-ylmethyl)glycerol (monomer P) in two complementary 8mer DNA strands (intercalating nucleic acids) opposite to each other resulted in the formation of an easily denaturing duplex, which had lower thermal stability (21.0 degrees C) than the wild-type double-stranded DNA (dsDNA, 26.0 degrees C), but both modified oligodeoxynucleotides had increased binding affinity toward complementary single-stranded DNA (ssDNA) (41.5 and 39.0 degrees C). Zipping of pyrene moieties in an easily denaturing duplex gave formation of a strong excimer band at 480 nm upon excitation at 343 nm in the steady-state fluorescence spectra. The excimer band disappeared upon addition of a similar short dsDNA, but remained when adding a 128mer dsDNA containing the same sequence. When P was inserted into 2'-OMe-RNA strands, the duplex with zipping P was found to be more stable (42.0 degrees C) than duplexes with the complementary ssDNAs (31.5 and 19.5 degrees C). The excimer band observed in the ds2'-OMe-RNA with zipping P had marginal changes upon addition of both 8 and 128mer dsDNA. Synthesized oligonucleotides were tested in a transcriptional inhibition assay for targeting of the open complex formed by Escherichia coli RNA polymerase with the lac UV-5 promoter using the above mentioned 128mer dsDNA. Inhibition of transcription was observed for 8mer DNAs possessing pyrene intercalators and designed to target both template and non-template DNA strands within the open complex. The observed inhibition was partly a result of unspecific binding of the modified DNAs to the RNA polymerase. Furthermore, the addition of 8mer DNA with three bulged insertions of P designed to be complementary to the template strand at the +36 to +43 position downstream of the transcription start resulted in a specific halt of transcription producing a truncated RNA transcript. This is to our knowledge the first report of an RNA elongation stop mediated by a small DNA sequence possessing intercalators. The insertions of P opposite to each other in ds2'-OMe-RNA showed inhibition efficiency of 96% compared with 25% for unmodified ds2'-OMe-RNA.

DNA↗
PubMed2001

Counterion association with native and denatured nucleic acids: an experimental approach.

The melting temperature of the poly(dA) . poly(dT) double helix is exquisitely sensitive to salt concentration, and the helix-to-coil transition is sharp. Modern calorimetric instrumentation allows this transition to be detected and characterized with high precision at extremely low duplex concentrations. We have taken advantage of these properties to show that this duplex can be used as a sensitive probe to detect and to characterize the influence of other solutes on solution properties. We demonstrate how the temperature associated with poly(dA) . poly(dT) melting can be used to define the change in bulk solution cation concentration imparted by the presence of other duplex and triplex solutes, in both their native and denatured states. We use this information to critically evaluate features of counterion condensation theory, as well as to illustrate "crosstalk" between different, non-contacting solute molecules. Specifically, we probe the melting of a synthetic homopolymer, poly(dA) . poly(dT), in the presence of excess genomic salmon sperm DNA, or in the presence of one of two synthetic RNA polymers (the poly(rA) . poly(rU) duplex or the poly(rU) . poly(rA) . poly(rU) triplex). We find that these additions cause a shift in the melting temperature of poly(dA) . poly(dT), which is proportional to the concentration of the added polymer and dependent on its conformational state (B versus A, native versus denatured, and triplex versus duplex). To a first approximation, the magnitude of the observed tm shift does not depend significantly on whether the added polymer is RNA or DNA, but it does depend on the number of strands making up the helix of the added polymer. We ascribe the observed changes in melting temperature of poly(dA) . poly(dT) to the increase in ionic strength of the bulk solution brought about by the presence of the added nucleic acid and its associated counterions. We refer to this communication between non-contacting biopolymers in solution as solvent-mediated crosstalk. By comparison with a known standard curve of tm versus log[Na+] for poly(dA) . poly(dT), we estimate the magnitude of the apparent change in ionic strength resulting from the presence of the bulk nucleic acid, and we compare these results with predictions from theory. We find that current theoretical considerations correctly predict the direction of the t(m) shift (the melting temperature increases), while overestimating its magnitude. Specifically, we observe an apparent increase in ionic strength equal to 5% of the concentration of the added duplex DNA or RNA (in mol phosphate), and an additional apparent increase of about 9.5 % of the nucleic acid concentration (mol phosphate) upon denaturation of the added DNA or RNA, yielding a total apparent increase of 14.5 %. For the poly(rU) . poly(rA) . poly(rU) triplex, the total apparent increase in ionic strength corresponds to about 13.6% of the amount of added triplex (moles phosphate). The effect we observe is due to coupled equilibria between the solute molecules mediated by modulations in cation concentration induced by the presence and/or the transition of one of the solute molecules. We note that our results are general, so one can use a different solute probe sensitive to proton binding to characterize subtle changes in solution pH induced by the presence of another solute in solution. We discuss some of the broader implications of these measurements/results in terms of nucleic acid melting in multicomponent systems, in terms of probing counterion environments, and in terms of potential regulatory mechanisms.

Animals↗
PubMed2005

Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins.

We study protein and nucleic acid structure and dynamics using single-molecule FRET and alternating-laser excitation. Freely diffusing molecules are sorted into subpopulations based on single-molecule signals detected within 100 micros to 1 ms. Distance distributions caused by fluctuations faster than 100 micros are studied within these subpopulations by using time-correlated single-photon counting. Measured distance distributions for dsDNA can be accounted for by considering fluorophore linkers and fluorophore rotational diffusion, except that we find smaller fluctuations for internally labeled dsDNA than DNA with one of the fluorophores positioned at a terminal site. We find that the electrostatic portion of the persistence length of short single-stranded poly(dT) varies approximately as the ionic strength (I) to the -1/2 power (I(-1/2)), and that the average contribution to the contour length per base is 0.40-0.45 nm. We study unfolded chymotrypsin inhibitor 2 (CI2) and unfolded acyl-CoA binding protein (ACBP) even under conditions where folded and unfolded subpopulations coexist (contributions from folded proteins are excluded by using alternating-laser excitation). At lower denaturant concentrations, unfolded CI2 and ACBP are more compact and display larger fluctuations than at higher denaturant concentrations where only unfolded proteins are present. The experimentally measured fluctuations are larger than the fluctuations predicted from a Gaussian chain model or a wormlike chain model. We propose that the larger fluctuations may indicate transient residual structure in the unfolded state.

Diazepam Binding Inhibitor↗
PubMed1984

Denaturation of nucleic acids induced by intercalating agents. Biochemical and biophysical properties of acridine orange-DNA complexes.

At high binding densities acridine orange (AO) forms complexes with ds DNA which are insoluble in aqueous media. These complexes are characterized by high red- and minimal green-luminescence, 1:1 (dye/P) stoichiometry and resemble complexes of AO with ss nucleic acids. Formation of these complexes can be conveniently monitored by light scatter measurements. Light scattering properties of these complexes are believed to result from the condensation of nucleic acids induced by the cationic, intercalating ligands. The spectral and thermodynamic data provide evidence that AO (and other intercalating agents) induces denaturation of ds nucleic acids; the driving force of the denaturation is high affinity and cooperativity of binding of these ligands to ss nucleic acids. The denaturing effects of AO, adriamycin and ellipticine were confirmed by biochemical studies on accessibility of DNA bases (in complexes with these ligands) to the external probes. The denaturing properties of AO vary depending on the primary structure (sugar- and base-composition) of nucleic acids.

Acridine Orange↗
PubMed1998

[Interaction of derivatives of short oligonucleotides with nucleic acids. VIII. Characteristics of target DNA modification by alkylating oligonucleotide derivatives in tandem complexes].

The influence of effectors [octanucleotides and their 3',5'-di-N-(2-hydroxyethyl)phenazinium derivatives] on the modification of a target DNA by alkylating oligonucleotide derivatives forming duplexes of different stability with the target ws studied. It is shown that, being in tandem complexes immediately adjacent to the reactive group of an oligonucleotide reagent possessing a high hybridization capacity, the effector, on the one hand, enhances the stability of the reagent target duplex, and on the other hand, changes the site-specificity of alkylation and decreases the efficiency of the target modification at temperatures that provide a high extent of the target association with the reagent. Conversely, in the case of oligonucleotide reagents forming weak complexes with the target, effectors enhance both the stability of the target.reagent duplex and the extent of the target throughout the temperature range tested. The data indicate that the varying influence of effectors on the target modification by reagents with different hybridization capacities is due to conformational features of the target reagent duplexed regions. Increasing the rigidity of the target.reagent duplex reduces the efficiency of the target modification in tandem complexes.

Alkylating Agents↗
PubMed2004

Preferential interactions of glycine betaine and of urea with DNA: implications for DNA hydration and for effects of these solutes on DNA stability.

Interactions of the solutes glycine betaine (GB) and urea with mononucleosomal calf thymus DNA in aqueous salt solutions are characterized by vapor pressure osmometry (VPO). Analysis of osmolality as a function of solute and DNA concentration yields the effect of the solute on the chemical potential, mu(2), of the DNA. Although both GB and urea generally are nucleic acid denaturants and therefore must interact favorably with the nucleic acid surface exposed upon melting, VPO demonstrates that neither interacts favorably with duplex DNA. Addition of GB greatly increases mu(2) of DNA, indicating that the average local concentration of GB in the vicinity of the double helix is much less than its bulk concentration. By contrast, addition of urea has almost no effect on mu(2) of duplex DNA, indicating that the average local concentration of urea in the vicinity of duplex DNA is almost the same as in bulk solution. Qualitatively, we conclude that the nonuniform distribution of GB occurs primarily because duplex DNA and GB prefer to interact with water rather than with each other. Comparison with thermodynamic data for the interaction of GB with various protein surfaces (Felitsky et al., Biochemistry, 43, 14732-14743) shows that GB is excluded primarily from anionic DNA surface and that the hydration of anionic DNA phosphate oxygen surface (>or approximately 17 H(2)O per nucleotide or >or approximately 0.22 H(2)O A(-)(2)) involves at least two layers of water. From analysis of literature data for effects of urea and of GB on DNA melting, we propose that urea is an effective nonspecific nucleic acid denaturant because of its favorable interactions with the polar amide-like surface of G, C, and especially T or U bases exposed in denaturation, whereas GB is a specific GC denaturant because of its favorable interaction with G and/or C surface in the single-stranded state.

Betaine↗
PubMed1977

Sequence specific interaction of the chromosomal proteins with DNA.

Calf thymus chromatin was partially deproteinized by 0.6-1 M NaCl extraction. From the shape of the temperature derivative plot of its melting curve, the DNA in each chromatin was resolved into regions of exposed DNA, and DNA still complexed with proteins. The partially deproteinized chromatin was capable of directing in vitro RNA synthesis in an amount proportional to the fraction of exposed DNA. The in vitro RNA transcript was hybridized to denatured calf DNA under hybridization conditions where only RNA's transcribed from repetitive DNA sequences were able to form hybrids. From the results it is deduced that the in vitro RNA transcripts of 0.6 M NaC1-extracted chromatin after the removal of HI histones plus one-quater of the non histones may be transcribed from repetitive DNA sequences and those of 1 M NaCl-extracted chromatin contain more transcripts from the unique DNA sequences.

Amino Acids↗