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PubMed2005

Duplex PCR to differentiate between Mycoplasma synoviae and Mycoplasma gallisepticum on the basis of conserved species-specific sequences of their hemagglutinin genes.

We developed a duplex PCR assay targeting the hemagglutinin multigene families, vlhA and pMGA, of Mycoplasma synoviae and Mycoplasma gallisepticum, respectively. The assay proved to be specific and sensitive enough to justify its use for the simultaneous detection of the two major avian mycoplasma species from field isolates.

Animals↗
PubMed1986

CC/GG contacts facilitate the B to A transition of DNA in solution.

Self-complementary decadeoxynucleotides, CCGATATCGG, CCAGATCTGG, CCCTGCAGGG, GGGGGCCCCC, were designed and synthesized to estimate the A-philic free energy of CC/GG contacts. First, regions of temperature-stability of the double-stranded conformation were determined for each 10-mer. Then, circular dichroism spectra were recorded for the B-family forms at different temperatures, counter-ion concentrations and trifluoroethanol contents. A cooperative change typical of the B-A transition is observed in the CD spectra at a trifluoroethanol content specific for each duplex. The positions of half-transition points were functions not only of the nucleotide sequence but of the duplex length as well: the B to A transitions were hindered in these 10-mers in comparison with a lengthy DNA. The B-phility value was estimated to be 3 kcal/mol of 10-mer. The B-A transition point was shown to drop with an increase in the number of CC/GG contacts in a duplex. The designed 10-mers made it possible to estimate quantitatively the A-phility of CC/GG contact as compared with an average DNA: (FA-FB)CC = 0.2 Kcal/mol, (FA-FB)DNA = 0.7 Kcal/mol.

Base Composition↗
PubMed2006

Intra- and intermolecular interactions between cyclic-AMP receptor protein and DNA: ab initio fragment molecular orbital study.

The ab initio fragment molecular orbital (FMO) calculations were performed for the cAMP receptor protein (CRP) complexed with a cAMP and DNA duplex to elucidate their sequence-specific binding and the stability of the DNA duplex, as determined by analysis of their inter- and intramolecular interactions. Calculations were performed with the AMBER94 force field and at the HF and MP2 levels with several basis sets. The interfragment interaction energies (IFIEs) were analyzed for interactions of CRP-cAMP with each base pair, DNA duplex with each amino acid residue, and each base pair with each residue. In addition, base-base interactions were analyzed including hydrogen bonding and stacking of DNA. In the interaction between DNA and CRP-cAMP, there was a significant charge transfer (CT) from the DNA to CRP, and this CT interaction played an important role as well as the electrostatic interactions. It is necessary to apply a quantum mechanical approach beyond the "classical" force-field approach to describe the sequence specificity. In the DNA intramolecular interaction, the dispersion interactions dominated the stabilization of the base-pair stacking interactions. Strong, attractive 1,2-stacking interactions and weak, repulsive 1,3-stacking interactions were observed. Comparison of the intramolecular interactions of free and complexed DNA revealed that the base-pairing interactions were stronger, and the stacking interactions were weaker, in the complexed structure. Therefore, the DNA duplex stability appears to change due to both the electrostatic and the CT interactions that take place under conditions of DNA-CRP binding.

Algorithms↗
PubMed1988

The translation of DNA primary base sequence into three-dimensional structure.

A procedure is outlined to obtain a reliable computer-generated representation of the DNA duplex from its primary sequence of base pairs. The calculations are based on the potential energies of interaction of adjacent side groups. The methods are, however, completely general and can be adapted to any set of base sequence dependent conformational rules. Static representations of the DNA are compared with the distributions of conformations obtained from Monte Carlo simulation studies. Direct matrix generator calculations of the average (equilibrium) extension and orientation of various sequences and numerical estimates of the flexibility of the chains as a whole are also reported. The methods are applied to three short fragments of kinetoplast DNA from Crithidia fasciculata which exhibit dramatically different behavior on non-denaturing polyacrylamide gels.

Base Sequence↗
PubMed1993

HPLC photofingerprinting of conformational peculiarities and transitions in oligonucleotide duplexes.

Two self-complementary sequence-isomeric decadeoxyribonucleotides were exposed to UV light under conditions in which they assume duplex structures. After that they were analyzed in the denatured state by reversed-phase high-performance liquid chromatography (HPLC). Characterization of the separated photoproducts allowed localization of cyclobutane pyrimidine dimers in the sequences of the modified oligonucleotides. For [d(GGAAATTTCC)]2, which is known to contain in its central part a stretch of rigid B'-conformation with decreased mobility of constituent bases, lower yields of thymine dimers, as compared with that for ordinary B-form [d(CCTTTAAAGG)]2, were found. On the contrary, mixed thymine-cytosine heterodimers generated in the former oligonucleotide demonstrate the increase in photoreactivity of these residues at the B'-B junction. This is probably due to the peculiar conformation adopted by this decanucleotide. Stimulation of B'-B transition, by increasing the temperature before melting, reduced an inhibition of thymine photodimer formation. During the melting of both oligonucleotides yields of all identified photoinduced cyclobutadipyrimidines were reduced. Possible influences of some metal cations on the stability of the B'-form were also studied by this photoprobing technique. The present study demonstrates the feasibility of HPLC photofingerprinting as a new approach for structural analysis of nucleic acids.

Base Sequence↗
PubMed1980

Cleavage of single strand oligonucleotides and bacteriophage phi X174 DNA by Msp I endonuclease.

Type II restriction endonucleases cleave duplex DNA at nucleotide sequences displaying 2-fold symmetry. Our data show that Msp I cleaves single strand oligonucleotides, d(G-A-A-C-C-G-G-A-G-A) and d(T-C-T-C-C-G-G-T-T) at 4 degrees, 25 degrees, and 37 degrees C reaction temperatures. The rate of cleavage of d(G-A-A-C-C-G-G-A-G-A) is several-fold faster than that of d(T-C-T-C-C-G-G-T-T). Single strand phi X174 DNA is also, cleaved by Msp I endonuclease giving well defined fragments. 5'-Nucleotide analysis of the fragments generated from single strand and replicating form DNA suggest that cleavage occurs at the recognition sequence d(C-C-G-G). The data show that Msp I endonuclease cleaves single strand oligonucleotides and prefers a recognition sequence surrounded by purine nucleotides. A general model for endonuclease cleavage of single strand and duplex DNA is presented.

Bacteriophage phi X 174↗
PubMed1984

Sequence dependence of conformations of self-complementary duplex tetradeoxynucleotides containing cytosine and guanine.

The four self-complementary tetradeoxynucleotides which contain only cytosine and guanine are 5'-d-(CpGpCpG)-3', 5'-d(CpCpGpG)-3', 5'-d(GpCpGpC)-3', and 5'-d(GpGpCpC)-3'. The Raman spectra of aqueous solutions (about 0.05 M in monomer) of these tetranucleotides at pH 7 and 2 degrees C show clearly that these self-complementary tetranucleotides form double-stranded duplex structures of the canonical B type when the NaCl concentration is 0.5 M NaCl. If the temperature is raised to 50 degrees C, the Raman spectra show that in each case the double-helical B form melts in a non-cooperative way to a disordered single-chain form. On the other hand, if the salt concentration is raised to saturation, the Raman spectrum of only one of these four tetranucleotide solutions at 2 degrees C is changed in any substantial way. The Raman spectrum of the tetranucleotide 5'-d(CpGpCpG)-3' at 2.2 degrees C and at 4 M or higher salt concentration strongly resembles that of double-helical Z-form poly(dC-dG) taken under similar conditions. We conclude that the tetramer 5'-d(CpGpCpG)-3' is the only self-complementary double-helical tetranucleotide containing only cytosine and guanine in which the B-Z transition can be induced by increasing the salt concentration. This tetramer has several types of stacking interactions which differ markedly from stacking interactions in the other tetramers and may account for the enhanced stabilization of its Z conformation.

Base Sequence↗
PubMed1996

The structure of an RNA/DNA hybrid: a substrate of the ribonuclease activity of HIV-1 reverse transcriptase.

The structure of a complementary hybrid duplex of RNA and DNA has been determined by X-ray crystallography. A ten residue DNA oligonucleotide of sequence 5'-G-G-C-G-C-C-C-G-A-A-3' was annealed to complementary RNA (5'-u-u-c-g-g-g-c-g-c-c-3') and crystallized, producing tetragonal crystals that diffract to 2.3 A resolution. The hybrid adopts a geometry that is neither strictly A nor B-form, rather the helix possesses qualities of both, reminiscent of spectroscopic descriptions of a hybrid conformation, or H-form. All of the ribonucleotides maintain the C3'-endo conformation seen in A-form, while both C3'-endo and C2'-endo conformations are found in the deoxyribonucleotides. The minor groove width (8.5 to 10.5 A) is intermediate between standard values for A (11 A) and B-form (7.4 A) DNA. The global parameters rise and base-pairs tilt (or inclination) are like that of A-DNA, however the slide and x displacement (Dx) are more like that of A-RNA, thus giving the hybrid a unique conformation. In addition, the 10-mer crystallizes in a manner that allows the formation of dimers that stack end-to-end, thereby providing a glimpse of how an extended (20 base-pair) helix of RNA-DNA hybrid might appear. This duplex sequence was selected for study because it is specifically recognized by the ribonuclease H function of HIV reverse transcriptase. A structure of a substrate of this enzyme is of potential value in understanding requirements for the selectivity of this important drug target. The minor groove of the hybrid duplex, lined with the 2-OH of the ribose rings, is the single distinguishing characteristic of the RNA/DNA hybrid, undoubtedly an important structural feature conferring selectivity.

Crystallography, X-Ray↗
PubMed2003

Structural competition involving G-quadruplex DNA and its complement.

Structural competition between the G-quadruplex, the I-motif, and the Watson-Crick duplex has been implicated for repetitive DNA sequences, but the competitive mechanism of these multistranded structures still needs to be elucidated. We investigated the effects of sequence context, cation species, and pH on duplex formation by the G-quadruplex of dG(3)(T(2)AG(3))(3) and its complement the I-motif of d(C(3)TA(2))(3)C(3), using ITC, DSC, PAGE, CD, UV, and CD stopped-flow kinetic techniques. ITC and PAGE experiments confirmed Watson-Crick duplex formation by the complementary strands. The binding constant of the two DNA strands in the presence of 10 mM Mg(2+) at pH 7.0 was shown to be 5.28 x 10(7) M(-1) at 20 degrees C, about 400 times larger than that in the presence of 100 mM Na(+) at pH 5.5. The dynamic transition traces of the duplex formation from the equimolar mixture of G-/C-rich complementary sequences were obtained at both pH 7.0 and pH 5.5. Fitting to a single-exponential function gave an observed rate of 8.06 x 10(-3) s(-1) at 20 degrees C in 10 mM Mg(2+) buffer at pH 7.0, which was about 10 times the observed rate at pH 5.5 under the same conditions. Both of the observed rates increased as temperature rose, implying that the dissociation of the single-stranded structured DNAs is the rate-limiting step for the WC duplex formation. The difference between the apparent activation energy at pH 7.0 and that at pH 5.5 reflects the fact that pH significantly influences the structural competition between the G-quadruplex, the I-motif, and the Watson-Crick duplex, which also implies a possible biological role for I-motifs in biological regulation.

Animals↗
PubMed1995

Solution conformation of the (-)-trans-anti-benzo[c]phenanthrene-dA ([BPh]dA) adduct opposite dT in a DNA duplex: intercalation of the covalently attached benzo[c]phenanthrenyl ring to the 3'-side of the adduct site and comparison with the (+)-trans-anti-[BPh]dA opposite dT stereoisomer.

This paper reports on NMR-molecular mechanics structural studies of the (-)- trans-anti-benzo[c]phenanthrene-dA adduct positioned opposite dT in the sequence context of the d(C1-T2-C3-T4-C5-[BPh]A6-C7-T8-T9-C10-C11).d(G12- G13-A14-A15-G16-T17-G18-A19-G20-A21- G22) duplex (designated as the (-)-trans-anti-[BPh]dA.dT 11-mer duplex). This adduct is derived from the covalent binding of (-)-1,2-dihydroxy-3,4-epoxy-1,2,3,4-tetrahydro-benzo[c]phenanthrene [(-)-anti-BPhDE] to N6 of dA6 in this duplex sequence. The benzo[c]phenanthrenyl and nucleic acid exchangeable and nonexchangeable protons were assigned in the predominant conformation following analysis of two-dimensional NMR data sets in H2O and D2O buffer solution. The solution structure of the (-)-trans-anti-[BPh]dA.dT 11-mer duplex has been determined by incorporating intramolecular and carcinogen-DNA proton-proton distances defined by lower and upper bounds deduced from NOESY data sets as restraints in molecular mechanics computations in torsion angle space. The results show that the [BPh]dA6.dT17 base pair propeller twists and buckles slightly to permit the covalently attached benzo[c]phenanthrenyl ring to intercalate between the [BPh]dA6.dT17 and dC7.dG16 base pairs to the 3'-side of the [BPh]dA6 lesion site without disrupting the Watson-Crick hydrogen bond alignments in the modified duplex. The strain in the highly sterically hindered fjord region of the benzo[c]phenanthrenyl moiety is relieved by the propeller-like nonplanar geometry of the aromatic phenanthrenyl ring system, which stacks predominantly with the dG16 and dT17 bases on the unmodified strand. The benzylic ring adopts a distorted half-chair form, in which the H1 and H2 protons are pseudo-diequatorial and the H3 and H4 protons are pseudodiaxial. The current observation that the (-)-trans-anti-[BPh]dA positioned opposite dT intercalates to the 3'-side of the intact modified base pair contrasts with our previous demonstration that the stereoisomeric (+)-trans-anti-[BPh]dA adduct positioned opposite dT intercalates to the 5'-side of the intact modified base pair [Cosman, M., et al. (1993b) Biochemistry 32, 12488-12497]. These stereochemically induced structural differences between isomeric [BPh]dA lesions derived from the binding of chiral (+)- and (-)-anti-BPhDE enantiomers may in turn profoundly influence the interactions of the carcinogen-modified DNA with repair and replication enzymes in the cell.

Base Sequence↗
PubMed1989

Substrate properties of 25-nt parallel-stranded linear DNA duplexes.

Four 25-nt oligonucleotides consisting of sequences of dA and dT (D1-4) have been synthesized. As shown in a companion paper (Rippe et al., 1989), the two combinations D1.D3 and D2.D4 form normal antiparallel duplexes, whereas the pairs D1.D2 and D3.D4 constitute duplexes with the same sequences, but with the two strands parallel to each other. The activities of the following DNA processing enzymes and chemical reagents on the parallel stranded (ps) and antiparallel stranded (aps) duplexes were tested. (i) The restriction endonucleases DraI, SspI, and MseI do not cut the ps duplexes. (ii) DNase I and exonuclease III exhibit a much lower activity with the ps duplexes. (iii) The nuclease activities of S 1 nuclease, micrococcal nuclease (S 7), phage lambda 5'-exonuclease, and the 3'-5' nuclease activity of Escherichia coli DNA polymerase I and its large fragment are higher with the ps than with the aps substrates. (iv) Bal 31 nuclease and the chemical nuclease 1,10-phenanthroline-copper ion [(OP)2Cu+] degrade ps-DNA and aps-DNA at approximately the same rate but show preferred cutting sites only with the aps molecules. (v) The iron(II)-EDTA complex has equivalent nuclease activities with the ps and the aps molecules. (vi) The ps duplex is not a substrate for blunt-end ligation with phage T4 DNA ligase.

Base Composition↗
PubMed1992

The estimation of distances between specific backbone-labeled sites in DNA using fluorescence resonance energy transfer.

A series DNA helices of twenty-four base pairs has been prepared for the study of fluorescence resonance energy transfer. Each of the DNA helices contains two phosphorothioate diesters (one in each strand) at pre-selected sites for introduction of the desired donor and acceptor fluorophores. The phosphorothioate-containing oligodeoxynucleotides have been prepared as pure Rp or Sp derivatives or as deastereomeric mixtures. Fluorescein and eosin are employed as the respective donor and acceptor fluorophores. A series of donor-acceptor pairs was generated by labeling of the appropriate phosphorothioate diester with the desired fluorophore and annealing the two complementary DNA strands (one containing the acceptor and one containing the donor fluorophore) to form the double-stranded helix. The 24-mer helices containing two covalently attached fluorophores exhibited some thermal destabilization and the extent of this destabilization was dependent upon the stereochemical orientation of the fluorophore. The Sp derivatives direct the fluorophore out, away from the the DNA helix, while the Rp derivatives direct the fluorophore toward the major groove. As expected, the Sp labeled duplexes were more stable than the corresponding Rp labeled sequences. However, all of the duplex structures formed were stable under the conditions used to measure energy transfer. Energy transfer could be observed with these complexes from the quenching of the donor fluorescence in the presence of the acceptor fluorophore. Using Förster's theories, distances separating the fluorophores could be calculated that were generally in reasonable agreement with the distances expected in an idealized B-form DNA helix. However anomalous results were obtained for one donor/acceptor pair where the expected distance was less than 20 A. Fluorescence anisotropy values determined in solutions of varying viscosity were quite high suggesting that the fluorophores did not experience complete freedom of movement when attached to the DNA helix.

Base Composition↗
PubMed1990

Interstrand duplexes in hnRNA: the occurrence of retrotransposon antisense sequences.

We have previously identified a class of non-polyadenylated hnRNA molecules (CLASS IA-) which hydrogen bond to polyadenylated hnRNA molecules. Characterization of clones from a Drosophila Class IA- cDNA library reveals that 18S rRNA and transcripts derived from intermediate repeat DNA constitute the bulk of such molecules. The most abundant non-ribosomal clone in the library derives from the antisense strand of the Copia retrotransposon and occurs in Drosophila Kc cell Class IA- hnRNA as non-polyadenylated transcripts of 10 and 15 kb.

Animals↗
PubMed1998

The impact of an exocyclic cytosine adduct on DNA duplex properties: significant thermodynamic consequences despite modest lesion-induced structural alterations.

The exocyclic base adduct 3,N4-deoxyethenocytosine (epsilonC) is a common DNA lesion that can arise from carcinogen exposure and/or as a biproduct of cellular processes. We have examined the thermal and thermodynamic impact of this lesion on DNA duplex properties, as well as the structural alterations imparted by the lesion. For these studies, we used calorimetric and spectroscopic techniques to investigate a family of 13-mer DNA duplexes of the form (5'CGCATGNGTACGC3')x(3'GCGTACNCATGCG5'), where the central NxN base pair represents the four standard Watson-Crick base pairs (corresponding to four control duplexes), and where either one of the N bases has been replaced by epsilonC, yielding eight test duplexes. Studies on these 12 duplexes permit us to assess the impact of the epsilonC lesion as a function of sequence context. Our spectroscopic and calorimetric data allow us to reach the following conclusions: (i) The epsilonC lesion imparts a large penalty on duplex stability, with sequence context only modestly modulating the extent of this lesion-induced destabilization. This result contrasts with our recent studies of duplexes with abasic sites, where sequence context was found to be the predominant determinant of thermodynamic damage. (ii) For the epsilonC-containing duplexes, sequence context effects are most often observed in the enthalpic contribution to lesion-induced duplex destabilization. However, due to compensating entropies, the free energy changes associated with this lesion-induced duplex destablization are nearly independent of sequence context. (iii) Despite significant lesion-induced changes in duplex energetics, our spectroscopic probes detect only modest lesion-induced changes in duplex structure. In fact, the overall duplex maintains a global B-form conformation, in agreement with NMR structural data. We discuss possible interpretations of the apparent disparity between the severe thermodynamic and relatively mild structural impacts of the epsilonC lesion on duplex properties. We also note and discuss the implications of empirical correlations between biophysical and biological properties of lesion-containing duplexes.

Base Sequence↗
PubMed2004

Sequence-dependent thermodynamic parameters for locked nucleic acid (LNA)-DNA duplex formation.

The design of modified nucleic acid probes, primers, and therapeutics is improved by considering their thermodynamics. Locked nucleic acid (LNA) is one of the most useful modified backbones, with incorporation of a single LNA providing a substantial increase in duplex stability. In this work, the hybridization DeltaH(o), DeltaS(o), and melting temperature (T(M)) were measured from absorbance melting curves for 100 duplex oligonucleotides with single internal LNA nucleotides on one strand, and the results provided DeltaDeltaH(o), DeltaDeltaS(o), DeltaDelta, and DeltaT(M) relative to reference DNA oligonucleotides. LNA pyrimidines contribute more stability than purines, especially A(L), but there is substantial context dependence for each LNA base. Both the 5' and 3' neighbors must be considered in predicting the effect of an LNA incorporation, with purine neighbors providing more stability. Enthalpy-entropy compensation in DeltaDeltaH(o) and DeltaDeltaS(o) is observed across the set of sequences, suggesting that LNA can stabilize the duplex by either preorganization or improved stacking, but not both simultaneously. Singular value decomposition analysis provides predictive sequence-dependent rules for hybridization of singly LNA-substituted DNA oligonucleotides to their all-DNA complements. The results are provided as sets of DeltaDeltaH(o), DeltaDeltaS(o), and DeltaDelta parameters for all 32 of the possible nearest neighbors for LNA+DNA:DNA hybridization (5' MX(L) and 5' X(L)N, where M, N, and X = A, C, G, or T and X(L) represents LNA). The parameters are applicable within the standard thermodynamic prediction algorithms. They provide T(M) estimates accurate to within 2 degrees C for LNA-containing oligonucleotides, which is significantly better accuracy than previously available.

Algorithms↗
PubMed1990

Recognition between mitomycin C and specific DNA sequences for cross-link formation.

An extensive series of oligodeoxyribonucleotides was reacted with reductively activated mitomycin C (MC), and the resulting cross-linked drug-oligonucleotide complexes were isolated by reverse-phase HPLC and characterized by nucleoside and MC-nucleoside adduct analysis. HPLC also served for assay of the yield of cross-linked oligonucleotides. AT-rich duplex oligonucleotides, containing a single central CG.CG, gave high yields of cross-links between the two guanines while those having GC.GC, instead, gave none. In another series, the central sequences CGC.GCG and CGC.ICG both yielded 50% cross-link while CGC.GCI was completely resistant. Cross-linking was conducted also in two steps: Oligonucleotides substituted monofunctionally by MC at guanine at either a CG or GC sequence were annealed with their complementary strands followed by reductive reactivation of the bound MC to form a cross-link. The CG oligomers were cross-linked quantitatively while the GC ones were again resistant. These results show unambiguously that the MC cross-link is absolutely specific to the CG.CG duplex sequence, confirming our previous finding [Chawla, A.K., Lipman, R., & Tomasz, M. (1987) in Structure and Expression, Volume 2: DNA and Its Drug Complexes (Sarma, R.H., & Sarma, M.H., Eds.) Adenine Press, Guilderland, NY]. Evidence is presented that this specificity is due to the specific orientation of the monofunctionally attached MC in the minor groove. Superimposed on the CG.CG requirement, a four-base-pair sequence preference was observed at PuCGPyr.PuCGPyr sequences. This suggests that the guanine N2 atom of GpPyr is more reactive toward the drug than that of GpPu, due to the favorable effect of the negative dipole of the O2 of the Pyr on the reaction; in accordance, GpT was more reactive than GpC.(ABSTRACT TRUNCATED AT 250 WORDS)

Base Sequence↗
PubMed2006

Effects of halogenated WNA derivatives on sequence dependency for expansion of recognition sequences in non-natural-type triplexes.

Triplex-forming oligonucleotides (TFOs) are sequence-specific DNA-binding agents, but their target duplexes are limited to homopurine/homopyrimidine sequences because of interruption of the pyrimidines bases in the purine region. This problem has not been fully solved despite a wide variety of studies. Recently, we have developed a bicyclic system as a novel scaffold for nucleoside analogues (WNA, W-shaped nucleoside analogues) and determined two useful compounds, WNA-betaT (2) and WNA-betaC (5), for highly stable and selective triplex formation at a TA and a CG interrupting site, respectively. However, subsequent investigations have shown that the triplex formation using WNA is dependent on the neighboring bases of the TFOs. In this study, we have synthesized new WNA derivatives having halogenated recognition bases or benzene rings and evaluated the effects of the modifications on the triplex stability as well as selectivity. It has been found that the WNA-betaT analogues holding 5-halogenated pyrimidine bases (WNA-beta(Br)U (3) and WNA-beta(F)U (4)) exhibit high CG-selectivity. On the other hand, the WNA-betaT derivatives having the bromo-substituted benzene ring (mBr-WNA-betaT (10) and oBr-WNA-betaT (11)) have shown high selectivity to a TA interrupting site with high stability in the sequences to which the original WNA-betaT do not bind. Thus, sequence-dependency has been overcome by the sequence-dependent use of WNA-betaT, mBr-WNA-betaT, and oBr-WNA-betaT.

Base Sequence↗