RESUMO
During the past few years, NMR methodology for the study of nucleic acids has benefited from new developments that greatly improved state-of-the-art technology for the precise determination of three-dimensional structures. Substantial progress has been made in designing experimental protocols for the measurement of residual dipolar couplings, in sensitivity optimization of triple-resonance experiments and in detection of hydrogen bonds and in developing computational methods for structure refinement using NMR restraints.
Assuntos
DNA/química , Espectroscopia de Ressonância Magnética/métodos , RNA/química , DNA/metabolismo , Ligação de Hidrogênio , Ácidos Nucleicos/química , RNA/metabolismo , Sensibilidade e Especificidade , SolventesRESUMO
Unique chiroptical properties of poly[d(A-T)] X poly[d(A-T)] observed in CsF solutions (Vorlícková et al., 1980) were specified by circular dichroism, ultraviolet light and 31P nuclear magnetic resonance spectroscopy. It was found that up to a 3 M concentration of the salt, caesium cations induced a gradual rearrangement of the polynucleotide double helix during which the phosphodiester bonds in one sequence changed the geometry and the base stacking decreased. At higher CsF concentrations poly[d(A-T)] X poly[d(A-T)] underwent a transition toward a novel conformation which had phosphodiester bonds in both sequences in considerably different non-B-DNA geometries.
Assuntos
Fluoretos , Conformação de Ácido Nucleico , Poli dA-dT , Polidesoxirribonucleotídeos , Césio , Dicroísmo Circular , Poli dA-dT/análogos & derivados , Espectrofotometria UltravioletaRESUMO
Monovalent cation binding sites on nucleic acids in solution can be localized using the isotopically labeled ammonium ion (15NH4+) as a probe in high resolution NMR spectroscopy experiments. The application of this technique to a series of DNA duplexes reveals a preference for the binding of ammonium cations in the minor groove of A-tract sequences. These results are consistent with a recent report which indicates that some solvent electron densities previously identified as water molecules in DNA X-ray crystal structures are partially occupied by sodium ions. The sequence-specific nature of monovalent cation binding sites demonstrated here for A-tract DNA provides an explanation for the origin of sequence-directed bending.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Compostos de Amônio Quaternário/metabolismo , Sítios de Ligação/genética , Cátions/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Isótopos de Nitrogênio , Oligodesoxirribonucleotídeos/metabolismo , Água/metabolismoRESUMO
Two-dimensional 1H n.m.r. spectroscopy has been used to study the 31-base DNA oligonucleotide 5'-dAGAGAGAACCCCTTCTCTCTTTTTCTCTCTT-3', which folds to form a stable intramolecular triplex in solution at acidic pH. This structure is considerably more difficult to assign than short B-DNA duplexes and requires new assignment methods. The assignment strategy and assignments of almost all of the exchangeable and nonexchangeable resonances are presented. Seven base triplets and one Watson-Crick base-pair form the core of the structure and are connected by a four C and four T loop at either end. The second pyrimidine "strand" (bases 24 to 31) in this intramolecular pyrimidine-purine-pyrimidine triplex binds via Hoogsteen base-pairs in the major groove and is parallel to the purine "strand" (bases 1 to 8). Analysis of the sugar puckers reveals that, contrary to widely accepted belief, the triplex sugars are not predominantly in the N-type (close to C3'-endo) conformation. Except for some of the C nucleotides, all sugars are predominantly S-type (close to C2'-endo). Thus, the duplex DNA does not assume N-type sugar conformations to accommodate a third strand in the major groove. A preliminary model of the triplex structure is presented.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Sequência de Bases , Desoxirribose/química , Glicosídeos/química , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/química , Prótons , TemperaturaRESUMO
Guanine quartets are readily formed by guanine nucleotides and guanine-rich oligonucleotides in the presence of certain monovalent and divalent cations. The quadruplexes composed of these quartets are of interest for their potential roles in vivo, their relatively frequent appearance in oligonucleotides derived from in vitro selection, and their inhibition of template directed RNA polymerization under proposed prebiotic conditions. The requirement of cation coordination for the stabilization of G quartets makes understanding cation-quadruplex interactions an essential step towards a complete understanding of G quadruplex formation. We have used 15NH4+ as a probe of cation coordination by the four G quartets of the DNA bimolecular quadruplex [d(G4T4G4)]2, formed from oligonucleotides with the repeat sequence found in Oxytricha nova telomeres. 1H and 15N heteronuclear NMR spectroscopy has allowed the direct localization of monovalent cation binding sites in the solution state and the analysis of cation movement between the binding sites. These experiments show that [d(G4T4G4)]2 coordinates three ammonium ions, one in each of two symmetry related sites and one on the axis of symmetry of the dimeric molecule. The NH4+ move along the central axis of the quadruplex between these sites and the solution, reminiscent of an ion channel. The residence time of the central ion is determined to be 250 ms. The 15NH4+ is shown to be a valuable probe of monovalent cation binding sites and dynamics.
Assuntos
DNA/metabolismo , Compostos de Amônio Quaternário/metabolismo , Telômero , Sítios de Ligação , Cátions , Ressonância Magnética Nuclear Biomolecular , Prótons , ÁguaRESUMO
We have used CD spectroscopy, NMR spectroscopy and unrestrained molecular dynamics to study conformational properties of a DNA duplex formed by the self-complementary octamer d(GGGGCCCC). Its unusual CD spectrum contains features indicating A-like stacking of half of the bases, whereas the other half stack in a B-like fashion. Unrestrained molecular dynamics simulations converged to a stable B-like double-helix of d(GGGGCCCC). However, the double-helix contained a central hole whose size was half of that occurring in structure A. In the canonical structure B, the hole does not exist at all because the base-pairs cross the double-helix centre. The cytosine bases were stacked in the duplex of d(GGGGCCCC) as in structure B, while stacking of the guanine bases displayed features characteristic for structure A. NMR spectroscopy revealed that the A-like guanine-guanine stacking was accompanied by an increased tendency of the deoxyribose rings attached to the guanine bases to be puckered in an A-like fashion. Otherwise, the duplex of d(GGGGCCCC) showed no clash, no bend and no other significant deviation from structure B. The present analysis demonstrates a remarkable propensity of the guanine runs to stack in an A-like fashion even within the B-DNA framework. This property explains why the oligo(dG). oligo(dC) tracts switch into structure A so easily. Secondly, this property may influence replication, because structure A is replicated more faithfully than structure B. Thirdly, the oligo(dG) runs might have played an important role in early evolution, when DNA took on functions that originally evolved on RNA. Fourthly, the present study extends the vocabulary of DNA secondary structures by the heteronomous duplex of d(GGGGCCCC) in which the B-like strand of oligo(dC) is bound to the A-like strand of oligo(dG).
Assuntos
Guanina/química , Oligodesoxirribonucleotídeos/química , Dicroísmo Circular , Simulação por Computador , Modelos Moleculares , Movimento (Física) , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , ÁguaRESUMO
DNA usually adopts structure B in aqueous solution, while structure A is preferred in mixtures of trifluoroethanol (TFE) with water. However, the octamer d(CCCCGGGG) and other d(C(n)G(n)) fragments of DNA provide CD spectra that suggest that the base-pairs are stacked in an A-like fashion even in aqueous solution. Yet, d(CCCCGGGG) undergoes a cooperative TFE-induced transition into structure A, indicating that an important part of the aqueous duplex retains structure B. NMR spectroscopy shows that puckering of the deoxyribose rings is of the B-type. Hence, combination of the information provided by CD spectroscopy and NMR spectroscopy suggests an unprecedented double helix of DNA in which A-like base stacking is combined with B-type puckering of the deoxyribose rings. In order to determine whether this combination is possible, we used molecular dynamics to simulate the duplex of d(CCCCGGGG). Remarkably, the simulations, completely unrestrained by the experimental data, provided a very stable double helix of DNA, exhibiting just the intermediate B/A features described above. The double helix contained well-stacked guanine bases but almost unstacked cytosine bases. This generated a hole in the double helix center, which is a property characteristic for A-DNA, but absent from B-DNA. The minor groove was narrow at the double helix ends but wide at the central CG step where the Watson-Crick base-pairs were buckled in opposite directions. The base-pairs stacked tightly at the ends but stacking was loose in the duplex center. The present double helix, in which A-like base stacking is combined with B-type sugar puckering, is relevant to replication and transcription because both of these phenomena involve a local B-to-A transition.
Assuntos
DNA/química , DNA/metabolismo , Desoxirribose/química , Desoxirribose/metabolismo , Conformação de Ácido Nucleico , Pareamento de Bases/efeitos dos fármacos , Pareamento de Bases/genética , Sequência de Bases , Dicroísmo Circular , Simulação por Computador , Citosina/metabolismo , DNA/genética , Desoxirribose/genética , Guanina/metabolismo , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico/efeitos dos fármacos , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Rotação , Soluções , Temperatura , Termodinâmica , Trifluoretanol/metabolismo , Trifluoretanol/farmacologiaRESUMO
A new NMR method is described for the generation of absorption mode two-dimensional NOE spectra of oligonucleotides in H2O solution. The method yields spectra that are free of baseline distortions with excellent suppression of the intense H2O resonance. The method is demonstrated for a sample of the dodecamer d(CGCGAATTCGCG)2. All exchangeable base protons are identified and a number of new types of NOE connectivities are observed.
Assuntos
Oligodesoxirribonucleotídeos , Aminas , Fenômenos Químicos , Físico-Química , Iminas , Espectroscopia de Ressonância Magnética , Prótons , ÁguaRESUMO
The use of new 1H-detected heteronuclear 1H-31P shift correlation experiments is demonstrated for oligonucleotides of 12 and 40 base pairs. The methods give unambiguous assignments of the 31P resonances and also permit identification of the C4' and C5' sugar protons. Use of the new methods enables one to make sequence-specific resonance assignments without reference to a known or assumed conformation of the DNA fragment.
Assuntos
Oligonucleotídeos/análise , DNA/análise , Hidrogênio , Espectroscopia de Ressonância Magnética , FósforoRESUMO
The results of systematic ab initio calculations of (15)N and (1)H chemical shielding tensors in the GC base pair as a function of hydrogen bond length are presented for the first time. The hydrogen bond length characterized by the distance r(N...N) between purine N1 and pyrimidine N3 was varied between 2.57 and 3.50 A and the chemical shift tensors were calculated by the sum-over-states density functional perturbation theory. It is shown that the hydrogen bond length has a strong effect on the chemical shielding tensor of both imino proton and nitrogen, on their orientation, and, as a consequence, on the relaxation properties of both nuclei. For a nitrogen nucleus not involved in hydrogen bonding, the shielding tensor is nearly axially symmetric and almost collinear with the bond vector. As the length of the hydrogen bond decreases, the least shielding component sigma(11) deflects from the N-H vector and the shielding tensor becomes increasingly asymmetric. The significance of the presented results for the analysis of relaxation data and the efficiency of TROSY effects together with a summary of the relevant shielding parameters are presented and discussed.
Assuntos
Pareamento de Bases , Citosina/química , Guanina/química , Hidrogênio , Ligação de Hidrogênio , Espectroscopia de Ressonância Magnética/métodos , Isótopos de NitrogênioRESUMO
Application of heteronuclear magnetic resonance pulse methods to 13C, 15N-labeled nucleic acids is important for the accurate structure determination of larger RNA and DNA oligonucleotides and protein-nucleic acid complexes. These methods have been applied primarily to RNA, due to the availability of labeled samples. The two major differences between DNA and RNA are at the C2' of the ribose and deoxyribose and the additional methyl group on thymine versus uracil. We have enzymatically synthesized a 13C,15N-labeled 32 base DNA oligonucleotide that folds to form an intramolecular triplex. We present two- and three-dimensional versions of a new HCCCH-TOCSY experiment that provides intraresidue correlation between the thymine H6 and methyl resonances via the intervening carbons (H6-C6-C5-Cme-Hme).
Assuntos
DNA/química , Ressonância Magnética Nuclear Biomolecular/métodos , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Isótopos de Carbono , DNA/síntese química , Desoxirribose/química , Ligação de Hidrogênio , Isótopos de Nitrogênio , Oligodesoxirribonucleotídeos/síntese química , Timina/químicaRESUMO
Methyl 6-O-(6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl)-2,3,4-tri- O-benzyl-alpha-D-glucopyranoside (5) was formed with high stereoselectivity when the condensation of methyl 2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl (1) with 6-O-acetyl-2,3,4-tri-O-benzyl-alpha-D-glucopyranosyl chloride in ether was promoted with silver perchlorate in the presence of 2,4,6-trimethylpyridine. O-Deacetylation of 5, followed by treatment of the formed 6, containing only HO-6' unsubstituted, with diethylaminosulfur trifluoride (DAST) or dimethylaminosulfur trifluoride (methyl DAST) gave the per-O-benzyl derivative (9) of methyl 6'-deoxy-6'-fluoro-alpha-isomaltoside. Compound 9 was also obtained by condensation of 1 with 2,3,4-tri-O-benzyl-6-deoxy-6-fluoro-beta-D-glucopyranosyl fluoride (4) in the presence of silver perchlorate and anhydrous stannous chloride. The fully benzylated methyl alpha-glycoside (15) of 6-deoxy-6-fluoro-isomaltotriose, was obtained by condensation of 6 with 4. Hydrogenolysis of 9 and 15 gave the methyl alpha-glycosides of isomaltose and isomaltotriose fluorinated at C-6 of their (nonreducing) D-glucosyl group. Fluoride-ion displacements involving DAST and methyl DAST gave practically identical results, but mixtures arising from reactions involving the latter reagent were lighter-colored and easier to resolve by chromatography. The isolation of methyl alpha-glycosides of 6'-deoxy-6'-fluorogentiobiose and of 6'-O-(6-deoxy-6-fluoro-beta-D-glucopyranosyl) isomaltose is also described.
Assuntos
Glicosídeos/síntese química , Trissacarídeos/síntese química , Configuração de Carboidratos , Indicadores e Reagentes , Espectroscopia de Ressonância MagnéticaRESUMO
DNA fragments d(TATATATA) and d(TATATA) were studied in low-salt aqueous solutions and found to coexist in more than one conformer. 1H-n.m.r. demonstrates that single-stranded and double-stranded states are involved in the conformational coexistence. Circular dichroism spectroscopy indicates a global B-DNA stacking of bases in the fragments. 31P-n.m.r. resonances of the TpA and ApT phosphodiester bonds are substantially separated in the spectra of both d(TATATATA) and d(TATATA) duplexes to suggest an alternating architecture of their backbones. In fact, the oligonucleotide duplexes are much more alternating than the corresponding polynucleotide under the same solution conditions. The alternating character of the d(TATATATA) double helix is further enhanced in molar caesium fluoride solutions. The oligonucleotide isomerization into X-DNA is, however, accompanied by gel formation, which makes high resolution n.m.r. measurements impossible.
Assuntos
DNA/química , Oligonucleotídeos/química , Sequência de Bases , Dicroísmo Circular , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Soluções , TemperaturaRESUMO
1H-1H and 31P-1H nuclear Overhauser effects and 31P NMR spin-lattice relaxation times were measured for a synthetic DNA poly(dA-dT).poly(dA-dT) in a low-salt aqueous solution. The results have shown that all bases in the double helix are anti-orientated with respect to deoxyribose residues and that the sugar-phosphate backbone has an alternating architecture.
Assuntos
DNA/análise , Poli dA-dT/análise , Polidesoxirribonucleotídeos/análise , Sequência de Bases , DNA de Cadeia Simples/análise , Cinética , Espectroscopia de Ressonância Magnética , Difração de Raios XRESUMO
Spread-plating belongs to traditional microbiological methods employed for quantification of subsurface microflora during bioremediation projects in the Czechia. Concentration of degrading organisms is estimated from the number of colonies grown on agar plates supplied with contaminant as the sole carbon source. The data obtained during in situ bioremediation of the Dacice site contaminated by cutting oil suggests that changes in the composition of the carbon source in the subsurface may cause a discrepancy between laboratory data and situation in subsurface.
Assuntos
Bactérias/isolamento & purificação , Técnicas Bacteriológicas , Microbiologia Ambiental , Bactérias/metabolismo , Biodegradação Ambiental , Carbono , Meios de Cultura , Óleos/metabolismoRESUMO
In the South Moravian region which is the area with the lowest prevalence of bacillary tuberculosis in the Czech Republic (6.6 per 100,000 population in 1996) in 1993-1996 a total of six local and family microepidemics of tuberculosis were detected. For their identification the RFLP fingerprinting method was used based on evidence of repeated sequence of IS6110 in the chromosomal DNA of the examined strains of Mycobacterium tuberculosis predigested with restrictive enzyme PvuII. The profiles of fingerprints of each microepidemic were included by means of a computer programme into the hierarchy of the fingerprint dendrogram of 184 strains of M. tuberculosis which made it possible to identify possible identical profiles of strains from patients from remote places in the Czech Republic. In three family microepidemics involving always two members no identical fingerprint profiles of other Czech strains of M. tuberculosis were revealed. To the fourth cluster formed by six members of one family an identical RFLP profile of a female patient living in a nearby locality was added. In another microepidemic recorded in three brothers identical fingerprints were found another four patients from the South Moravian region and in one from the Central Bohemian region. The last cluster of two brothers was surprisingly enlarged by six identical RFLP profiles of patients from the West Bohemian region and one from Prague. These findings suggest that in areas with a low prevalence tuberculosis persists more frequently than in areas with a high prevalence as familial or local microepidemics.