RESUMO
The repetitive DNA sequences found at telomeres and centromeres play a crucial role in the structure and function of eukaryotic chromosomes. This role may be related to the tendency observed in many repetitive DNAs to adopt non-canonical structures. Although there is an increasing recognition of the importance of DNA quadruplexes in chromosome biology, the co-existence of different quadruplex-forming elements in the same DNA structure is still a matter of debate. Here we report the structural study of the oligonucleotide d(TCGTTTCGT) and its cyclic analog d
Assuntos
Quadruplex G , Pareamento de Bases , Dimerização , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Desnaturação de Ácido Nucleico , Motivos de Nucleotídeos , Oligonucleotídeos/química , PrótonsRESUMO
Some DNA oligonucleotides can fold back and self-associate forming dimeric structures stabilized by intermolecular base pairs. The resulting antiparallel dimer is a tightly packed four-stranded structure formed by a core of minor groove tetrads connected by short loops of unpaired nucleotides. We have explored the sequential requirements for the loop residues and have found that this family of structures is only stable with one- and two-residue loops, with the stability of the former ones being only marginal. Two-residue loops with purines in the first position give rise to the most stable structures due to their enhanced stacking interaction with the adjacent minor groove tetrad. On the other hand, pyrimidines confer more stability than purines in the second position of the loop.
Assuntos
Oligonucleotídeos/química , Dimerização , Modelos Moleculares , Conformação de Ácido Nucleico , TemperaturaRESUMO
The glmS ribozyme is the first natural self-cleaving ribozyme known to require a cofactor. The d-glucosamine-6-phosphate (GlcN6P) cofactor has been proposed to serve as a general acid, but its role in the catalytic mechanism has not been established conclusively. We surveyed GlcN6P-like molecules for their ability to support self-cleavage of the glmS ribozyme and found a strong correlation between the pH dependence of the cleavage reaction and the intrinsic acidity of the cofactors. For cofactors with low binding affinities, the contribution to rate enhancement was proportional to their intrinsic acidity. This linear free-energy relationship between cofactor efficiency and acid dissociation constants is consistent with a mechanism in which the cofactors participate directly in the reaction as general acid-base catalysts. A high value for the Brønsted coefficient (ß ~ 0.7) indicates that a significant amount of proton transfer has already occurred in the transition state. The glmS ribozyme is the first self-cleaving RNA to use an exogenous acid-base catalyst.
Assuntos
Ácidos/química , Álcalis/química , Proteínas de Bactérias/química , RNA Catalítico/química , Catálise , Cinética , Modelos MolecularesRESUMO
Active-site guanines that occupy similar positions have been proposed to serve as general base catalysts in hammerhead, hairpin, and glmS ribozymes, but no specific roles for these guanines have been demonstrated conclusively. Structural studies place G33(N1) of the glmS ribozyme of Bacillus anthracis within hydrogen-bonding distance of the 2'-OH nucleophile. Apparent pK(a) values determined from the pH dependence of cleavage kinetics for wild-type and mutant glmS ribozymes do not support a role for G33, or any other active-site guanine, in general base catalysis. Furthermore, discrepancies between apparent pK(a) values obtained from functional assays and microscopic pK(a) values obtained from pH-fluorescence profiles with ribozymes containing a fluorescent guanosine analogue, 8-azaguanosine, at position 33 suggest that the pH-dependent step in catalysis does not involve G33 deprotonation. These results point to an alternative model in which G33(N1) in its neutral, protonated form donates a hydrogen bond to stabilize the transition state.
Assuntos
Guanina/metabolismo , RNA Catalítico/metabolismo , Bacillus anthracis/química , Bacillus anthracis/metabolismo , Biocatálise , Domínio Catalítico , Guanina/química , Modelos Moleculares , RNA Catalítico/químicaRESUMO
In addition to the better known guanine-quadruplex, four-stranded nucleic acid structures can be formed by tetrads resulting from the association of Watson-Crick base pairs. When such association occurs through the minor groove side of the base pairs, the resulting structure presents distinctive features, clearly different from quadruplex structures containing planar G-tetrads. Although we have found this unusual DNA motif in a number of cyclic oligonucleotides, this is the first time that this DNA motif is found in linear oligonucleotides in solution, demonstrating that cyclization is not required to stabilize minor groove tetrads in solution. In this article, we have determined the solution structure of two linear octamers of sequence d(TGCTTCGT) and d(TCGTTGCT), and their cyclic analogue d
Assuntos
Citosina/química , DNA/química , Quadruplex G , Guanina/química , Pareamento de Bases , Dimerização , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Desnaturação de Ácido Nucleico , Oligodesoxirribonucleotídeos/químicaRESUMO
Minor groove aligned tetrads resulting from the association of Watson-Crick base pairs stabilize a distinct class of four-stranded DNA structures, different from G-quadruplexes or i-motifs. These tetrads can be formed by several arrangements of G-C or A-T base pairs. Here we prove that minor groove tetrads can be also formed by G-T mismatches. In this manuscript we describe the dimeric solution structures of two cyclic oligonucleotides stabilized by intermolecular G-T non-canonical base pairs. In the dimeric structure of d
Assuntos
Pareamento de Bases , Quadruplex G , Oligonucleotídeos/química , Dimerização , Conformação de Ácido NucleicoRESUMO
The stability of 2'-deoxy-2'-fluoroarabinonucleic acid (2'F-ANA) to hydrolysis under acidic and basic conditions was compared to that of DNA, RNA and 2'F-RNA. In enzyme-free simulated gastric fluid (pH approximately 1.2), 2'F-ANA was found to have dramatically increased stability (virtually no cleavage observed after 2 days) with respect to both DNA (t(1/2) approximately 2 min) and RNA (t(1/2) approximately 3 h (PO) or 3 days (PS)). These results were observed for both phosphodiester and phosphorothioate backbones and with multiple mixed-base sequences. Under basic conditions, 2'F-ANA also showed good stability. In 1 M NaOH at 65 degrees C, 2'F-ANA had a t(1/2) of approximately 20 h, while RNA was entirely degraded in a few minutes. Furthermore, the nuclease cleavage of phosphorothioate 2'F-ANA and DNA by snake venom phosphodiesterase was studied in detail. One diastereomer of the PS-2'F-ANA linkage was found to be much more vulnerable to enzymatic cleavage than the other, which is parallel to the properties observed for PS-DNA. Additional studies of 2'F-ANA-containing oligonucleotides are warranted based on the excellent stability properties described here.
Assuntos
Arabinonucleotídeos/química , 2',3'-Nucleotídeo Cíclico Fosfodiesterases/metabolismo , Ácidos , Álcalis , Animais , Arabinonucleotídeos/metabolismo , Sequência de Bases , Catálise , Crotalus/metabolismo , DNA/química , DNA/metabolismo , Hidrólise , Estrutura Molecular , RNA/química , RNA/metabolismo , Estereoisomerismo , Venenos de Víboras/enzimologiaRESUMO
We have studied the structure and stability of the cyclic dodecamer d
RESUMO
We have investigated the molecular interaction between cyclic and linear oligonucleotides. We have found that short cyclic oligonucleotides can induce hairpinlike structures in linear DNA fragments. By using NMR and CD spectroscopy we have studied the interaction of the cyclic oligonucleotide d