RESUMO
The cross-peaks of 1H-NOESY spectra at different time delays are compared to a mode-coupling diffusion (MCD) calculation, including the evaluation of the full 1H relaxation matrix, in the case of a 23 nucleotide fragment of the stem-loop SL1 domain of HIV-1Lai genomic RNA mutated in a single position. The MCD theory gives significant agreement with 1H relaxation experiments enabling a thorough understanding of the differential local dynamics along the sequence and particularly of the dynamics of nucleotides in the stem and in the loop. The differential dynamics of this hairpin structure is important in directing the dimerization of the retroviral genome, a fundamental step in the infectious process. The demonstration of a reliable use of time dependent NOE cross-peaks, largely available from NMR solution structure determination, coupled to MCD analysis, to probe the local dynamics of biological macromolecules, is a result of general interest of this paper.
Assuntos
Genoma Viral , HIV-1/genética , Conformação de Ácido Nucleico , RNA Viral/química , Simulação por Computador , Matemática , Mutação , Ressonância Magnética Nuclear Biomolecular , Nucleotídeos/química , RNA Viral/genéticaRESUMO
Retroviruses contain dimeric RNA consisting of two identical copies of the genomic RNA. The interaction between these two RNA molecules occurs near their 5' ends. A region upstream from the splice donor comprising an auto-complementary sequence has been identified as being responsible for the initiation of the formation of dimeric HIV-1(Lai) RNA. This region (SL1), part of the PSI encapsidation domain, can adopt a stem-loop structure. It has already been shown that this stem-loop structure can initiate the formation of two distinct dimers differing in their thermostability: a loop-loop dimer or 'kissing complex' and an extended dimer. We report here a study using UV and 1D NMR spectroscopy of the dimerization of a short oligoribonucleotide (23 nucleotides) spanning nucleotides 248-270 of the HIV-1(Lai) SL1 sequence, in order to derive the thermodynamic parameters associated with the transition from the loop-loop complex to the extended dimer. The temperature dependence of the UV absorbency shows an hypochromicity for this transition with a small enthalpy change equal to - 29.4 +/- 5 kcal x mol-1, together with a concentration independent transition which implies a monomolecular reaction. On the other hand, our NMR results don't indicate a dissociation of the GCGCGC sequence engaged in the loop-loop interaction during the rearrangement of the loop-loop complex into the extended dimer. Our data suggest that the loop-loop interaction is maintained during the temperature dependent conformational change while the intramolecular base-pairing of the stems is disrupted and then reconstituted to form an intermolecular base-pairing leading to an extended dimer.
Assuntos
HIV-1/genética , RNA Viral/química , Sequência de Bases , Primers do DNA , Dimerização , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , RNA Viral/genética , Espectrofotometria Ultravioleta , TermodinâmicaRESUMO
The DNA duplex d(CTCTCG*AGTCTC).d(GAGAC-TC*GAGAG) containing a single trans- diammine-dichloroplatinum(II) interstrand cross-link (where G* and C* represent the platinated bases) has been studied by two-dimensional NMR. All the exchangeable and non-exchangeable proton resonance lines were assigned (except H5'/H5") and the NOE intensities were transformed into distances via the RELAZ program. By combining the NOESY and COSY data (330 constraints) and NMR-constrained molecular mechanics using JUMNA, a solution structure of the cross-linked duplex has been determined. The duplex is distorted over two base pairs on each side of the interstrand cross-link and exhibits a slight bending of its axis ( approximately 20 degrees ) towards the minor groove. The platinated guanine G* adopts a syn conformation. The rotation results in a Hoogsteen-type pairing between the complementary G(6)* and C(19)* residues which is mediated by the platinum moiety and is stabilized by a hydrogen bond between O6(G(6)*) and N4H(C(19)*). The rise between the cross-linked residues and the adjacent residues is increased owing to the interaction between these adjacent residues and the ammine groups of the platinum moiety. These results are discussed in relation to the slow rate of closure of the monofunctional adducts into interstrand cross-links.
Assuntos
Cisplatino/metabolismo , Reagentes de Ligações Cruzadas/metabolismo , DNA/química , DNA/metabolismo , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , Pareamento de Bases , Sequência de Bases , Nucleotídeos de Citosina/química , Nucleotídeos de Citosina/genética , Nucleotídeos de Citosina/metabolismo , DNA/genética , Adutos de DNA/química , Adutos de DNA/genética , Adutos de DNA/metabolismo , Nucleotídeos de Guanina/química , Nucleotídeos de Guanina/genética , Nucleotídeos de Guanina/metabolismo , Ligação de Hidrogênio , Cinética , Modelos Moleculares , Oligodesoxirribonucleotídeos/química , Oligodesoxirribonucleotídeos/genética , Oligodesoxirribonucleotídeos/metabolismo , Platina/metabolismo , Prótons , Software , SoluçõesRESUMO
The genome of all retrovirus consists of two copies of genomic RNA which are noncovalently linked near their 5' end. A sequence localized immediately upstream from the splice donor site inside the HIV-1 psi-RNA region was identified as the domain responsible for the dimerization initiation. It was shown that a kissing complex and a stable dimer are both involved in the HIV-1Lai RNA dimerization process in vitro. The NCp7 protein activates the dimerization by converting a transient loop-loop complex into a more stable dimer. The structure of this transitory loop-loop complex was recently elucidated by Mujeeb et al. In work presented here, we use NMR spectroscopy to determine the stable extended dimer structure formed from a 23 nucleotides RNA fragment, part of the 35 nucleotides SL1 sequence. By heating to 90 degrees C, then slowly cooling this sequence, we were able to show that an extended dimer is formed. We present evidence for the three dimensional structure of this dimer. NMR data yields evidence for a zipper like motif A8A9.A16 existence. This motif enables the surrounding bases to be positioned more closely and permit the G7 and C17 bases to be paired. This is different to other related sequences where only the kissing complex is observed, we suggest that the zipper like motif AA.A could be an important stabilization factor of the extended duplex.
Assuntos
HIV-1/genética , RNA Viral/química , Dimerização , Genoma Viral , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Conformação de Ácido Nucleico , RNA Líder para Processamento/química , SoluçõesRESUMO
A strategy is presented for searching which atoms can be regrouped within rigid sub-units during the time course of Molecular Dynamics simulations of biopolymers. The root mean square fluctuations of the interatomic distances are used as a criterion. The number of rigid sub-units which are found depends on the tolerance rc for the definition of a rigid body, i.e. until which value the fluctuations can be neglected. The method is applied to two self-complementary oligonucleotides belonging to the B-form family which give identical results. With rc = 0.027 nm each nucleotide may be described as 3 rigid sub-units: the sugar ring, the base and the backbone (PO4 + C5' atoms). With rc = 0.01 nm, the same sub-units are obtained except that C5' can no more be regrouped with the PO4 atoms. It is shown that the variation of the coulombic potential owing to the deformation of the sub-units during the time course of the simulation is on the same order of magnitude as the inaccuracy due to the choice of the force field parameters.
Assuntos
Simulação por Computador , DNA/química , Conformação de Ácido Nucleico , Modelos Moleculares , Oligodesoxirribonucleotídeos/químicaRESUMO
In order to study some internal dynamic processes of the lac operator sequence, the 13C-labeled duplex 5'd(C0G1C2T3C4A5C6A7A8T9T10).d(A10A9T8T7G6T5G4A3G2C1G0)3' was used. The spreading of both the 1H1' and 13C1' resonances brought about an excellent dispersion of the 1H1'-13C1' correlations. The spin-lattice relaxation parameters R(Cz), R(Cx,y) and R(Hz --> Cz) were measured for each residue of the two complementary strands, except for the 3'-terminal residues which were not labeled. Variation of the relaxation rates was found along the sequence. These data were analyzed in the context of the model-free formalism proposed by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546-4570] and extended to three parameters by Clore et al. [(1990) Biochemistry, 29, 7387-7401; and (1990) J. Am. Chem. Soc., 112, 4989-4991]. A careful analysis using a least-squares program showed that our data must be interpreted in terms of a three-parameter spectral density function. With this approach, the global correlation time was found to be the same for each residue. All the C1'-H1' fragments exhibited both slow (tau s = 1.5 ns) and fast (tau f = 20 ps) restricted libration motions (Ss2 = 0.74 to 1.0 and Sf2 = 0.52 to 0.96). Relaxation processes were described as governed by the motion of the sugar relative to the base and in terms of bending of the whole duplex. The possible role played by the special structure of the AATT sequence is discussed. No evident correlation was found between the amplitude motions of the complementary residues. The 5'-terminal residues showed large internal motions (S2 = 0.5), which describe the fraying of the double helix. Global examination of the microdynamical parameters Sf2 and Ss2 along the nucleotide sequence showed that the adenine residues exhibit more restricted fast internal motions (Sf2 = 0.88 to 0.96) than the others, whereas the measured relaxation rates of the four nucleosides in solution were mainly of dipolar origin. Moreover, the fit of both R(Cz) and R(Hz --> Cz) experimental relaxation rates using an only global correlation time for all the residues, gave evidence of a supplementary relaxation pathway affecting R(Cx,y) for the purine residues in the (5' --> 3') G4A3 and A10A9T8T7 sequences. This relaxation process was analyzed in terms of exchange stemming from motions of the sugar around the glycosidic bond on the millisecond time scale. It should be pointed out that these residues gave evidence of close contacts with the protein in the complex with the lac operator [Boelens et al. (1987) J. Mol. Biol., 193, 213-216] and that these motions could be implied in the lac-operator-lac-repressor recognition process.
Assuntos
Proteínas de Bactérias/genética , DNA/genética , Proteínas de Escherichia coli , Óperon Lac/genética , Proteínas Repressoras/genética , Análise de Sequência de DNA , Radioisótopos de Carbono , DNA/química , Repressores Lac , Espectroscopia de Ressonância MagnéticaRESUMO
A 10 base pairs double-stranded oligonucleotide with the sequence d(CCTCG.CTCTC). d(GAGAG.CGAGG) containing a single interstrand cross-link resulting from chelation of the N7 position of two guanine residues on the opposite strands of DNA at the d(G.C/G.C) site by a cis-diammineplatinum(II) residue was analyzed by 1H NMR spectroscopy. All the exchangeable and nonexchangeable protons resonance lines (except some H5'-H5") were assigned. NOESY spectra and chemical shifts indicated that the cross-linkage of the guanines of G.5 and G.6 induced extrahelicity of C5 and C6. Moreover, several unusual proximities were observed such as: (i) NOE cross-peaks between the H2'-H2" of G.5 or G.6 and the aromatic proton of their 5' neighbor C4 or A7 (ii) the absence of cross-peak for the steps G.5-C6, C6-T7 and C5-G4 (iii) a strong NOESY connectivity between H8(G.5) and H2(A7). All these data allowed us to describe the head to tail arrangement of the two cross-linked guanines as well as their stacking with flanking neighbor nucleotides (G.5 with T7.A7 base pair and G.6 with C4.G4 base pair). Using all the NOESY and TOCSY data (208 constraints), we have obtained a solution structure of the cross-linked duplex by using the NMR-constrained molecular mechanics program JUMNA. The reversal position of the two cross-linked guanines placed the cis-diammineplatinum(II) residue in the minor groove. The stacking of the two cross-linked guanines with the surrounding bases induced a bend of 40 degrees toward the minor groove. The locally left-helix formation, the extrusion of the cytosines and the stacking of the platinated guanines led to an unwinding of 76 degrees. This value is in good agreement with the values deduced from gel electrophoresis experiments.
Assuntos
Antineoplásicos/química , Cisplatino/química , Reagentes de Ligações Cruzadas/química , DNA/química , Antineoplásicos/metabolismo , Cisplatino/metabolismo , DNA/metabolismo , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Conformação de Ácido Nucleico , Ácidos Nucleicos Heteroduplexes/química , Ácidos Nucleicos Heteroduplexes/metabolismo , SoluçõesRESUMO
Psoralen (pso) was attached via its C-5 position to the 5'-phosphate group of an oligodeoxynucleotide d(TAAGCCG) by a hexamethylene linker (m6). Complex formation between pso-m6-d(TAAGCCG) and the complementary strands d(CGGCTTA)[7-7mer] or d(CGGCTTAT)[7-8mer] was investigated by nuclear magnetic resonance in aqueous solution. Structural informations derived from DQF-COSY and NOESY maps, revealed that the mini double helix adopts a B-form conformation and that the deoxyriboses preferentially adopt a C2'-endo conformation. The nOe connectivities observed between the protons of the bases or the sugars in each duplex, and the protons of the psoralen and the hexamethylene chain, led us to propose a model involving an equilibrium between two conformations due to different locations of the psoralen. Upon UV-irradiation, the psoralen moiety cross-linked the two DNA strands at the level of 5'TpA3' sequences. NMR studies of the single major photo-cross-linked duplex pso-m6-d(TAAGCCG) and d(CGGCTTA) were performed. The stereochemistry of the diadduct is indeed cis-syn at both cyclobutane rings. In addition, the effects of this diadduct on the helical structure are analyzed in detail.
Assuntos
Furocumarinas/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Furocumarinas/efeitos da radiação , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , SoluçõesRESUMO
The three-dimensional structure of an intramolecular triple helix whose three strands have been linked by a hexaethylene glycol chain, and selectively 13C-enriched in position C1' on the third strand was investigated by NMR spectroscopy and constrained molecular mechanics calculations. Starting from different initial conformations, we show that the NOE constraints determined by the complete relaxation matrix calculation and iterative back-calculations allowed us to reach the same final restrained triple helix, taking into account implicitly the solvent effect. We conclude that this triplex adopted a B-type conformation rather than a A-type. The sugar pucker was found predominantly in the S-type conformation, in the range of C2'-endo geometry.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Sequência de Bases , Configuração de Carboidratos , Carboidratos/química , Desoxirribose/química , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Cloreto de Sódio/química , Soluções/química , Água/químicaRESUMO
In order to examine the internal dynamic processes of the dodecamer d(CGCAAATTTGCG)2, the 13C-enriched oligonucleotide has been synthesized. The three central thymines were selectively 13C-labeled at the C1' position and their spin-lattice relaxation parameters R(CZ), R(CX,Y), R(HZ-->CZ), R(2HZCZ), R(2HZCX,Y) and R(HZC) were measured. Density functions were computed for two models of internal motions. Comparisons of the experimental data were made with spin-lattice relaxation rates rather than with the density functions, whose values were altered by accumulation of the uncertainties of each relaxation rate measurement. The spin-lattice relaxation rates were computed with respect to the motions of the sugar around the C1'-N1 bond. A two-state jump model between the anti- and syn-conformations with P(anti)/P(syn) = 91/9 or a restricted rotation model with delta chi = 28 degrees and an internal diffusion coefficient of 30 x 10(7) s-1 gave a good fit with the experimental data. Twist, tilt or roll base motions have little effect on 13C1' NMR relaxation. Simulation of spin-relaxation rates with the data obtained at several temperatures between 7 and 32 degrees C, where the dodecamer is double stranded, shows that the internal motion amplitude is independent of the temperature within this range, as expected for internal motion. Using the strong correlation which exists in a B-DNA structure between the chi and delta angle, we suggest that the change in the glycosidic angle value should be indicative of a sugar puckering between the C1'-exo and C2'-endo conformations.
Assuntos
DNA/química , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos/química , Sequência de Bases , Simulação por Computador , Temperatura Alta , Espectroscopia de Ressonância Magnética/métodos , Modelos Químicos , Dados de Sequência Molecular , MovimentoRESUMO
We present NMR studies of an intramolecular triple helix, the three strands of which have been linked by a hexaethylene glycol chain. To overcome the generally encountered difficulties of assignment in the homo-pyrimidine strands, the carbon Cl' of the pyrimidines were selectively 13C-enriched. Assignments of the aromatic and sugar protons were obtained from NOESY-HMQC and TOCSY-HMQC spectra. We show that the recognition of a DNA duplex by a third strand via triplex formation is easily carried out in solution by observing the changes of the 1Hl'-13Cl' connectivities as a function of pH. Furthermore, the conformation of the sugars has been found to be C2'-endo, on the basis of the coupling constant values directly measured in an HSQC spectrum.
Assuntos
DNA/química , Espectroscopia de Ressonância Magnética , Conformação de Ácido Nucleico , Sequência de Bases , Isótopos de Carbono , Reagentes de Ligações Cruzadas , Etilenoglicóis , Hidrogênio , Marcação por Isótopo , Dados de Sequência MolecularRESUMO
The structure difference between the free operator of the lac system d(GCTCACAAT).d(ATTGTGAGC) and the same operator complexed to the headpiece of the lac repressor has been investigated by 2-D-1H NMR spectroscopy in conjunction with molecular modelling in internal coordinates (JUMNA). The free and complexed operator adopt both a right-handed B helical conformation, but a more detailed analysis of the conformational parameters using the Curves program shows striking differences in the groove geometries, the rises, the twists and the total bending.
Assuntos
Óperon Lac , Fatores de Lactose/química , Proteínas Repressoras/química , Sequência de Bases , Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência MolecularRESUMO
A new and simple model enabling a chemical species to be brought to a preselected site in single strand DNA is reported. Two oligonucleotides containing a propanediol linkage were hybridized to their complementary sequences with an extra-base opposite the propanediol derivative. Absorption studies results shown that the addition of a bisacridine derivative strongly increased the stabilities of both duplexes when added in a 1:1 ratio. NMR studies on one of these duplexes brought evidence of the intercalation of the bisacridine at the position involving the propanediol linkage. These results suggest that this system could be used to target a specific reaction at a preselected position using the bisacridine derivative as carrier for the reactive species.
Assuntos
DNA de Cadeia Simples/química , Oligodesoxirribonucleotídeos/química , Acridinas , Sequência de Bases , Substâncias Intercalantes , Espectroscopia de Ressonância Magnética/métodos , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Desnaturação de Ácido NucleicoRESUMO
15N uniformly labeled lac repressor and lac repressor headpiece were prepared. 15N NMR spectra of lac repressor were shown resolution inadequate for detailed study while the data showed that the 15N labeled N-terminal part of the protein is quite suitable for this type of study allowing future investigation of the specific interaction of the lac repressor headpiece with the lac operator. We report here the total assignment of proton 1H and nitrogen 15NH backbone resonances of this headpiece in the free state. Assignments of the 15N resonances of the protein were obtained in a sequential manner using heteronuclear multiple quantum coherence (HMQC), relayed HMQC nuclear Overhauser and relayed HMQC-HOHAHA spectroscopy. More than 80 per cent of residues were assigned by their 15NH(i)-N1H(i + 1) and 15NH(i)-N1H(i - 1) connectivities. Values of the 3JNH alpha splitting for 39 of the 51 residues of the headpiece were extracted from HMQC and HMQC-J. The observed 15NH(i)-C beta H cross peaks and the 3JNH alpha coupling constants values are in agreement with the three alpha-helices previously described [Zuiderweg, E.R.P., Scheek, R.M., Boelens, R., van Gunsteren, W.F. and Kaptein, R., Biochimie 67, 707 (1985)]. The 3JNH alpha coupling constants can be now used for a more confident determination of the lac repressor headpiece. From these values it is shown that the geometry of the ends of the second and third alpha-helices exhibit deviation from the canonical alpha-helix structure. On the basis of NOEs and 3JNH alpha values, the geometry of the turn of the helix-turn-helix motif is discussed.
Assuntos
Proteínas Repressoras/química , Sequência de Aminoácidos , Espectroscopia de Ressonância Magnética , Dados de Sequência Molecular , Conformação ProteicaRESUMO
The three-dimensional structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAACTC) covalently linked to an acridine derivative (m5Acr) with its natural and parallel complementary sequence beta-d(AGATTTGAG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. All the nonexchangeable and exchangeable resonances were assigned in this duplex. The structure was refined by using interproton distances determined by NOE measurements. The NOE values were converted into distances by using the complete 190 x 190 relaxation matrix. The unnatural duplex Acrm5-alpha-d(TCTAAACTC)-beta-d(AGATTTGAG) forms a parallel right-handed helix with Watson-Crick base pairing; the alpha and beta deoxyriboses adopt a 3'-exo conformation. The acridine moiety was found stacked up the C9-G9 base pair. The structure of the first seven base pairs of this duplex was found similar to that of the duplex alpha-d(TCTAAAC)-beta-d(AGATTTG), which we had already investigated [Lancelot, G., et al. (1989) Biochemistry 28, 7871-7878]. Since these structures were generated by using experimental NOE values obtained independently on macromolecules whose global correlation time was different (3.8 and 2.2 ns), we conclude that this comparison is a good test of the viability of our method to generate three-dimensional structures of oligonucleotides in solution. Starting from different initial conformations, we show that the NOE constraints allow one to reach the same final restrained conformation, taking into account implicitly the solvent effect.
Assuntos
Oligodesoxirribonucleotídeos , Aminoacridinas , Sequência de Bases , Corantes Fluorescentes , Espectroscopia de Ressonância Magnética , Modelos Moleculares , Dados de Sequência Molecular , Conformação de Ácido NucleicoRESUMO
The solution structure of the duplex formed by the association of the unnatural oligonucleotide alpha-d(TCTAAAC) with its natural and parallel complementary sequence beta-d(AGATTTG) was investigated by nuclear magnetic resonance spectroscopy and constrained molecular mechanics calculations. The structure was refined on the basis of interproton distances determined by NOE measurements for a series of mixing times. The NOE values were converted to distances by using the complete 134 x 134 relaxation matrix including all proton dipole-dipole interactions and spin diffusion. The computation of the relaxation matrix requires the Cartesian coordinates of the oligonucleotide, which are not known, a priori. To avoid this ambiguity, we used an iterative procedure in which the new distance constraints are obtained by using the complete relaxation matrix calculated from the previous structure. After three iterations, the process converged. The unnatural duplex alpha-d(TCTAAAC)-beta-d(AGATTTG) adopts in solution a right-helical structure with Watson-Crick base pairing, an anti conformation on the glycosyl linkage on the beta-strand, a syn conformation on the alpha-strand, and a 3'-exo conformation of the deoxyriboses for both sugar anomers. The three-dimensional structure obtained allowed us to describe the local heterogeneity of the duplex.
Assuntos
Espectroscopia de Ressonância Magnética/métodos , Matemática , Conformação de Ácido Nucleico , Oligonucleotídeos , Sequência de Bases , DNA , Modelos MolecularesRESUMO
Using proton magnetic resonance, we have investigated the structure and the base-pair opening kinetics of the d-(AATTGCAATT) self-complementary duplex. All the non-exchangeable (except H5',5") and most exchangeable proton resonances have been assigned. The structure belongs to the B family. Imino proton exchange, measured by line broadening, longitudinal relaxation and magnetization transfer from water, is catalyzed by proton acceptors. The base-pair lifetimes, obtained by extrapolation of the exchange times to infinite concentration of ammonia are 2 and 3 milliseconds for internal A.Ts and 18 ms for G.C at 15 degrees C. In the absence of added catalysts, the imino proton of the first A.T base pair exchanges faster than that of the unpaired thymidine of the duplex formed by the sequence d-(AATTGCAATTT). This gives strong evidence for intrinsic exchange catalysis. The exchange of adenine amino protons from the closed state has been observed. Hence amino proton exchange is ill-suited for the investigation of base-pair opening kinetics.
Assuntos
Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , Aminas , Catálise , Deutério , Ligação de Hidrogênio , Iminas , Cinética , Espectroscopia de Ressonância Magnética , PrótonsRESUMO
The oligodeoxynucleotide d(TTCTGT) was covalently attached to the 9-amino group of 2-methoxy-6-chloro-9-aminoacridine (Acr) through its 3'-phOsphate via a pentamethylene linker (m5). In order to avoid its hydrolysis by nucleases inside the cel., one of its phosphates (TpT) was substituTed with a neopentyl group. Complex formation between each of the two purified isomers and the complementary strand d(GCACAGAA) was investigated by nuclear magnetic resonance. The COSY and NOESY connectivities allowed us to assign all the proton resonances of the bases, the sugars (except the overlapping 5'-5'' resonances), the acridine, and the pentamethylene chain. Structural information derived from the relative intensity of COSY and NOESY maps revealed that the duplex d(T*TCTGT).d(GCACAGAA) adopts a B-type conformation and that the deoxyriboses preferentially adopt a 2'-endo conformation. The NOE connectivities observed between the protons of the bases or the sugars and the protons of the dye show the intercalation of the acridine between the base pairs. NOE connectivities as well as imino proton resonances show that, at room temperature, the C7 base and the G8 base belonging to two different duplexes are paired. The pseudoaxial and pseudoequatorial isomers were assigned, and the differences in stability of their complex with the complementary strand are discussed.
Assuntos
Substâncias Intercalantes , Conformação de Ácido Nucleico , Oligodesoxirribonucleotídeos , Composição de Bases , Espectroscopia de Ressonância Magnética/métodos , Modelos MolecularesRESUMO
The binding of oligopeptides Lys-Trp-Gly-Lys OtBu, Lys-Gly-Trp-Lys OtBu and Lys-Trp-Lys to Polyadenylic, Polycytidylic and Polyuridylic acid has been studied by Proton NMR at 90 MHz and 500 MHz at oligopeptide/Polynucleotide ratios ranging from 0.01 to 0.20 at 275-365 K. Downfield shift of 0.01-0.2 ppm at 296 K of the H2, H8 and H1' resonances of Poly A due to binding with oligopeptides is accompanied by a marked narrowing of resonance lines of Poly A. The ring protons of tryptophan shift upfield by 0.3-0.6 ppm at 296 K on binding to Poly A. Changes in chemical shift of both adenine and tryptophan protons on binding are much smaller at 355 K than that at 275 K. These observations are ascribed to intercalation of the tryptophan ring in the adenine bases resulting in partial destacking of adenine bases in Poly A. Using the magnetic anisotropy ring current shifts, an overlap geometry of tryptophan ring in the adenine has been proposed. Addition of oligopeptides to Poly C and Poly U, on the other hand, suggests that tryptophan ring does not stack in Poly U and Poly C.
Assuntos
Oligopeptídeos , Poli A , Poli C , Poli U , Polirribonucleotídeos , Triptofano , Espectroscopia de Ressonância Magnética/métodos , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
The unnatural oligonucleotide alpha-d(TCTAAAC) was synthesized and was found more resistant towards endonucleases than its beta-analog. 2D-NMR experiments allowed the assignment of all non-exchangeable aromatic and sugar protons except for the overlapping 5' -5" resonances, as well as the exchangeable imino protons of the parallel hybrid duplex alpha-d (TCTAAAC)-beta-d(AGATTTG). NMR studies show that the strength of the association between the alpha-strand and the beta parallel strand is equivalent to that between their anti-parallel complementary beta-analogs beta-d(CAAATCT) and beta-d(AGATTTG). NOE data provide evidence that both duplexes form stable right-helical duplexes with an anti-conformation on the glycosyl linkages and a Watson-Crick pairing. NOESY and COSY spectra allowed us to determine that alpha and beta deoxyriboses adopt a 3' -exo conformation.