STRUCTURE AND DYNAMICS OF A DNA-CENTER-DOT-RNA HYBRID DUPLEX WITH A CHIRAL PHOSPHOROTHIOATE MOIETY - NMR AND MOLECULAR-DYNAMICS WITH CONVENTIONAL AND TIME-AVERAGED RESTRAINTS

The three-dimensional structure of two thiophosphate-modified DNA . RN A hybrid duplexes d(GCTATAA(ps)TGG). r(CCAUUAUAGC), one with R-thiopho sphate chirality and one with S-thiophosphate chirality, have been det ermined by restrained molecular dynamics simulations (rMD). As the two yielded almost identical results, a description of results can be pre sented in the singular. The conformational flexibility of this hybrid has been investigated by employing time-averaged constraints during th e molecular dynamics simulations (MD-tar). A set of structural restrai nts, comprising 322 precise interproton distance constraints obtained by a complete relaxation matrix analysis of the 2D NOE intensities as well as J coupling constants obtained from quantitative simulations of DQF-COSY cross-peaks in deoxyriboses, was reported in our previous pa per [Gonzalez, C., Stec, W., Kobylanska, A., Hogrefe, R. I., Reynolds, M., and James, T. L. (1994) Biochemistry 33, 11062-11072]. Multiple c onformations of the deoxyribose moieties were evident from the scalar coupling constant analysis. Accurate distance constraints, obtained fr om complete relaxation matrix analysis, yielded a time-averaged soluti on structure via conventional restrained molecular dynamics which is n ot compatible with the experimental J coupling constants (root-mean-sq uare deviation in J value similar to 2 Hz). However, vicinal coupling constant information can be reproduced when time-averaged constraints are used during the molecular dynamics calculations instead of the con ventional restraints (J(rms) similar to 0.6 Hz). MD-tar simulations al so improve the NMR R factors. This improvement is more evident in the DNA than in the RNA strand, where no indication of conformational flex ibility had been obtained. Analysis of the MD-tar trajectories confirm s that deoxyriboses undergo pucker transitions between the S and N dom ain, with the major conformer in the S domain. The ribose moieties in the RNA strand, however, remain in the N domain during the entire simu lation. Conformations of deoxyriboses in the intermediate domain near O4'-endo are obtained when the average structure is calculated with co nventional NMR restraints. Since these conformations cannot account fo r the experimental J coupling information, and they only appear in a v ery low population in the MD-tar ensemble, we conclude that intermedia te E sugar puckers are artifacts produced by the attempt to fit all th e structural constraints simultaneously when in reality more than one conformer is present. Most structural features of the duplex remain th e same in the average structure and in the MD-tar ensemble, e.g., the minor groove width, exhibiting an intermediate value compared with tho se of canonical A- and B-like structures. Calculations with the two du plexes of differing chirality in the modified phosphate have been carr ied out. Only minor effects in the backbone close to the thiophosphate have been detected.