STRUCTURAL STUDY OF A DNA-CENTER-DOT-RNA HYBRID DUPLEX WITH A CHIRAL PHOSPHOROTHIOATE MOIETY BY NMR - EXTRACTION OF DISTANCE AND TORSION ANGLE CONSTRAINTS AND IMINO PROTON-EXCHANGE RATES

The solution structure of the thiophosphate-modified DNA.RNA hybrid du plex d(GCTATAA(ps)-TGG).r(CCAUUAUAGC) has been studied by NMR. Two sam ples with pure stereochemistry in the modified phosphate have been inv estigated. Two-dimensional NMR (2D NMR) methods have been applied to a ssign nearly all the resonances in both duplexes. Scalar coupling cons tants have been determined by comparing quantitative simulations with experimental double-quantum filtered COSY (DQF-COSY) cross-peaks. More than 300 distance constraints have been obtained from two-dimensional nuclear Overhauser spectroscopy (2D NOE) spectra recorded in D2O and H2O by using a complete relaxation matrix analysis as implemented in t he program MARDIGRAS. This hybrid duplex presents a heteronomous struc ture. Riboses in the RNA strand are found in a N-type conformation typ ical of the A-form family as shown by the lack of H1'-H2' cross-peaks in DQF-COSY spectra and confirmed by the measured interproton distance s. In contrast, the DNA strand adopts a different conformation with su gar puckers partially in the S-type domain, which is not in agreement with the A-family of structures. Coupling constants in deoxyriboses ar e not consistent with any single sugar conformation. Therefore, sugar pucker pseudorotation parameters are calculated according to a two-sta te dynamic equilibrium between N- and S-type conformers. In general, t he population of major S conformer is lower than in double-stranded DN A duplexes, indicating that hybrid duplexes may be more flexible than pure DNA or RNA. The only differences observed in the spectra between the two stereoisomers studied originate from resonances of protons loc ated near the modified phosphate. No significant differences in interp roton distance have been detected, and only a slight difference of sug ar pucker in the 5' neighbor has been found. The sulfur atom appears t o be well-accommodated without further changes in the structure of the hybrid.