No abstract

The solution structure of the thiophosphate-modified DNA.RNA hybrid duplex d(GCTATAApsTGG).r(CCAUUAUAGC) has been studied by NMR. Two samples with pure stereochemistry in the modified phosphate have been investigated. Two-dimensional NMR (2D NMR) methods have been applied to assign nearly all the resonances in both duplexes. Scalar coupling constants 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 the program MARDIGRAS. This hybrid duplex presents a heteronomous structure. Riboses in the RNA strand are found in a N-type conformation typical 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 distances. In contrast, the DNA strand adopts a different conformation with sugar puckers partially in the S-type domain, which is not in agreement with the A-family of structures. Coupling constants in deoxyriboses are not consistent with any single sugar conformation. Therefore, sugar pucker pseudorotation parameters are calculated according to a two-state dynamic equilibrium between N- and S-type conformers. In general, the population of major S conformer is lower than in double-stranded DNA 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 located near the modified phosphate. No significant differences in interproton distance have been detected, and only a slight difference of sugar pucker in the 5' neighbor has been found. The sulfur atom appears to be well-accommodated without further changes in the structure of the hybrid.

Proteins that bind to specific sites on DNA often do so in order to carry out catalysis or specific protein-protein interaction while bound to the recognition site. Functional specificity is enhanced if this second function is coupled to correct DNA site recognition. To analyze the structural and energetic basis of coupling between recognition and catalysis in EcoRI endonuclease, we have studied stereospecific phosphorothioate (PS) or methylphosphonate (PMe) substitutions at the scissile phosphate GpAATTC or at the adjacent phosphate GApATTC in combination with molecular-dynamics simulations of the catalytic center with bound Mg2+. The results show the roles in catalysis of individual phosphoryl oxygens and of DNA distortion and suggest that a "crosstalk ring" in the complex couples recognition to catalysis and couples the two catalytic sites to each other.

The stability of stereoregular oligo(nucleoside phosphorothioate)s (PS-oligos) in human plasma has been studied. 3'-Exonuclease present in human plasma appeared to be RP specific, that is, it cleaves internucleotide phosphorothioate linkages of [RP]-configuration and not those of [SP]-configuration. Therefore, PS-oligos containing all phosphorothioate internucleotide linkages of [RP]-configuration [RP-PS-oligos]) are more effectively degraded by the enzyme than PS-oligos prepared via nonstereo-controlled methods (so-called random mixture of diastereomers [Mix-PS-oligos]), whereas oligo(nucleoside phosphorothioate)s of [S(P)]-configuration remain intact. The enzyme activity depends on the sequence of nucleobases. The presence of deoxycytidine units (three or more residues) at the 3'-end of PS-oligo substrate significantly inhibits the enzyme activity.