Dynamic NMR structures of [Rp]- and [Sp]-phosphorothioated DNA-RNA hybrids: is flexibility required for RNase H recognition?
Chemically modified DNA oligonucleotides have been crucial to the development of antisense therapeutics. High-resolution structural studies of pharmaceutically relevant derivatives have been limited to only a few molecules. We have used NMR to elucidate the structure in solution of two DNA-RNA hybrids with the sequence d(CCTATAATCC).r(GGAUUAUAGG). The two hybrids contain an unmodified RNA target strand, whereas the DNA strand contains one of two different stereoregular sugar-phosphate backbone linkages at each nucleotide: 1), [Rp]-phosphorothioate or 2), [Sp]-phosphorothioate. Homonuclear two-dimensional spectroscopy afforded nearly complete nonlabile proton assignments. Distance bounds, calculated from the nuclear Overhauser effect (NOE) crosspeak intensities via a complete relaxation matrix approach with the program MARDIGRAS, were used to restrain the structure of the two hybrids during simulations of molecular dynamics. Analysis of restrained molecular dynamics trajectories suggests that both hybrids are flexible, requiring the use of molecular dynamics with time-averaged restraints (MDtar) to generate ensembles of structures capable of satisfying the NMR data. In particular, the deoxyribose sugars of the DNA strand show strong evidence of repuckering. Furthermore, deoxyribose sugar repuckering is accompanied by increased flexibility of overall helical geometry. These observations, together with the analysis of the crystal structure of a hybrid duplex in complex with ribonuclease H (RNase H), suggested that this flexibility may be required for recognition by RNase H.
D001483:Base Sequence; D001665:Binding Sites; D003198:Computer Simulation; D004247:DNA / Q000737:chemistry*; D004789:Enzyme Activation; D046911:Macromolecular Substances; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009038:Motion; D009690:Nucleic Acid Conformation; D009693:Nucleic Acid Hybridization / Q000379:methods*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D016376:Oligonucleotides, Antisense / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry; D012313:RNA / Q000737:chemistry*; D016914:Ribonuclease H / Q000737:chemistry*; D012996:Solutions; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013873:Thionucleotides / Q000737:chemistry*
D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D009943:Organophosphorus Compounds; D012996:Solutions; D013873:Thionucleotides; D012313:RNA; D004247:DNA; D016914:Ribonuclease H