This Article Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Tonelli, M. Articles by James, T. L. Search for Related Content PubMed PubMed Citation Articles by Tonelli, M. Articles by James, T. L.

Dynamic NMR Structures of [Rp]- and [Sp]-Phosphorothioated DNA-RNA Hybrids: Is Flexibility Required for RNase H Recognition?

Marco Tonelli ^*, Nikolai B. Ulyanov ^*, Todd M. Billeci ^*, Boleslaw Karwowski , Piotr Guga , Wojciech J. Stec  and Thomas L. James ^*

^* Department of Pharmaceutical Chemistry, University of California, San Francisco, San Francisco, California 94143-2280 USA; and Department of Bioorganic Chemistry, Centre of Molecular and Macromolecular Studies, Polish Academy of Sciences, 90-363 Lód'z, Sienkiewicza 112, Poland

Correspondence: Address reprint requests to T. L. James, Dept. of Pharmaceutical Chemistry, University of California, San Francisco, 600 16^th St., San Francisco, CA 94143-2280. Tel.: 415-476-1916; Fax: 415-502-8298; E-mail: james{at}picasso.ucsf.edu.

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.