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|Title:||Structural effect of complete [Rp]-Phosphorothioate and substitutions in the DNA strand of a model antisense inhibitor-target RNA complex||Authors:||P., Furrer
T. L., James
|Keywords:||antisense; RNA·DNA hybrid structure; phosphorodithioate; RNase H||Issue Date:||1999||Project:||None||Journal:||JOURNAL OF MOLECULAR BIOLOGY||Abstract:||
Chemically modified DNA oligonucleotides have been crucial to the success of antisense therapeutics. Although such modifications are ubiquitous in the clinic, high-resolution structural studies of pharmaceutically relevant derivatives have been limited to only a few molecules. We have completed a high-resolution NMR structural study of three DNA·RNA hybrids with the sequence d(CCTATAATCC)·r(GGAUUAUAGG). All hybrids contain an unmodified RNAstrand, whereas the DNAstrand of each hybrid contains one of three different sugar-phosphate backbone linkages at each nucleotide: (1) phosphate, (2) [Rp]-phosphorothioate, or (3) phosphorodithioate. The UV and NMR melting profiles revealed that the normal hybrid is more stable than the [Rp]-phosphorothioate, which in turn is more stable than the phosphorodithioate. Homonuclear two-dimensional nuclear Overhauser effect spectroscopy and double quantum-filtered correlation spectroscopy afforded nearly complete non-labile proton assignments. The three molecules show nearly equivalent chemical shifts, with the exception of H3′ protons, which are shifted downfield in a manner that appears correlated with the degree of sulfur substitution at phosphate. All three hybrids exhibit unusually broad linewidths for deoxyribose protons H2′ and H2″.Distance restraints were calculated from NOE cross-peak intensities viaacomplete relaxation matrix approach using the program RANDMARDI. Detailed comparison of interproton distances from each hybrid indicates that the three molecules share a common structure, with neither strand in canonical A or B form. Correlation of R factors, calculated using the program CORMA with DNA H2′-base and H3′-base distances, revealed a relative increase in the population of B-type sugar conformations for deoxyriboses in the A+T-rich center of the hybrid sequence. It is widely known that the activity of enzymes which act upon DNA·RNA hybrid substrates (e.g. ribonuclease H) is impacted when the hybrids contain phosphorothioate or phosphorodithioate substitutions. The structural similarity of the three hybrids examined here suggests that factors other than global structure may mediate the activity of these enzymes.
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