Structural effect of complete [Rp]-phosphorothioate and phosphorodithioatesubstitutions in the DNA strand of a model antisense inhibitor-target RNA complex

Chemically modified DNA oligonucleotides have been crucial to the success o f antisense therapeutics. Although such modifications are ubiquitous in the clinic, high-resolution structural studies of pharmaceutically relevant de rivatives have been limited to only a few molecules. We have completed a hi gh-resolution NMR structural study Of three DNA RNA hybrids with the sequen ce d(CCTATAATCC) .r(GGAUUAUAGG). All hybrids contain an unmodified RNA stra nd, whereas the DNA strand of each hybrid contains one of three different s ugar-phosphate backbone linkages at each nucleotide: (1) phosphate, (2) [Rp ]-phosphorothioate, or (3) phosphorodithioate. The UV and NMR melting profi les revealed that the normal hybrid is more stable than the [Rp]-phosphorot hioate, which in turn is more stable than the phosphorodithioate. Homonucle ar two-dimensional nuclear Overhauser effect spectroscopy and double quantu m-filtered correlation spectroscopy afforded nearly complete non-labile pro ton assignments. The three molecules show nearly equivalent chemical shifts , with the exception of H3' protons, which are shifted downfield in a manne r that appears correlated with the degree of sulfur substitution at phospha te. All three hybrids exhibit unusually broad linewidths for deoxyribose pr otons H2' and H2 ". Distance restraints were calculated from NOE cross-peak intensities via a c omplete relaxation matrix approach using the program RANDMARDI. Detailed co mparison 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 D NA H2'-base and H3'-base distances, revealed a relative increase in the pop ulation of B-type sugar conformations for deoxyriboses in the A + T-rich ce nter of the hybrid sequence. It is widely known that the activity of enzyme s which act upon DNA.RNA hybrid substrates (e.g. ribonuclease H) is impacte d when the hybrids contain phosphorothioate or phosphorodithioate substitut ions. The structural similarity of the three hybrids examined here suggests that factors other than global structure may mediate the activity of these enzymes. (C) 1999 Academic Press.