THE INFLUENCE OF ANTISENSE OLIGONUCLEOTIDE-INDUCED RNA STRUCTURE ON ESCHERICHIA-COLI RNASE H1 ACTIVITY

The ability of Escherichia coli RNase H1 to hydrolyze structured subst rates containing antisense oligonucleotides preannealed to a 47 mer RN A was compared with its ability to hydrolyze unstructured substrates c ontaining antisense oligonucleotides duplexed with 13-mer RNA. These r esults demonstrate that when antisense oligonucleotides were bound to structured RNA, the resultant duplexes were cleaved at rates significa ntly slower than when the same oligonucleotides were bound to unstruct ured oligoribonucleotides. Structured substrates exhibited fewer cleav age sites, and each cleavage site was cleaved less rapidly than in uns tructured substrates. Furthermore, the enzymatic activity of E. coli R Nase H1 for the structured substrates was most affected when the cleav age sites corresponding to the enzymatically most active sites on the unstructured substrates were blocked in the structured substrates. Mol ecular modeling suggests that the observed ablation of RNase H activit y was due to the steric hindrance of the enzyme by the structured RNA, i.e. steric interference of the phosphate groups on the substrate and /or the binding site of the enzyme. When chimeric oligonucleotides com posed of a five-base deoxynucleotide sequence flanked by chemically mo dified nucleotides were bound to structured RNA, the resultant duplexe s were even worse substrates for RNase H. These results offer further insights into the role of antisense-induced RNA structure on RNase H a ctivity and may facilitate the design of effective antisense oligonucl eotides.