BINDING-AFFINITY AND SPECIFICITY OF ESCHERICHIA-COLI RNASE H1 - IMPACT ON THE KINETICS OF CATALYSIS OF ANTISENSE OLIGONUCLEOTIDE-RNA HYBRIDS

In this study we report for the first time the binding affinity of RNa se H1 for oligonucleotide duplexes. We used a previously described 17- mer antisense sequence [Monia, B. P., Johnston, J. F., Ecker, D. J., Z ounes, M. A., Lima, W. F., & Freier, S. M. (1992) J. Biol. Chem. 267, 19954-19962] hybridized to a complementary oligoribonucleotide to eval uate both the binding affinity and the catalytic rate of RNase H1. The dissociation constants (K-d) of RNase H1 for the various substrates t ested were determined by inhibition analysis using chemically modified noncleavable oligonucleotide heteroduplexes. Catalytic rates were det ermined using heteroduplex substrates containing chimeric antisense ol igonucleotides composed of a five-base deoxynucleotide sequence flanke d on either side by chemically modified nucleotides. We find that the enzyme preferentially binds A-form duplexes: RNase PI bound A-form dup lexes (RNA:RNA and DNA:RNA) approximately 60-fold tighter than B-form duplexes (DNA:DNA) and approximately 300-fold tighter than single-stra nd oligonucleotides. The enzyme exhibited equal affinity for both the wild type (RNA:DNA) oligonucleotide substrate and heteroduplexes conta ining various 2'-sugar modifications, while the cleavage rates for the se chemically modified substrates were without exception slower than f or the wild type substrate. The introduction of a single positively ch arged 2'-propoxyamine modification into the chimeric antisense oligonu cleotide portion of the heteroduplex substrate resulted in both decrea sed binding affinity and a slower rate of catalysis by RNase H. The cl eavage rates for heteroduplexes containing single-base mismatch sequen ces within the chimeric oligonucleotide portion varied depending on th e position of the mismatch but had no effect on the binding affinity o f the enzyme. These results offer further insights into the physical b inding properties of the RNase PI-substrate interaction as well as the design of effective antisense oligonucleotides.