Received for publication April 11, 2006.
Revised October 4, 2006.
Accepted for publication October 4, 2006.

Human RNase H1 discriminates between subtle variations in the structure of the heteroduplex substrate

Abstract

In a previous study we demonstrated that the sugar conformation and helical geometry of the heteroduplex substrate at the catalytic site of human RNase H1 directs the selective recognition of the substrate by the enzyme (Lima, W. F., Nichols, J. G., Wu, H-J., Prakash, T. P., Migawa, M. T., Wyrzykiewicz, T. K., Bhat, B., Crooke, S. T. (2004) J. Biol. Chem. 279, 36317-36326). Here, we systematically introduced 2'-methoxyethoxy (MOE) nucleotides into the antisense oligodeoxyribonucleotide (ASO) of the heteroduplex to alter the helical geometry of the substrate. The MOE substitutions at the 3' and 5'-poles of the ASO resulted in fewer cleavage sites and slower cleavage rates compared to the unmodified substrates. Further, a greater reduction in cleavage activity was observed for MOE substitutions at the 5'-pole of the ASO. The 3'-most and 5'-most cleavage sites were positioned, respectively, 2 and 4-5 base-pairs from the nearest MOE residues suggesting a conformational transmission of the MOE/RNA helical geometry into the DNA/RNA portion of the heteroduplex. Similar conformational transmission was observed for Okazaki-like substrates containing deoxyribonucleotide substitutions at the 3'-pole of the oligoribonucleotide. Finally, the heteroduplex substrates exhibited preferred cleavage sites, which were cleaved 2 to 3-fold faster than other sites in the substrate and these sites exhibited the greatest influence on the initial cleavage rates. The data presented here offer further insights into the role substrate structure plays in directing human RNase H1 activity as well as the design of effective ASOs.

Key words: Enzymology, Regulation - post-transcriptional, Antisense

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