Insertion of a methylene group into the backbone of an antisense oligonucleotide reveals the importance of deoxyribose recognition by RNase H.

RNase H acts as a key effector in gene knockdown by antisense oligonucleotides (ASOs). Although various chemical modifications have been developed to regulate RNase H-mediated cleavage, precise control is yet to be achieved. In this study, we tried to address the question of whether the interaction of phosphate groups or deoxyriboses is more important in the recognition of DNA/RNA duplex by RNase H. To answer this question, we investigated the effect of methylene group insertion at the 5'-upstream or 3'-downstream phosphorothioate groups on RNase H-mediated cleavage. By inserting a methylene group at the 5'-upside or 3'-downside, the distance between phosphates or deoxyriboses could be changed in a different pattern. Maximum suppression of the cleavage reaction was observed when a methylene group was inserted at the 5'-phosphate group of the nucleoside which is known to distinguish ribose and deoxyribose via stacking of the W221 residue in RNase H. This effect was observed in a different sequence as well as mismatched duplexes, suggesting the interaction of deoxyribose rings with RNase H is more important than that of phosphate groups. Our results will contribute to the designing of further molecular modifications that improve the selectivity of RNase H-mediated cleavage reactions which allows for the development of allele-specific ASOs.