The RNase H affinity and cleavage of the target RNA in the antisense–RNA hybrid duplexes containing various 3 -tethered substituents in the antisense strand

The kinetics of RNase H promoted cleavage of the RNA strand in the antisense oligonucleotide (AON)–RNA hybrid duplexes, with the 3 -end of the AON strand tethered with cholic acid (2), its triacetate (3), cholesterol (4) or dipyridophenazine (5), have been investigated by changing the concentration of both the AON and the RNA strands, while keeping the enzyme and the buffer concentration constant. It has been shown that the extent of the cleavage of the target RNA by RNase H in the conjugated AON (2–5)–RNA (6) hybrid duplexes, at saturation conditions for RNA in the presence of an excess of AON, is higher than in the native 9mer AON (1)–RNA (6) duplex. The RNA concentration-dependent kinetics of the RNase H promoted cleavage reaction gave values for Km and Vmax for RNA (6)–AON (1–5) duplexes. The Vmax values for all 3 -tethered AON–RNA duplexes were ∼30% less efficient, and their Km were also 4–14 times less, than the native counterpart, which means that the 3 -tethered substituent decreases the catalytic activity of RNase H owing to the increased affinity toward the enzyme. Since the values of Vmax and Km change in a compensating manner at a fixed enzyme concentration, the Vmax : Km ratio for all 3 -tethered AONs in the corresponding AON–RNA duplexes showed the unique nature of the 3 -tethered substituent, dictating the 3 -substituent-dependent enzyme affinity of the heteroduplex, in comparison with the native. Accordingly, 3 -tethered Dppz AON (5)–RNA duplex has a maximum affinity for RNase H, ∼6-fold more compared to the native 9mer (1), and ∼2-fold more compared to 3 -tethered cholic acid (2), its triacetate (3) or cholesterol (4) containing AON–RNA duplexes.