Kinetic analysis of the RNA cleavage of the conformationally-constrained oxetane-modified antisense-RNA hybrid duplex by RNase H

Abstract

The kinetics of the complementary 15mer RNA (3) cleavage by RNase H in the conformationally-constrained [North-East type sugar constraint] triple oxetane modified antisense oligonucleotide AON (2)–RNA (3) hybrid duplexes (P. I. Pradeepkumar and J. Chattopadhyaya, J. Chem. Soc., Perkin Trans. 2, 2001, 2074) have been investigated, in comparison with the native 15mer counterpart AON (1)–RNA (3), by changing both AON and RNA concentrations, while keeping the enzyme and buffer concentrations constant. The RNA concentration-dependent kinetics of the RNase H promoted cleavage reaction gave values for K_m and V_max for both substrates: AON (1)–RNA (3) and AON (2)–RNA (3) heteroduplexes. The V_max and the K_m values were respectively ∼2 and ∼10 times greater for the AON (2)–RNA (3) duplex than those for the native counterpart, which means that the incorporation of the North-East type sugar constrained triple oxetane modifications in the AON increases the catalytic activity of RNase H by almost ca. 2-fold owing to the decreased affinity of the substrate toward the enzyme. The T_m for the AON (2)–RNA (3) hybrid duplex was 18 °C less than that of the native. Thus, an inverse correlation between AON–RNA hybrid duplex thermostability and RNase H activity has been found under the high substrate concentration conditions. Under high substrate concentration conditions, the RNase H activity of the AON (2)–RNA (3) hybrid duplex is however higher than the native because of a more rapid turn over (N_max) of RNase H. Conversely, under low substrate concentration conditions, the RNase H activity of AON (2)–RNA (3) hybrid duplex dramatically drops compared to the native because of the less effective turn over (N_eff) of enzyme. This is because the N_eff, under low substrate concentration conditions, depends on maximal turn over, N_max, as well as on the extent of saturation of enzyme by substrate, which in turn depends upon the value of the V_max/K_m ratio, which for the AON (2)–RNA (3) hybrid duplex has been found to be ∼4-fold less than the native.