Antisense oligonuclotides with oxetane-constrained cytidine enhance heteroduplex stability, and elicit satisfactory RNase H response as well as showing improved resistance to both exo and endonucleases

Abstract

Antisense oligonucleotides (AONs) with single and double oxetane modifications [1′,2′-oxetane constrained cytidine, 1-(1′,3′-O-anhydro-β-D-psicofuranosyl)cytosine] have been evaluated, in comparison with the corresponding -modified AONs, for their antisense potentials by targeting to a 15mer complementary RNA. Although the modified mixmer AONs show ∼3 °C drop per modification in melting temperature (T_m) of their hybrid AON–RNA duplexes, they are found to be good substrates for RNase H, in comparison with the native AON–RNA duplex. An AON with double modifications along with 3′-DPPZ (dipyridophenazine) conjugation shows the T_m of the hybrid duplexes as high as that of the native, and the RNase H activity as good as its unconjugated counterpart. A detailed Michaelis–Menten kinetic analysis of RNase H cleavage showed that the single and double modified AON–RNA duplexes as well as double modifications along with 3′-DPPZ have catalytic activities (k_cat) close to the native. However, the RNase H binding affinity (1/K_m) showed a slight decrease with increase in the number of modifications, which results in less effective enzyme activity (k_cat/K_m) for modified AON–RNA duplexes. All oxetane modified AON–RNA hybrids showed a correlation of T_m with the 1/K_m, V_max, or V_max/K_m. The modified AONs (with 3′-DPPZ), as in the counterpart, showed an enhanced tolerance towards the endonuclease and exonuclease degradation compared to the native (the oxetane-sugar and the DPPZ based AONs are non-toxic to K562 cell growth, ref. ). Thus a balance has been found between exo and endonuclease stability vis-a-vis thermostability of the heteroduplex and the RNase H recruitment capability and cleavage with the oxetane-constrained cytidine incorporated AONs as potential antisense candidates with a fully phosphate backbone for further biological assessment.