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 endonucleasesElectronic supplementary information (ESI) available: RNA cleavage kinetics. See http://www.rsc.org/suppdata/ob/b2/b210163g/

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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 (Tm) 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 Tm 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 (kcat) close to the native. However, the RNase H binding affinity (1/Km) showed a slight decrease with increase in the number of modifications, which results in less effective enzyme activity (kcat/Km) for modified AON–RNA duplexes. All oxetane modified AON–RNA hybrids showed a correlation of Tm with the 1/Km, Vmax, or Vmax/Km. 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.
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