Extending the cleavage rules for the hammerhead ribozyme: Mutating adenosine15.1 to inosine15.1 changes the cleavage site specificity from N16.2U16.1H17 to N16.2C16.1H17

Abstract

In this paper, we show that an adenosine to inosine mutation at position 15.1 changes the substrate specificity of the hammerhead ribozyme from N 16.2 U 16.1 H 17 to N 16.2 C 16.1 H 17 (H represents A, C or U). This result extends the hammerhead cleavage triplet definition from N 16.2 U 16.1 H 17 to the more general N 16.2 Y 16.1 H 17. Comparison of cleavage rates using I 15.1 ribozymes for NCH triplets and standard A 15.1 ribozymes for NUH triplets under single turnover conditions shows similar or slightly enhanced levels of reactivity for the I 15.1-containing structures. The effect of I 15.1 substitution was also tested in nuclease-resistant 2′-O-alkyl substituted derivatives (oligozymes), showing a similar level of activity for the NUH and NCH cleaving structures. The availability of NCH triplets that can be targeted without loss of efficiency increases the flexibility of ribozyme targeting strategies. This was demonstrated by an efficient cleavage of an HCV transcript at a previously inaccessible GCA site in codon 2.