EXTENDING THE CLEAVAGE RULES FOR THE HAMMERHEAD RIBOZYME - MUTATING ADENOSINE(15.1) TO INOSINE(15.1) CHANGES THE CLEAVAGE SITE-SPECIFICITY FROM (NUH17)-U-16.2-H-16.1 TO (NCH17)-C-16.2-H-16.1

In this paper, we show that an adenosine to inosine mutation at positi on 15.1 changes the substrate specificity of the hammerhead ribozyme f rom (NUH17)-U-16.2-H-16.1 to (NCH17)-C-16.2-H-16.1 (H represents A, C or U). This result extends the hammerhead cleavage triplet definition from (NUH17)-U-16.2-H-16.1 to the more general (NYH17)-Y-16.2-H-16.1. Comparison of cleavage rates using I-15.1 ribozymes for NCH triplets a nd standard A(15.1) ribozymes for NUH triplets under single turnover c onditions shows similar or slightly enhanced levels of reactivity for the I-15.1-containing structures. The effect of I-15.1 substitution wa s also tested in nuclease-resistant 2'-Oalkyl substituted derivatives (oligozymes), showing a similar level of activity for the NUH and NCH cleaving structures. The availability of NCH triplets that can be targ eted without loss of efficiency increases the flexibility of ribozyme targeting strategies. This was demonstrated by an efficient cleavage o f an HCV transcript at a previously inaccessible GCA site in codon 2.