EXTENDED TARGET-SITE SPECIFICITY FOR A HAMMERHEAD RIBOZYME

In vitro mutagenesis has been used to systematically mutate the GUC target site cleaved by a synthetic ribozyme based on the catalytic domain of the satellite RNA of tobacco ringspot virus. Amongst the spectrum of changes, it is found that GUC, UUC, CUC, GUA and GUU targets show equivalent rates of cleavage. An AUC target does not cleave, in contrast to observations from other studies. For a GUG target site, the normal ribozyme cannot induce cleavage, but an alteration of the stem-loop in the catalytic domain leads to the formation of a weakly active ribozyme. Certain double mutations, not previously studied, showed slow but discernable cleavage. This mutational approach shows that general rules for cleavage at NUY triplets for the target site of hammerhead ribozymes should be modified. Not all NUY targets cleave under all circumstances, and there are some targets with nucleotides other than U in the centre position which show significant, discernable cleavage.