Multitarget ribozyme against the S1 genome segment of reovirus possesses novel cleavage activities and is more efficacious than its constituent mono-ribozymes

Abstract

Two hammerhead motif containing ribozymes (Rzs) were constructed through recombinant techniques that were directed to cleave at the conserved sites of the reovirus S1 gene segment which encodes the cell attachment protein sigma1. The two mono-ribozymes 553 and 984 cleaved the target RNA in a sequence specific manner, and Rz-553 being the more efficient. When the mono-Rzs were combined in direct tandem to make it a multitarget-Rz, very efficient cleavage of the S1 RNA was achieved that retained the specificity of the two mono-ribozymes. This cleavage was, as expected, Mg(++)-dependent but protein-independent. Almost complete cleavage of the S1 RNA was observed with multitarget ribozyme alone. Although S1-Rz-984 cleaved the short S1 synthetic RNA, it failed to cleave the full length S1 RNA (1.4 kb). On the contrary, Rz-553 cleaved the short synthetic RNA as well as the full length S1 RNA with equal efficiency. Full length S1 RNA was, however, cleaved efficiently by the multitarget-ribozyme-S1-Rz-984-553 that cleaved at both the target sites. Thus, hybridization of one ribozyme (Rz-553) to a full length S1 RNA potentially opened up the 984-Rz target site that was otherwise inaccessible to the mono-Rz-984. Multitarget ribozyme expressing mammalian cells showed reduced amounts of S1 RNA that correlated well with the levels of reovirus sigma1 protein. Potential uses of such multitarget-Rzs are discussed.