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Group I ribozymes can repair mutant RNAs via trans-splicing. Unfortunately, substrate specificity is quite low for the trans-splicing reaction catalyzed by the group I ribozyme from Tetrahymenathermophila. We have used a systematic approach based on biochemical knowledge of the function of the Tetrahymena ribozyme to optimize its ability to discriminate against nonspecific substrates in vitro. Ribozyme derivatives that combine a mutation which indirectly slows down the rate of the chemical cleavage step by weakening guanosine binding with additional mutations that weaken substrate binding have greatly enhanced specificity with short oligonucleotide substrates and an mRNA fragment derived from the p53 gene. Moreover, compared to the wild-type ribozyme, reaction of a more specific ribozyme with targeted substrates is much less sensitive to the presence of nonspecific RNA competitors. These results demonstrate how a detailed understanding of the biochemistry of a catalytic RNA can facilitate the design of customized ribozymes with improved properties for therapeutic applications.

One largely unexplored question about group I intron splicing is how the cleavage and ligation steps of the reaction are coordinated. We describe a simple in vitro trans-splicing model system in which both steps take place, including the exchange of ligands in the guanosine-binding site that must occur between the two steps. Using this model system, we show that the switch is accomplished by modulating the relative affinity of the binding site for the two ligands. While the terminal guanosine of the intron (omegaG) and exogenous guanosine compete for binding during the first step of splicing, no competition is apparent during the second step, when omegaG is bound tightly. These results help explain how the ribozyme orchestrates progression through the splicing reaction. In addition to providing a new tool to ask basic questions about RNA catalysis, the trans-splicing model system will also facilitate the development of therapeutically useful group I ribozymes that can repair mutant mRNAs.