Optimizing the substrate specificity of a group I intron ribozyme

Group I ribozymes can repair mutant RNAs via trans-splicing. Unfortunately, substrate specificity is quite low for the trans-splicing reaction catalyz ed by the group I ribozyme from Tetrahymena thermophila. We have used a sys tematic approach based on biochemical knowledge of the function of the Tetr ahymena ribozyme to optimize its ability to discriminate against nonspecifi c substrates in vitro. Ribozyme derivatives that combine a mutation which i ndirectly slows down the rate of the chemical cleavage step by weakening gu anosine binding with additional mutations that weaken substrate binding hav e greatly enhanced specificity with short oligonucleotide substrates and an mRNA fragment derived from the p53 gene. Moreover, compared to the wild-ty pe ribozyme, reaction of a more specific ribozyme with targeted substrates is much less sensitive to the presence of nonspecific RNA competitors. Thes e results demonstrate how a detailed understanding of the biochemistry of a catalytic RNA can facilitate the design of customized ribozymes with impro ved properties for therapeutic applications.