Optimizing the substrate specificity of a group I intron ribozyme.

Journal Article

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.

Full Text

Duke Authors

Cited Authors

  • Zarrinkar, PP; Sullenger, BA

Published Date

  • March 16, 1999

Published In

Volume / Issue

  • 38 / 11

Start / End Page

  • 3426 - 3432

PubMed ID

  • 10079089

International Standard Serial Number (ISSN)

  • 0006-2960

Digital Object Identifier (DOI)

  • 10.1021/bi982688m

Language

  • eng

Conference Location

  • United States