Optimizing the substrate specificity of a group I intron ribozyme

被引:18
作者
Zarrinkar, PP
Sullenger, BA
机构
[1] Duke Univ, Med Ctr, Dept Surg, Ctr Genet & Cellular Therapies, Durham, NC 27710 USA
[2] Duke Univ, Med Ctr, Dept Genet, Durham, NC 27710 USA
关键词
D O I
10.1021/bi982688m
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 Tetrahymena thermophila. 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.
引用
收藏
页码:3426 / 3432
页数:7
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