Design of highly specific cytotoxins by using trans-splicing ribozymes

被引:41
作者
Ayre, BG
Köhler, U
Goodman, HM
Haseloff, J
机构
[1] MRC, Mol Biol Lab, Cambridge CB2 2QH, England
[2] Harvard Univ, Sch Med, Dept Genet, Boston, MA 02114 USA
[3] Massachusetts Gen Hosp, Dept Mol Biol, Boston, MA 02114 USA
关键词
D O I
10.1073/pnas.96.7.3507
中图分类号
O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];
学科分类号
07 ; 0710 ; 09 ;
摘要
We have designed ribozymes based on a self-splicing group I intron that can trans-splice exon sequences into a chosen RNA target to create a functional chimeric mRNA and provide a highly specific trigger for gene expression. We have targeted ribozymes against the coat protein mRNA of a widespread plant pathogen, cucumber mosaic virus, The ribozymes were designed to trans-splice the coding sequence of the diphtheria toxin A chain in frame with the viral initiation codon of the target sequence. Diphtheria toxin A chain catalyzes the ADP ribosylation of elongation factor 2 and can cause the cessation of protein translation. In a Saccharomyces cerevisiae model system, ribozyme expression was shown to specifically inhibit the growth of cells expressing the virus mRNA. A point mutation at the target splice site alleviated this ribozyme-mediated toxicity. Increasing the extent of base pairing between the ribozyme and target dramatically increased specific expression of the cytotoxin and reduced illegitimate toxicity invivo. Trans-splicing ribozymes may provide a new class of agents for engineering virus resistance and therapeutic cytotoxins.
引用
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页码:3507 / 3512
页数:6
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