Design of highly specific cytotoxins by using trans-splicing ribozymes.
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 in vivo. Trans-splicing ribozymes may provide a new class of agents for engineering virus resistance and therapeutic cytotoxins.
Ayre
B G
BG
Medical Research Council Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, England.
Köhler
U
U
Goodman
H M
HM
Haseloff
J
J
D001483:Base Sequence; D002213:Capsid / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D017799:Cucumovirus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D003603:Cytotoxins / Q000138:chemical synthesis*; D017931:DNA Primers; D015195:Drug Design; D005091:Exons; D007438:Introns; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D012326:RNA Splicing; D016337:RNA, Catalytic / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae
D003603:Cytotoxins; D017931:DNA Primers; D016337:RNA, Catalytic; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins