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 functiona l chimeric mRNA and provide a highly specific trigger for gene expression. We have targeted ribozymes against the coat protein mRNA of a widespread pl ant pathogen, cucumber mosaic virus, The ribozymes were designed to trans-s plice the coding sequence of the diphtheria toxin A chain in frame with the viral initiation codon of the target sequence. Diphtheria toxin A chain ca talyzes the ADP ribosylation of elongation factor 2 and can cause the cessa tion of protein translation. In a Saccharomyces cerevisiae model system, ri bozyme expression was shown to specifically inhibit the growth of cells exp ressing the virus mRNA. A point mutation at the target splice site alleviat ed this ribozyme-mediated toxicity. Increasing the extent of base pairing b etween the ribozyme and target dramatically increased specific expression o f the cytotoxin and reduced illegitimate toxicity invivo. Trans-splicing ri bozymes may provide a new class of agents for engineering virus resistance and therapeutic cytotoxins.