Optimization of trans-splicing ribozyme efficiency and specificity by in vivo genetic selection.
Trans-splicing ribozymes are RNA-based catalysts capable of splicing RNA sequences from one transcript specifically into a separate target transcript. In doing so, a chimeric mRNA can be produced, and new gene activities triggered in living cells dependent on the presence of the target mRNA. Based on this ability of trans-splicing ribozymes to deliver new gene activities, a simple and versatile plating assay was developed in Saccharomyces cerevisiae for assessing and optimizing constructs in vivo. Trans-splicing ribozymes were used to splice sequences encoding a GAL4-derived transcription activator into a target transcript from a prevalent viral pathogen. The transcription activator translated from this new mRNA in turn triggered the expression of genes under the regulatory control of GAL4 upstream-activating sequences. Two of the activated genes complemented metabolic deficiencies in the host strain, and allowed growth on selective media. A simple genetic assay based on phenotypic conversion from auxotrophy to prototrophy was established to select efficient and specific trans-splicing ribozymes from a ribozyme library. This simple assay may prove valuable for selecting optimal target sites for therapeutic agents such as ribozymes, antisense RNA and antisense oligodeoxyribonucleotides, and for optimizing the design of the therapeutic agents themselves, in higher eukaryotes.
Ayre
Brian G
BG
Plant Biology Department, Cornell University, Ithaca, NY 14853, USA and. Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge CB2 3EA, UK. bga2@cornell.edu
Köhler
Uwe
U
Turgeon
Robert
R
Haseloff
Jim
J
D002455:Cell Division / Q000235:genetics; D004268:DNA-Binding Proteins; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014170:Transformation, Genetic
D004268:DNA-Binding Proteins; C052130:GAL4 protein, S cerevisiae; D008164:Luminescent Proteins; D016337:RNA, Catalytic; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins