The aim of this research is the development of a new PCR based approach for the generation of novel self-cleaving ribonucleic acids, or ribozymes. This scheme will be used to select for highly efficient ribozymes with cleavage activities greater than that of currently known, naturally occurring ribozymes. Ribozymes of increased activity will be more effective for use in ribozyme-mediated antisense gene inhibition, and therefore will be better suited for use as anti-viral/anti-cancer therapeutics. For selection of new self-cleaving RNA molecules degenerate RNA transcripts will be prepared by in vitro transcription of appropriate DNA templates and incubated under suitable cleavage conditions. Transcripts that are incapable of self-cleaving will be reversed transcribed and subsequently amplified using PCR. The amplification products will be used for subtractive hybridization to eliminate templates encoding non- cleaving transcripts, from the original degenerate template mixture. The resulting difference template mixture will contain templates encoding self-cleaving RNAs. Identification and characterization of these novel ribozymes will further our understanding of RNA mediated catalysis and lead to the design of more effective ribozymes for use in antisense mediated gene inhibition. This method uses a form of negative selection to identify novel ribozymes and provides significant advantages over previous methods based on positive selection.
