In Vitro Selection of RNase P Ribozymes That Efficiently Cleave a Target mRNA

An in vitro selection procedure for identifying highly efficient RNase P ribozyme (M1GS RNA) variants is presented as a model system for engineering ribozymes to improve their catalytic efficiency. Detailed protocols as well as the rationale for setting up such a system are included, using the mRNA sequence that encodes the thymidine kinase (TK) of herpes simplex virus 1 (HSV 1) as a target substrate of choice. Using the selection system, we have successfully generated M1GS RNA variants that more efficiently cleave the TK mRNA in vitro and more effectively inhibit the TK expression in cultured cells than the ribozyme derived from the wild-type RNase P ribozyme sequence. The in vitro selection system represents a novel and effective approach for engineering highly active RNase P ribozymes that can be used in both basic research and clinical therapeutic settings.