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Abstract: Ribonuclease P (RNase P) complexed with an external guide sequence (EGS) represents a novel nucleic acid-based gene interference approach to modulate gene expression. This enzyme is a ribonucleoprotein complex for tRNA processing. In Escherichia coli, RNase P contains a catalytic RNA subunit (M1 ribozyme) and a protein subunit (C5 cofactor). EGSs, which are RNAs derived from natural tRNAs, bind to a target mRNA and render the mRNA susceptible to hydrolysis by RNase P and M1 ribozyme. When covalently linked with a guide sequence, M1 can be engineered into a sequence-specific endonuclease, M1GS ribozyme, which cleaves any target RNAs that base pair with the guide sequence. Studies have demonstrated efficient cleavage of mRNAs by M1GS and RNase P complexed with EGSs in vitro. Moreover, highly active M1GS and EGSs were successfully engineered using in vitro selection procedures. EGSs and M1GS ribozymes are effective in blocking gene expression in both bacteria and human cells, and exhibit promising activity for antimicrobial, antiviral, and anticancer applications. In this review, we highlight some recent results using the RNase P-based technology, and offer new insights into the future of using EGS and M1GS RNA as tools for basic research and as gene-targeting agents for clinical applications. [Copyright &y& Elsevier]

Ribozymes are promising gene-targeting agents for regulation of gene expression. In our recent studies, RnaseP (M1GS) ribozymes were constructed to target the overlapping region (IE mRNA) of IE1 and IE2 mRNAs of human cytomegalovirus (HCMV) and the mRNA (TK mRNA) coding for thymidine kinase (TK) of herpes simplex virus 1 (HSV-1). Our results indicate that RNase P ribozymes efficiently cleaved the IE mRNA and TK mRNA sequences in vitro. Significant inhibitions (∼75–85%) of HCMV IE1/IE2 and HSV-1 TK expression were observed in cells that expressed these ribozymes while a reduction of less than 10% was found in cells that did not express the ribozymes or expressed a disabled one that contained mutations abolishing catalytic activity. Ribozyme variants, which cleaved a TK mRNA sequence in vitro more efficiently than the ribozyme derived from the wildtype RNase P sequence, were selected by an in vitro selection system. When the selected ribozymes were expressed in cultured cells, they were more effective in inhibiting viral IE1/IE2 and TK expression and viral growth than the wildtype ribozyme sequence. Our results provide the first direct evidence that RNase P ribozymes are highly effective in inhibiting HCMV gene expression and growth. Moreover, a selection system was developed for generating novel ribozyme variants that cleave a mRNA substrate efficiently in vitro. These results suggest that M1GS ribozyme-mediated inhibition of expression of viral genes can be used as a new approach for the studies of HCMV gene function and the treatment of HCMV infection. [Copyright &y& Elsevier]