The objective of this proposal is to study the catalytic mechanisms of ribonuclease P ribozymes and their applications for inhibition of viral gene expression. The applicant has constructed a ribozyme engineered from the catalytic RNA subunit of RNase P from E. coli. This new ribozyme cleaves RNA substrates, including viral mRNAs, that base pair to its guide sequence. It order to increase the cleavage efficiency, the applicant will conduct experiments to elucidate the mechanism of catalysis and substrate recognition. He will also test whether viral replication can be abolished by targeting an essential viral gene, ICP4. Ribozyme recognition of target mRNA will be studied by both in vitro kinetic and physical mapping analyses. Efficient ribozymes will be generated by either site-directed mutagenesis or in vitro selection. The requirements for efficient ribozyme activity in tissue culture will be defined and the sequence specificity determined. Finally, whether the engineered ribozyme can inhibit HSV-1 viral replication by abolishing ICP4 expression will be investigated. The long term goal is to develop a system to study RNA catalysis and its use as a gene-targeting tool in basic research and clinical applications.
| Trang, Phong; Liu, Fenyong (2004) RNase P ribozyme as an antiviral agent against human cytomegalovirus. Methods Mol Biol 252:437-50 |
