(A) synthetic ribozyme activity in vivo

Abstract

A class of ribozymes, small RNA molecules forming hammerhead secondary structure, has been known to cleave themselves catalytically. Catalytic domain of the RNAs has been identified and shown to cleave target RNA molecules at a specific site in vitro. We have designed a ribozyme for specifically cleaving mRNA for Hepatitis B viral X gene in E. coli JM109(DE3) $F^-$. There are no $\beta$ -galactosidase expression in E. coli JM109(DE3) $F^-$ and Hepatitis B viral X gene is fused with $\beta$ -galactosidase gene. So, by checking $\beta$ -galactosidase activity, we can detect the ribozyme activity indirectly. When the plasmid carrying $\phi$10 promoter was introduced to the cell harvoring the plasmid carrying the $\beta$ -galactosidase gene, the transformants were rarely obtained. But in the case of the plasmid carrying T7 lysozyme gene as well as $\phi$10, the transformants grew normally. The results of $\beta$-galactosidase assay indicate that the activity of the designed ribozyme was minimal, if any, in the presence of high level target mRNA.