Published online Aug 15, 2002. doi: 10.3748/wjg.v8.i4.694
Revised: November 20, 2001
Accepted: November 27, 2001
Published online: August 15, 2002
AIM: To explore whether HDV ribozymes have the ability to trans-cleave HCV RNA.
METHODS: Three HDV genomic ribozymes were designed and named RzC1, RzC2 and RzC3. The substrate RNA contained HCV RNA 5’-noncoding region and 5'-fragment of C region (5'-NCR-C). All the ribozymes and HCV RNA 5'-NCR-C were obtained by transcription in vitro from their DNA templates, and HCV RNA 5'-NCR-C was radiolabelled at its 5’-end. Under certain pH, temperature, appropriate concentration of Mg2+ and deionized formamide, these ribozymes were respectively or simultaneously mixed with HCV RNA 5'-NCR-C and reacted for a certain time. The trans-cleavage reaction was stopped at different time points, and the products were separated with polyacrylamide gel electrophoresis (PAGE), displayed by autoradiography. Percentage of trans-cleaved products was measured to indicate the activity of HDV ribozymes.
RESULTS: RzC1 and RzC2 could trans-cleave 26% and 21.8% of HCV RNA 5'-NCR-C under our reaction conditions with 2.5 mol•L-1 deionized formamide respectively. The percentage of HCV RNA 5'-NCR-C trans-cleaved by RzC1, RzC2 or combined usage of the three ribozymes increased with time, up to 24.9%, 20.3% and 37.3% respectively at 90 min point. Almost no product from RzC3 was observed.
CONCLUSION: HDV ribozymes are able to trans-cleave specifically HCV RNA at certain sites under appropriate conditions, and combination of several ribozymes aiming at different target sites can trans-cleave the substrate more efficiently than using only one of them.