Activity of HDV ribozymes to trans-cleave HCV RNA

AIM: To explore whether HDV ribozymes have the abilityto trans-cleave HCVRNA.METHODS: Three HDV genomic ribozymes weredesigned and named RzC1, RzC2 and RzC3. Thesubstrate RNA contained HCVRNA 5'-noncoding regionand 5'-fragment of C region (5'-NCR-C). All theribozymes and HCV RNA 5'-NCR-C were obtained bytranscription in vibo from their DNA templates, and HCVRNA 5'-NCR-C was radiolabelled at its 5'-end Undercertain pH, temperature, appropriate concentration ofMg2+ and deionized formamide, these ribozymes wererespectively or simultaneously mixed with HCVRNA 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 polyacrylamidegel electrophoresis (PAGE), displayed byautoradiography. Percentage of trans-deaved productswas measured to indicate the activity of HDV ribozymes.RESULTS: RzC1 and RzC2 could trans-cleave 26 % and21.8 % of HCV RNA 5'-NCR-C under our reactionconditions with 2.5 mol. L-1 deionized formamiderespectively. The percentage of HCV RNA 5'-NCR-Ctrans-cleaved by RzC1, RzC2 or combined usage of thethree ribozymes increased with time, up to 24.9 %, 20.3 %and 37.3 % respectively at 90 min point. Almost noproduct from RzC3 was observed.CONCLUSION: HDV ribozymes are able to trans-cleavespecifically HCV RNA at certain sites under appropriateconditions, and combination of several ribozymesaiming at different target sites can trans-cleave thesubstrate more efficiently than using only one of them.

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 Mg^(2+)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 rain 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.