Yue-Cheng Yu1, Qing Mao, Chang-Hai Gu, Qi-Fen Li, Yu-Ming Wang. 1. Institute of Infectious Diseases of Chinese PLA, Southwest Hospital, Third Military Medical University, Chongqing 400038,China. yuechengyu212001@163.net
Abstract
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 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.
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 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.
Authors: A Ludwig; G Saretzki; P S Holm; F Tiemann; M Lorenz; T Emrich; C B Harley; T von Zglinicki Journal: Cancer Res Date: 2001-04-01 Impact factor: 12.701
Authors: M Kruger; C Beger; Q X Li; P J Welch; R Tritz; M Leavitt; J R Barber; F Wong-Staal Journal: Proc Natl Acad Sci U S A Date: 2000-07-18 Impact factor: 11.205