Cleavage of HCV by HCV specific deoxyribozyme in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the cleavage activity of specific deoxyribozyme to hepatitis C virus in vitro.</p><p><b>METHODS</b>Three deoxyribozymes were designed to cleave at sites 157, 168, 173 in HCV 5'-noncoding region with the active region of 5'-GGCTAGCTACAACGA-3' respectively. Plasmid pCMV/T7-NCRC -Delta Luc was completely linearized with restriction endonuclease Xba I. HCV RNA5'-NCRC was transcribed in vitro from the linearized products and radiolabelled with [alpha-32P] UTP. Under the conditions of 37 degrees C, pH7.5, Mg2+ 10 mmol/L, the three deoxyribozymes were mixed with substrate RNA individually for 120 minutes and then the reactions were terminated. The cleavaged products were separated with 8% denaturated polyacrylamide gel electrophoresis and displayed by autoradiography. DRz3 was mixed with the substrate RNA at different Mg2+ concentrations. The cleavage efficiency was analyzed with a gel document action analyzing systems.</p><p><b>RESULTS</b>Under the adopted conditions the three deoxyribozymes efficiently cleaved to the target RNA in vitro and the cleavage activity of DRz3 was increased with the increase of Mg2+ concentration.</p><p><b>CONCLUSION</b>The designed deoxyribozymes can cleave 5'-NCR mRNA of HCV efficiently in vitro and it is dose-respondent to Mg2+ concentration.</p>

Study on the cleavage activity of U1 small nuclear RNA chimeric ribozyme against HCV RNA in vitro / 中华肝脏病杂志

<p><b>OBJECTIVE</b>To study the cleavage activity on the HCV RNA of a chimeric recombinant of HCV specific ribozyme and U1 small nuclear RNA, which compartmentalizes within the nucleolus.</p><p><b>METHODS</b>The third stem-loop sequence of human U1 snRNA (position 95-116) within pBSIISK+ U1 was substituted by hammerhead ribozyme against HCV RNA by PCR and cloning methods, and the constructed plasmid was named pBSIISK+ (U1-Rz). Then the whole gene fragment of the chimeric ribozyme was cloned into a pGEM-T vector under the control of T7 promoter, and the constructed plasmid was named pGEM- (U1-Rz). The pGEM- (U1-Rz) and pGEM-Rz (containing the same ribozyme sequence as that in U1-Rz) transcripts as enzyme were transcribed in vitro. Also the (32)P-labeled pCMV/T7-NCRC luc (containing the gene sequence of the whole 5'-NCR and part core of HCV RNA) transcripts as target-RNAs were transcribed in vitro. The enzymes were incubated with the target RNAs under different conditions and autoradiographed after denaturing gel-electrophoresis.</p><p><b>RESULTS</b>The sequencing result showed that the construction of U1 snRNA chimeric ribozyme was correct. Compared with the ribozyme alone, both of them were active at 37 degree C and with Mg2+ (10 mmol/L) and TrisCl (10 mmol/L, pH7.9), and there was no remarkable difference between them. The cleavage activity of the chimeric ribozyme increased with the prolongation of reaction time and increment of enzyme concentration.</p><p><b>CONCLUSION</b>Both ribozyme and U1 snRNA chimeric ribozyme exhibited specifically catalytic activity against HCV RNA in vitro. There was no remarkable difference between their cleavage efficiencies.</p>