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

ABSTRACT

<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>