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Updated: Aug 14, 2026
A Protocol for Analyzing Hepatitis C Virus Replication
Published on: June 26, 2014
[Cleavage of HCV by HCV specific deoxyribozyme in vitro]
Xiao-yu Wen1, Wan-guo Bao, Xiu-yun Yang
1Department of Infectious Diseases, First Hospital of Jilin University, Changchun 130021, China.
Objective:
To study the cleavage activity of specific deoxyribozyme to hepatitis C virus in vitro.
Methods:
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.
Results:
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.
Conclusion:
The designed deoxyribozymes can cleave 5'-NCR mRNA of HCV efficiently in vitro and it is dose-respondent to Mg2+ concentration.
