[Cleavage of HCV by HCV specific deoxyribozyme in vitro].
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.


CONCLUSIONS
The designed deoxyribozymes can cleave 5'-NCR mRNA of HCV efficiently in vitro and it is dose-respondent to Mg2+ concentration.
Wen
Xiao-yu
XY
Department of Infectious Diseases, First Hospital of Jilin University, Changchun 130021, China.
Bao
Wan-guo
WG
Yang
Xiu-yun
XY
Tian
Mei-mei
MM
Wang
Feng
F
Niu
Jun-qi
JQ
D021881:DNA, Catalytic / Q000235:genetics*; D004277:DNA, Single-Stranded / Q000235:genetics*; D015316:Genetic Therapy; D016174:Hepacivirus / Q000235:genetics*; D006526:Hepatitis C / Q000628:therapy*; D006801:Humans; D012333:RNA, Messenger / Q000235:genetics
D021881:DNA, Catalytic; D004277:DNA, Single-Stranded; D012333:RNA, Messenger; C402582:RNA-cleaving DNA 10-23