Anti-HBV hairpin ribozyme-mediated cleavage of target RNA in vitro.
OBJECTIVE
To study the preparation and cleavage activity of HpRz directed against the transcript of HBV core gene in vitro.


METHODS
HpRz gene designed by computer targeting the transcript of HBV core gene was cloned into the vector p1.5 between 5'-cis-Rz and 3'-cis-Rz. 32p-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme in vitro. 32p-labeled pKC transcript containing HBV core region as target-RNA was transcribed using T7 RNA polymerase and purified by denaturing PAGE. Cold HpRz transcript was incubated with 32p-labeled target-RNAs under different conditions and radio autographed after denaturing polyacrylamide gel electrophoresis.


RESULTS
HpRz has the specific ability of cleavage of target RNA at 37 degrees and 12 mM MgCl2. Km=26.31 nmol/L, Kcat=0.18/min. These results revealed that the design of HpRz was correct.


CONCLUSIONS
HpRz prepared in this study possesses specific catalytic activity from the identification of cleavage activity. These results indicate that hairpin ribozyme may intracellularly inhibit the replication of HBV, therefore it may become a novel potent weapon for the treatment of hepatitis B.
Song
Yu-Hu
YH
Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
Lin
Ju-Sheng
JS
Liu
Nan-Zhi
NZ
Kong
Xin-Juan
XJ
Xie
Na
N
Wang
Nan-Xia
NX
Jin
You-Xin
YX
Liang
Kuo-Huan
KH
D001483:Base Sequence; D004789:Enzyme Activation; D015316:Genetic Therapy; D006509:Hepatitis B / Q000628:therapy / Q000821:virology; D006512:Hepatitis B Core Antigens / Q000235:genetics; D006515:Hepatitis B virus / Q000235:genetics*; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D016337:RNA, Catalytic / Q000378:metabolism*; D012367:RNA, Viral / Q000378:metabolism*; D014158:Transcription, Genetic
D006512:Hepatitis B Core Antigens; D016337:RNA, Catalytic; D012367:RNA, Viral; C418481:hairpin ribozyme