title: Anti-HBV hairpin ribozyme-mediated cleavage of target RNA in vitro.
abstract: 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.
authors.affiliation: Institute of Liver Diseases, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China.
authors.forename: Yu-Hu
authors.initials: YH
authors.lastname: Song
authors.forename: Ju-Sheng
authors.initials: JS
authors.lastname: Lin
authors.forename: Nan-Zhi
authors.initials: NZ
authors.lastname: Liu
authors.forename: Xin-Juan
authors.initials: XJ
authors.lastname: Kong
authors.forename: Na
authors.initials: N
authors.lastname: Xie
authors.forename: Nan-Xia
authors.initials: NX
authors.lastname: Wang
authors.forename: You-Xin
authors.initials: YX
authors.lastname: Jin
authors.forename: Kuo-Huan
authors.initials: KH
authors.lastname: Liang
mesh_terms: 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
chemical_list: D006512:Hepatitis B Core Antigens; D016337:RNA, Catalytic; D012367:RNA, Viral; C418481:hairpin ribozyme