[Clone, expression and cleavage activity of anti-caspase-7 hammerhead ribozyme in vitro].
OBJECTIVE
To design hammerhead ribozymes against mouse caspase-7 and to study their expression and cleavage activity in vitro.


METHODS
The secondary structures of ribozyme and caspase-7 genes were analyzed and simulated by computer. Ribozymes DNA sequences were synthesized by automatic synthetic apparatus. Caspase-7 DNA sequence was acquired by reverse transcription PCR. Ribozymes and caspase-7 DNA sequences were separately cloned into pBSKneo U6 and pGEM-T vectors. Ribozymes and caspase-7 mRNA were obtained by transcription in vitro, and ribozymes cleavage activity was identified by cleavage experiment in vitro.


RESULTS
Two ribozymes named Rz333 and Rz394 targeting 333 and 394 sites in caspase-7 mRNA were designed by computer software, and their DNA sequences were synthesized. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed by in vitro transcription. In vitro cleavage experiments showed that Rz333 cleaved caspase-7 mRNA and produced 243nt and 744nt segments. The cleavage efficiency is 67.98%, while Rz394 cannot cleave caspase-7 mRNA.


CONCLUSIONS
Rz333 can site-specifically cleave caspase-7 mRNA.
Zhang
Wei
W
Department of Infectious Diseases, Ruijin Hospital, Shanghai Second Medical University, Shanghai 200025, China.
Xie
Qing
Q
Zhou
Xia-qiu
XQ
Jiang
Shan
S
Jin
You-xin
YX
D000818:Animals; D001483:Base Sequence; D053179:Caspase 7; D061945:Caspase Inhibitors; D020169:Caspases / Q000235:genetics*; D003001:Cloning, Molecular; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics
D061945:Caspase Inhibitors; D016337:RNA, Catalytic; D012333:RNA, Messenger; C427204:hammerhead ribozyme; C505524:Casp7 protein, mouse; D053179:Caspase 7; D020169:Caspases