[Preparation and identification of hammerhead ribozyme in vitro against rat caspase-3 mRNA fragment].
OBJECTIVE
To study the transcript effect and cleavage activity in vitro of rat Caspase-3 specific ribozyme (Rz107 and Rz544).


METHODS
Rat caspase-3 gene fragment was cloned into T-vector under the control of T7 promoter. (32)P-labeled caspase-3 transcript was target-RNA. Rz107 and Rz544 genes against caspase-3 mRNA were cloned and transcribed in vitro. Cleavage reaction was detected.


RESULTS
It was found that Rz107 was active at 37 degrees C and more so at higher temperature within allowing temperature range. The optimal temperature was 50 degrees C. For Rz107, Km and kcat was 14.13 nmol/L and 2.31 min(-1), respectively. However, the Rz544 had no cleavage activity at all.


CONCLUSIONS
Rz107 prepared in vitro possesses the perfect specific catalytic cleavage activity. It is hopeful that Rz107 would be developed to be a new nucleic acid drug that could effectively inhibit the inflammation of hepatitis through cleaving the key gene, caspase-3, in apoptosis in vivo.
Xu
R
R
Department of Infectious Diseases, Ruijin Hospital of Shanghai Second Medical University, Shanghai 200025, China.
Zhou
X
X
Xie
Q
Q
Jin
Y
Y
Liao
D
D
D000818:Animals; D001483:Base Sequence; D053148:Caspase 3; D020169:Caspases / Q000235:genetics*; D008969:Molecular Sequence Data; D010957:Plasmids; D016337:RNA, Catalytic / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D013696:Temperature
D016337:RNA, Catalytic; D012333:RNA, Messenger; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases