[Preparation and characterization of ribozyme for tissue inhibitor of metalloproteinases-1 mRNA ribozymes in vitro].
To study the activity of the U6 driven ribozymes for tissue inhibitor of metalloproteinases-1(TIMP-1) mRNA and explore their use to cure hypertrophic scar, the ribozyme gene was designed according to the  hammerhead structure  described by Symons and then was cloned into pBSKneorU6, a vector containing mutant human U6 gene. TIMP-1 cDNA fragments were cloned into T-vector pGEM-T, to form the plasmid pTIMP-1. [(32)P]-labeled TIMP-1 transcripts as target RNAs and [(32)P]-labeled ribozyme, which was transcribed from the template that had been amplified from the corresponding cloning plasmids in vitro, were incubated together under various conditions for cleavage reactions. The reaction mixtures were electrophoresed on PAGE and autoradiographed. The results showed that the ribozyme(U6-Rz358) cleaved the mRNA successfully at 37 degrees; and the cleavage activity was best at 50 degrees ( K(m)=39.6 nmol/L, k(cat)=0.21 min(-1)), and the cleavage efficiencies were up to 76.34% at 50 degrees and 55.21% at 37 degrees. The designed ribozymes possessed perfect specific cleavage activity to TIMP-1. These findings suggested the potential application of this ribozyme as a new therapeutic agent against hypertrophic scar.
Wang
Zi-Min
ZM
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, the Chinese Academy of Sciences, Shanghai 200031, China. yxjin@sunm.shcnc.ac.cn
Wu
Jian-Ming
JM
Lin
Zi-Hao
ZH
Jiang
Hua
H
Song
Yu-Hu
YH
Jin
You-Xin
YX
D001483:Base Sequence; D002384:Catalysis; D004274:DNA, Recombinant / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D012342:RNA, Small Nuclear / Q000235:genetics; D013379:Substrate Specificity; D013696:Temperature; D013997:Time Factors; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics*
D004274:DNA, Recombinant; D016337:RNA, Catalytic; D012333:RNA, Messenger; D012342:RNA, Small Nuclear; D019715:Tissue Inhibitor of Metalloproteinase-1; C405486:U6 small nuclear RNA