Preparation
and Characterization of Ribozyme for Tissue Inhibitor of Metalloproteinases-1
mRNA in Vitro
WANG Zi-Min1,2, WU
Jian-Ming1, LIN Zi-Hao1, JIANG Hua1, SONG
Yu-Hu2, JIN You-Xin2*
( 1 Department of Plastic Surgery, Changzheng
Hospital, Second Military Medical University, Shanghai 200003, China;
2 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 )
Abstract 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 contains mutant human U6 gene. TIMP-1 cDNA fragments
were cloned into T-vector pGEM-T, to form the plasmid pTIMP-1. [32P]-labeled
TIMP-1 transcripts as target RNAs and [32P]-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 ºC; and the cleavage activity
was best at 50 ºC (K
nmol/L, k
efficiencies were up to 76.34% at 50 ºC and 55.21% at 37 ºC.
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.
Key
words TIMP-1;
ribozyme; transcript; clone
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