Preparation and Characterization of Ribozyme for Tissue Inhibitor of Metalloproteinases-1 mRNA in Vitro

WANG Zi-Min^1,2, 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_m=39.6 nmol/L, k_cat=0.21 min^-1), and the cleavage 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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