Colorectal Cancer
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jan 15, 2003; 9(1): 104-107
Published online Jan 15, 2003. doi: 10.3748/wjg.v9.i1.104
Design of a ribozyme targeting human telomerase reverse transcriptase and cloning of it’s gene
Zhi-Ming Hao, Jin-Yan Luo, Jin Cheng, Quan-Yin Wang, Guang-Xiao Yang
Zhi-Ming Hao, Department of Gastroenterology, 1st Hospital, Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Jin-Yan Luo, Jin Cheng, Quan-Yin Wang, Guang-Xiao Yang, Department of Gastroenterology, 2nd Hospital, Xi’an Jiaotong University, Xi’an 710061, Shaanxi Province, China
Author contributions: All authors contributed equally to the work.
Supported by the National Natural Science Foundation, No. 39800061. (Reversal of malignant proliferation of a telomerase-specific ribozyme on colonic carcinoma cells)
Correspondence to: Dr. Zhi-Ming Hao, Department of Gastroenterology, 1st Hospital, Xi’an Jiaotong University, 1 Jiankanglu, Xi’an 710061, Shaanxi Province, China. haozhm@public.xa.sn.cn
Telephone: +86-029-5265523
Received: April 13, 2002
Revised: April 19, 2002
Accepted: April 27, 2002
Published online: January 15, 2003
Abstract

AIM: To design a hammerhead ribozyme targeting human telomerase reverse transcriptase (hTERT) and clone it’s gene for future use in the study of tumor gene therapy.

METHODS: Using the software RNAstructure, the secondary structure of hTERT mRNA was predicted and the cleavage site of ribozyme was selected. A hammerhead ribozyme targeting this site was designed and bimolecular fold between the ribozyme and hTERT was predicted. The DNA encoding the ribozyme was synthesized and cloned into pGEMEX-1 and the sequence of the ribozyme gene was confirmed by DNA sequencing.

RESULTS: Triplet GUC at 1742 of hTERT mRNA was chosen as the cleavage site of the ribozyme. The designed ribozyme was comprised of 22 nt catalytic core and 17 nt flanking sequence. Computer-aided prediction suggested that the ribozyme and hTERT mRNA could cofold into a proper conformation. Endonuclease restriction and DNA sequencing confirmed the correct insertion of the ribozyme gene into the vector pGEMEX-1.

CONCLUSION: This fundamental work of successful designing and cloning of an anti-hTERT hammerhead ribozyme has paved the way for further study of inhibiting tumor cell growth by cleaving hTERT mRNA with ribozyme.

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