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Design of a ribozyme targeting human telomerase reverse transcriptase and cloning of it's gene
Zhi-Ming Hao1, Jin-Yan Luo, Jin Cheng
1Department of Gastroenterology, 1st Hospital, Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, China. haozhm@public.xa.sn.cn
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 RNA structure, 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 22nt catalytic core and 17nt 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.