Design of a ribozyme targeting human telomerase reverse transcriptase and cloning of it's gene.
OBJECTIVE
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.


CONCLUSIONS
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.
Hao
Zhi-Ming
ZM
Department of Gastroenterology, 1st Hospital, Xi'an Jiaotong University, Xi'an 710061, Shaanxi Province, China. haozhm@public.xa.sn.cn
Luo
Jin-Yan
JY
Cheng
Jin
J
Wang
Quan-Yin
QY
Yang
Guang-Xiao
GX
D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D015316:Genetic Therapy; D006801:Humans; D009690:Nucleic Acid Conformation; D016337:RNA, Catalytic / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D019098:Telomerase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D004268:DNA-Binding Proteins; D016337:RNA, Catalytic; C427204:hammerhead ribozyme; D019098:Telomerase