Construction and Cleavage Characterization of SingleTargeted Multi-ribozyme Expression Systems in vitro

Chemical Research in Chinese Universities ›› 2009, Vol. 25 ›› Issue (4): 506-512.

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Construction and Cleavage Characterization of SingleTargeted Multi-ribozyme Expression Systems in vitro

TIAN Sheng-Li^1*, XU Dong-ping² and OHNUMA Takao³

1. Shenzhen Key Laboratory of Microbiology and Gene Engineering, College of Life Sciences, Shenzhen University, Shenzhen 518060, P. R. China;
2. The 302 Hospital of Beijing, Beijing 100039, P. R. China;
3. Mount Sinai School of Medicine, New York 10029, USA

Received:2008-05-23 Revised:2008-06-19 Online:2009-07-25 Published:2009-10-16
Contact: TIAN Sheng-Li. E-mail: sltian@szu.edu.cn
Supported by:
Supported by Fund of “T. J. Martell Foundation for AIDS, Leukemia and Cancer Research”, and Fund of Shenzhen Bureau of Science and Technology, China(No.20008).

Abstract

Abstract:

In order to clarify cleavage efficiency of a multi-ribozyme expression system on their substrates, plasmids containing 20 cis-acting hammerhead ribozymes for self-cleavage and 10 trans-acting hammerhead ribozymes targeted on MDR1 and MRP1 substrates, the target plasmids pGEM-MDR1 and pGEM-MRP1 were constructed by employing PCR and DNA recombination. The endonuclease’s digestion and DNA sequencing confirmed exactness of the multi-ribozyme expression systems and the target plasmids. The repeated self-cleavage tests revealed that the cis-acting hammerhead ribozymes in the multi-ribozyme expression system were able to cleave themselves and trans-acting hammerhead ribozymes 196 Rz and 210 Rz were released. The cleavage efficiencies of liberated ribozymes on their targets were evaluated in vitro and the results indicate that 196 Rz and 210 Rz were able to cleave the MDR1 and MRP1 RNA substrates. The profile of cleavage reaction shows that the cleavage efficiencies were correlated with the numbers of trans-acting hammerhead ribozymes contained in the multi-ribozyme expression systems, as well as the multi-ribozyme system is much better than the single ribozyme. Test of various concentrations of Mg²⁺ reveals that ribozyme cleavage reactions were dependent on Mg²⁺ concentration. The plot of lg(k_obs) vs. lg[Mg²⁺] displays a linear relationship in the concentration range observed(2.5―20 mmol/L).

Key words: Multi-ribozyme expression systems; Hammerhead ribozyme; RNA substrate

TIAN Sheng-Li*, XU Dong-ping and OHNUMA Takao. Construction and Cleavage Characterization of SingleTargeted Multi-ribozyme Expression Systems in vitro[J]. Chemical Research in Chinese Universities, 2009, 25(4): 506-512.

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Construction and Cleavage Characterization of SingleTargeted Multi-ribozyme Expression Systems in vitro

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