http://www.abbs.info
E-mail: [email protected]
ISSN
1672-9145
Acta Biochim Biophys Sin
2005, 37(3): 210–214
CN 31-1940/Q
In Vitro Construction of Effective M1GS
Ribozymes Targeting HCMV UL54 RNA Segments
Yun-Zhen SU, Hong-Jian LI, Yue-Qin LI, Hao-Jun CHEN, Dong-Sheng
TANG, Xin ZHANG, Hong JIANG, and Tian-Hong
ZHOU*
College of Life Science and Technology, Jinan University, Guangzhou
510632, China
Abstract Seven sequence-specific ribozymes (M1GS RNAs) derived in vitro
from the catalytic RNA subunit of Escherichia coli RNase P and targeting
the mRNAs transcribed by the UL54 gene encoding the DNA polymerase of
human cytomegalovirus were screened from 11 ribozymes that were designed based
on four rules: (1) the NCCA-3' terminal must be unpaired with the
substrate; (2) the guide sequence (GS) must be at least 12 nt in length; (3)
the eighth nucleotide must be U, counting from the site –1; and (4) around the
cleavage site, the sites –1/+1/+2 must be U/G/C or C/G/C. Further investigation
of the factors affecting the cleavage effect and the optimal ratio for
M1GS/substrate was carried out. It was determined that the optimal ratio for
M1GS/substrate was 2:1 and too much M1GS led to substrate degrading. As
indicated above, several M1GS that cleaved HCMV UL54 RNA segments in
vitro were successfully designed and constructed. Our studies support the
use of ribozyme M1GS as antisense molecules to silence HCMV mRNA in vitro,
and using the selection procedure as a general approach for the engineering of RNase P ribozymes.
Key words ribonuclease P (RNase P); guide sequence; HCMV; DNA polymerase
-----------------
Received: November 9, 2004 Accepted: January 18,
2005
This work was supported by the grants from the National Natural
Science Foundation of China (No. 30370776) and the Natural Science Foundation
of Guangdong Province (36703, 021162 and 000718)
*Corresponding author: Tel, 86-20-85226386; E-mail, [email protected]