In Vitro Construction of Effective M1GS Ribozymes Targeting HCMV UL54 RNA Segments  

作　　者：Yun-ZhenSU Hong-JianLI Yue-QinLI Hao-JunCHEN Dong-ShengTANG XinZHANG HongJIANG Tian-HongZHOU 

机构地区：[1]CollegeofLifeScienceandTechnologyJinanUniversity,Guangzhou510632,China

出　　处：《Acta Biochimica et Biophysica Sinica》2005年第3期210-214,共5页生物化学与生物物理学报（英文版）

基　　金：This work was supported by the grants from the National Natural Science Foundation of China(No.30370776);the Natural Science Foundation of Guangdong Province(36703,021162 and 000718)

摘　　要：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 1ed 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.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 1ed 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.

关 键 词：ribonuclease P (RNase P) guide sequence HCMV DNA polymerase 

分 类 号：R373[医药卫生—病原生物学]