title: In vitro construction of effective M1GS ribozymes targeting HCMV UL54 RNA segments.
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.
authors.affiliation: College of Life Science and Technology, Jinan University, Guangzhou, China.
authors.forename: Yun-Zhen
authors.initials: YZ
authors.lastname: Su
authors.forename: Hong-Jian
authors.initials: HJ
authors.lastname: Li
authors.forename: Yue-Qin
authors.initials: YQ
authors.lastname: Li
authors.forename: Hao-Jun
authors.initials: HJ
authors.lastname: Chen
authors.forename: Dong-Sheng
authors.initials: DS
authors.lastname: Tang
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.forename: Hong
authors.initials: H
authors.lastname: Jiang
authors.forename: Tian-Hong
authors.initials: TH
authors.lastname: Zhou
mesh_terms: D003001:Cloning, Molecular / Q000379:methods; D003587:Cytomegalovirus / Q000235:genetics* / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D020868:Gene Silencing; D018390:Gene Targeting / Q000379:methods*; D005818:Genetic Engineering / Q000379:methods*; D006801:Humans; D016337:RNA, Catalytic / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D043262:Ribonuclease P / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*
chemical_list: D016337:RNA, Catalytic; D012333:RNA, Messenger; D012367:RNA, Viral; C109367:UL54 protein, Human herpesvirus 5; D014764:Viral Proteins; D004259:DNA-Directed DNA Polymerase; D043262:Ribonuclease P