| RNase P from E.coli is a ribonucleoprotein complex responsible for the 5' maturation of tRNAs. The smallest substrate contains two important parts: NCCA-3' terminal and RNA helix area. RNase P can be directed to cleave any RNA when the target mRNA is in a complex with a short, complementary oligonucleotide called an external guide sequence (EGS). The EGS can be covalently linked to Ml RNA, the catalytic RNA subunit of RNase P, forming a new kind of ribozyme, M1GS. M1GS can cleave any RNA segments in vitro when the RNA is in a complex with its guide sequence (GS). The requirement for Mg2+ in the reaction in vitro is indispensable.The UL54 mRNA sequence encoding HCMV DNA polymerase is very important for virus duplication. Construction of MIGSs may be a tool that allows us selectively to cleave HCMV UL54 mRNA with high specificity and effectiveness in vitro.pGEMSz, which contains a T7 promoter region, was chosen as the vector for gene UL54. The recombinated plasmid was named PU54. Based on this material, 4 recombinated plasmids PU54-A, PU54-B, PU54-C and PU54-D were construted, which contained different cloning segments of UL54 gene.11 M1GS ribozyme containing different GSs designed to the target sequences of UL54 mRNA were construted by PCR. GS sequences were covalent ligated to the 3' end of a "bridge RNA" which acting as a junction between Ml RNA and GS. Attachment to the substrate in vitro shew that 7 of these designed MIGSs can cleave their substrates respectively.Different MIGS/substrate reacting concentrations were tested, the best MIGS/substrate ratio was 2:1 and too much M1GS led to substrate degrading. It was also found that the secondary structure affected the action of M1GS.As indicated above, several M1GS that cleaved HCMV UL54 mRNA segments in vitro were successfully designed and constructed. Our studies demonstrates the utility of this ribozyme M1GS for antiviral application.
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