In Vitro Selection Of EGS For Directing RNase P-mediated Cleavage Of Human Cytomegalovirus (HCMV) UL54 MRNA Segments

RNase P is a ribonucleoprotein complex and catalyzes a hydrolysis reaction to remove the leader sequence of precursor tRNA. RNase P from Escherichia coli contains a catalytic RNA subunit of 377 nucleotides RNA and a single polypeptide of 119 amino acids known as C5 protein. In the presence of a high concentration of Mg²⁺, Ml RNA itself can hydrolyze tRNA precursors in vitro. EGS is a short, complementary oligonucleotide which can direct RNase P to cleave any RNA when it is in a complex with the target mRNA.Four specific sequences of human cytomegalovirus (HCMV) UL54 mRNA segments were selected as targets. RNA-based EGSs, M1 RNA and substrates labeled with [ α -³²P]UTP were all transcribed by T7 RNA polymerase in vitro. The substrates were incubated with appropriate amount of Ml RNA and EGS at 37℃ for 1 hour in buffer A (50mM Tris, pH7.5, 100mM NH₄C1, 100mM MgCl₂, 4% PEG 6000). Three specific and efficient RNA-based EGSs were selected. The same result was obtained from the assay of DNA-based EGS. Our results also suggested that the requirement of Mg²⁺ in this reaction may be much lower.In this study, we successfully achieved the cleavage of HCMV UL54 mRNA segments using RNase P/EGS technology. Design and construction of EGSs allowed it to be further applied in vivo. This RNase P/EGS-mediated inhibition of viral gene expression will facilitate the development of RNase P as a gene-targeting agent for antiviral applications.