Targeted Cleavage Of Human Cytomegalovirus (HCMV) UL97 MRNA Segments In Vitro By The Catalytic RNA Subunit Of RNaseP From Escherichia Coli

Background:RNase P from E.coli is a ribonucleoprotein complex responsible for the 5' maturation of tRNAs.The catalytic core of Ml RNA,composed of 377 nucleotides, can specifically cleavage RNA substrate under certain ion conditions in vitro.The feature of RNase P is their ability to recognize the structures.rather than the sequences of their substrates.The smallest substrate contains two important parts:NCCA-3' terminal and RNA helix area.The short,complementary sequence to the substrate is called external guide sequence.A sequence-specific mRNA-cleavaving ribozyme ,M1GS RNA,can be constructed by linking a guide sequence covalently to Ml RNA.ObjectiverTo screen the high specific and effective cleavage M1GS ribozymes,which target HCMV UL97 mRNA,and the foundation have been set up for the inhibition of viral genes transcripts by antisense RNA base on RNase P.Methods:8 M1GS ribozymes containing different GSs targetting sequences of UL97 mRNA were constructed by PCR.The latter 1/4 part of UL97 was subcloned to pGEM3z,due to its richment of target sites.M1GS ribozymes and their substrates were transcripted in vitro and the substrates were labeled by a-32P.The ribozymes and substrates were incubated in buffer B at 37癈, separated by polyacrylamide gels after reaction,then inspected by the autoradiograph.Results:Obtained 3 specific and effective cleavage M1GS ribozymes from 8 M1GS ribozymes.Confirmed the essential of NCCA-3' terminal and 88nt bridge by experiment.Conclusions:We constructed the sequence-specific mRNA-cleavage ribozymes M1GS by linking a guide sequence covalently to Ml RNA.The results suggest that M1GS ribozyme-mediated inhibition of viral genes transcripts can be used as a new approach for the studies of antiviral.