Construction of an effective m1 GS ribozyme targeting HCMV UL97 mRNA segment in vitro.

Abstract

A sequence-specific M1GS ribozyme (M1-T3) was constructed by covalently linking an oligonucleotide (guide sequence,GS) to the 3' terminus of M1 RNA ,the catalytic subunit of RNase P from Escherichia coli. The engineered ribozyme is targeted to the mRNA sequence encoding a protein kinase (UL97) of HCMV and could effectively cleave the mRNA segment in vitro. Further studies about the significance of some structural elements in the M1 GS (e.g. the 3' CCA tail sequence and a bridge sequence between the 3' terminus of M1 RNA and the 5' terminus of the GS) were carried out. The results showed that the bridge sequence of 88 nucleotides in a mutated M1 GS (i.e. M1-T3*) dramatically increased the cleavage activity to the substrate in vitro. Moreover, the 3'CCA tail sequence was confirmed to be a necessary element for the cleavage activity of M1 GS ribozyme. These data we got in the study will help in understanding the interaction between the M1 GS RNA and its substrate,and will markedly facilitate the research of a general gene targeting agent for anti-HCMV applications.