In vitro cleavage of bovine ephemeral fever virus mRNA species with synthetic hammerhead ribozymes. [Symposium paper]
1993
Cowley, J.A. | O'Keefe, K.J. | Walker, P.J. (Commonwealth Scientific and Industrial Research Organisation, Indooroopilly (Australia). Div. of Tropical Animal Production)
Ribozymes are RNA molecules with secondary structures that afford enzymatic, RNA-cleaving activities. By inserting the catalytic domain of a hammerhead ribozyme into RNA sequences complementary to any unique RNA sequence, ribozymes can be designed to associate specifically with a target via base-pairing, cleave the RNA substrate and subsequently dissociate to catalyse further cleavage events. We have constructed and targeted synthetic hammerhead ribozymes to the polymerase (L), nucleoprotein (N) and putative non-virion (NV3) mRNAs of bovine ephemeral fever (BEF) virus. BEF virus cDNA and oligonucleotides representing specific ribozyme RNA sequences were cloned into pGEM plasmids downstream of the T7 RNA promoter. Synthetic RNA transcripts of BEF virus RNA species and ribozymes were prepared using T7 RNA polymerase and reacted in vitro in the presence of magnesium ions. Specific cleavage was demonstrated with 3 ribozymes to a clone (m89) of 3'-L-gene mRNA, 2 to a clone (g238 X-E) of the NV3-gene mRNA. Factors that affect cleavage efficiency, including temperature, substrate RNA secondary structures and reaction conditions are discussed, together with ribozyme cleavage of native BEF virus mRNA in vitro, liposome delivery of ribozymes into BEF virus-infected cells and the potential application of ribozymes as therapeutic antiviral agents.
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