Design and analysis of hammerhead ribozyme activity against an artificial gene target.

James R Carter, Pruksa Nawtaisong, Velmurugan Balaraman, Malcolm J Fraser

Methods in molecular biology (Clifton, N.J.) 2014

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In vitro cleavage assays are routinely conducted to properly assess the catalytic activity of hammerhead ribozymes (HHR) against target RNA molecules like dengue virus RNA. These experiments are performed for initial assessment of HHR catalysis in a cell-free system and have been simplified by the substitution of agarose gel electrophoresis for SDS-PAGE. Substituting mobility assays enables the analysis of ribozymes in a more rapid fashion without radioisotopes. Here we describe the in vitro transcription of an HHR and corresponding target from T7-promoted plasmids into RNA molecules leading to the analysis of HHR activity against the RNA target by in vitro cleavage assays.

Citation

James R Carter, Pruksa Nawtaisong, Velmurugan Balaraman, Malcolm J Fraser. Design and analysis of hammerhead ribozyme activity against an artificial gene target. Methods in molecular biology (Clifton, N.J.). 2014;1103:57-66