Specific Hammerhead Ribozyme-mediated Cleavage of Mutant N-rasmRNAin Vitroandex Vivo

Michaela Scherr, Manuel Grez, Arnold Ganser, Joachim W. Engels
1997 Journal of Biological Chemistry  
Two hammerhead ribozymes targeted to point mutations in codon 13 of the N-ras oncogene were synthesized and their catalytic activity and substrate specificity evaluated in vitro and ex vivo. In vitro studies showed that these ribozymes were specific for the oncogenic form of N-ras, since cleavage was observed only in a 849-nucleotide-long transcript containing mutant but not wild-type N-ras sequences. For the ex vivo studies, the ribozymes were 2-modified to protect them against degradation by
more » ... nst degradation by nucleases. 2-Fluoro-2-deoxyuridine/ cytidine-substituted ribozymes were nearly as active as their unmodified counterparts, but had a prolonged stability in cell culture supernatant containing fetal calf serum. The stability of the modified ribozymes increased by introduction of terminal phosphorothioates groups without significant influence in their catalytic efficiency. A sensitive assay based on the use of N-ras/ luciferase fusion genes as a reporter system was established to detect ribozyme-mediated cleavage in HeLa cells. A reduction of nearly 60% in luciferase activity was observed in cells expressing mutant but not wild-type N-ras/luciferase fusion transcripts. Moreover, cleavage of N-ras transcripts in HeLa cells was directly confirmed by a semi-quantitative RT-PCR assay.
doi:10.1074/jbc.272.22.14304 pmid:9162065 fatcat:tv2ucqrtinhjxchr5kkuhsrala