Specific Hammerhead Ribozyme-mediated Cleavage of Mutant N-ras mRNA in Vitro and ex Vivo

Abstract

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 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.

Ribozyme and free alkylated base: a dual approach for sensitizing Mex+ cells to the alkylating antineoplastic drug

“…These results confirm that chemical stabilization reduces the cleavage activity of ribozymes. 14,15 Nevertheless, a nonspecific degradation of RNAs during the long cleavage incubation time was also observed (Fig 2B).…”

“…The ribozymes and short substrate sequences were chemically synthesized according to a method described previously. 14,15 Their activities were assessed by in vitro cleavage of a 13-mer synthetic substrate under multiple turnover conditions. Cleavage rates were quantified by high-performance liquid chromatography analysis.…”

Effective reversal of a transformed phenotype by retrovirus-mediated transfer of a ribozyme directed against mutant N-ras

“…23 Catalytic activity of pBabe-REC transcripts in HeLa cells expressing a N 13 -ras/luciferase fusion transcript The catalytic activity of pBabe-REC was evaluated in cell lines after retrovirus-mediated transfer of MRE763C in the HeLa cell clone C3 which expresses an N 13 -ras/luciferase fusion transcript. 20 In this setting, ribozyme-mediated cleavage of N 13 -ras transcripts led to a reduction in luciferase activity. 20 As a control, HeLa cells were transduced with the retroviral vector alone (pBabe-puro), or with a pBabe vector containing either a nonsense ribozyme (pBabe-NRE) or an inactive form of MRE763C (pBabe-IRE).…”

“…20 In this setting, ribozyme-mediated cleavage of N 13 -ras transcripts led to a reduction in luciferase activity. 20 As a control, HeLa cells were transduced with the retroviral vector alone (pBabe-puro), or with a pBabe vector containing either a nonsense ribozyme (pBabe-NRE) or an inactive form of MRE763C (pBabe-IRE). After transduction, puromycinresistant clones were isolated, expanded and tested for luciferase activity.…”

“…20 Exogenous delivery of MRE763C to HeLa cells expressing an N 13 -ras/luciferase fusion transcript led to almost 60% reduction in luciferase activity. The catalytic activity of MRE763C was specific, since no reduction in luciferase activity was observed when MRE763C was delivered to HeLa cells expressing a luciferase fusion transcript containing wild-type N-ras sequences.…”