SPECIFICITY OF RIBOZYME DESIGNED FOR MUTATED DHFR MESSENGER-RNA

When MOLT-3 human acute leukemia cells were exposed sequentially to tr imetrexate (TMQ) and then to methotrexate (MTX), the cells became resi stant to antifolate. We designated this subline MOLT-3/TMQ(800)-MTX(10 ,000). This cell line was found to contain two point mutations in the dihydrofolate reductase (DHFR) gene: a T-->C transition at nucleotide 95 in codon 31, and a T-->A transition at nucleotide 100 in codon 33. In an attempt to specifically inhibit these double-mutated cells, we s ynthesized a ribozyme which perfectly base-paired with the double-muta ted DHFR mRNA. We found that the ribozyme for the double-mutated DHFR mRNA not only cleaved the mutated DHFR RNA, but also efficiently cleav ed the wild-type RNA substrate. This observation suggests proceeding w ith caution when using a ribozyme against a mutated mRNA of an essenti al enzyme as a specific means of treatment.