Improvement of hammerhead ribozymes cleaving mdr-1 mRNA.

Overexpression of the mdr-1 gene is one of the mechanisms involved in therapy induced drug resistance. Gene-specific reduction of mdr-1 overexpression in human cancer using antisense technology may be an efficient tool for the reduction of multiple drug resistance (MDR). The application of catalyticly active RNA species--the so-called ribozymes--represents a possible improvement of this molecular strategy using oligonucleotides due to the catalytic potential of ribozymes. In the present paper we investigated the catalytic activity of ribozymes directed against three different cleavage sites on the mdr-1 mRNA. We designed ribozymes against cleavage sites at position 2408 (GUC, ribozyme III), 2429 (CUC, ribozyme I), and 2440 (GUC, ribozyme II). At all these cleavage sites we investigated ribozymes containing a 14 nucleotide complementary sequence to the target RNA (ribozymes I14-III14); at two sites (I and II) additional ribozymes with a 24 nucleotide hybridizing sequence have been tested. Catalytic activity was dependent on ribozyme to target ratio, pH, MgCl2 concentration, and incubation time. The highest cleavage activity was found with ribozyme II14, which cleaved 91% of an 292 nucleotides long in vitro transcript of the mdr-1 mRNA within 15 h in the presence of 10 mM MgCl2. Ribozymes I14 and II24 exhibited very similar but 6-fold reduced activity compared to ribozyme II14. Ribozyme III14 showed little and ribozyme I24 no cleavage activity.