INHIBITION OF C-HA-RAS GENE-EXPRESSION BY HAMMERHEAD RIBOZYMES CONTAINING A STABLE C(UUCG)G HAIRPIN LOOP

Catalytic RNAs recognize specific sequences of RNA and cleave at a spe cific site. In this study, we designed hammerhead ribozymes with a the rmodynamically stable loop of the sequence 5'C(UUCG)G3' to prevent the aggregation of ribozymes with hammerhead structures. The cleavage act ivities of these ribozymes were examined using a synthetic pentadecame r with the sequence for the c-Ha-ras mRNA mutated at codon 12 (GGU --> GUU). For in vivo studies, we constructed a plasmid which expressed a highly active ribozyme targeted against the mutated c-Ha-ras mRNA. Wh en this ribozyme-encoding gene and the activated c-Ha-ras gene were co transfected into NIH3T3 cells, morphologically normal cells were obtai ned. We also determined that the expression of the c-Ha-ras gene was i nhibited in these cells. These results show that ribozymes containing this stable hairpin loop are useful for the regulation of specific gen e expression in vivo.