Sequence specific inhibition of in vitro translation of mutated or normal ras p21.

Abstract

Antisense methylphosphonate-modified oligomers (ONMP) complementary to 8 nucleotides spanning the twelfth amino acid codon of human c-Ha-ras have been synthesized to explore their inhibitory effect on ras p21 translation. The ONMP Ras 0, perfectly complementary to the specific target region in normal c-Ha-ras, inhibits cell-free translation of p21 from a normal c-Ha-ras mRNA template in a dose-dependent manner. At 200uM Ras 0, p21 translation is inhibited by almost 90% in a rabbit reticulocyte lysate; at 100uM, p21 is reduced by over 60%. Ras I and Ras II contain, internally, a single and double base mismatch, respectively. The inhibitory activity of these ONMPs is reduced in proportion to the number of mismatches. When the target mRNA encodes the activated c-Ha-ras differing by a single nucleotide at the twelfth amino acid codon from normal c-Ha-ras, the magnitude of the inhibitory effect of Ras I increased significantly because Ras I is now perfectly complementary to its target mRNA. In turn, Ras 0, now only partially complementary, is considerably less effective at the same concentrations. Therefore, the inhibitory effect is exquisitely sensitive to the extent of the sequence complementarity between the antisense ONMP and the targeted mRNA.