Antisense PNA tridecamers targeted to the coding region of Ha-ras mRNA arrest polypeptide chain elongation

We have previously described the rational design of mutation-selective anti sense oligonucleotides targeted to codon 12 of oncogenic Ha-ras mRNA. in or der to further improve the biological efficacy of these unmodified oligonuc leotides, we have studied three different classes of modifications: peptide nucleic acid backbone (PNA), sugar modification (2'-O-methyl) and phosphor amidate Linkage (PN). We show that PNA is unique among the investigated ste ric blocking agents in its ability to specifically inhibit the translation of Ha-ras mRNA in vitro. The PNA RNA hybrid (T-m = 86 degrees C), which is not dissociated by cellular proteins and resists phenol extraction and urea denaturing conditions, specifically blocks the translation of mutated Ha-r as mRNA. A PNA tridecamer which forms with wild-type Ha-ras mRNA a duplex w ith a central mismatch had little effect on mRNA translation. Codon 12 is l ocated close to the translation initiation site and hybridization of the PN A at this position may interfere with the assembly of the translation initi ation complex. To test whether polypeptide chain elongation can also be blo cked, we have targeted PNA tridecamers to codons in the 74, 128 and 149 reg ions. These PNAs form equally stable duplexes as that formed by the PNA tar geted to the codon 12 region (ten G.C base-pairs out of 13). We show that P NA-RNA duplexes block the progression of the 80 S ribosome. Therefore, it i s possible to arrest translation with concomitant production of a truncated protein by using duplex-forming PNA oligonucleotides targeted to a G + C-r ich sequences. Our data demonstrate for the first time that a non-covalent duplex can arrest the translation machinery and polypeptide chain elongatio n. (C) 1999 Academic Press.