RNA Hairpin Invasion and Ribosome Elongation Arrest by Mixed Base PNA Oligomer

Recently, we have shown that peptide nucleic acid (PNA) tridecamers targeted to the codon 74, 128 and 149 regions of Ha- ras mRNA arrested translation elongation in vitro. Our data demonstrated for the first time that PNAs with mixed base sequence targeted to the coding region of a messenger RNA could arrest the translation machinery and polypeptide chain elongation. The peculiarity of the complexes formed with PNA tridecamers and Ha- ras mRNA rests upon the stability of PNA–mRNA hybrids, which are not dissociated by cellular proteins or multiple denaturing conditions. In the present study, we show that shorter PNAs such as a dodecamer or an undecamer targeted to the codon 74 region arrest translation elongation in vitro. The 13, 12, and 11-mer PNAs contain eight and the 10-mer PNA seven contiguous pyrimidine residues. Upon binding with parallel Hoogsteen base-pairing to the PNA–RNA duplex, six of the cytosine bases and one thymine base of a second PNA can form C·G∗C + and T·A∗T triplets. Melting experiments show two well-resolved transitions corresponding to the dissociation of the third strand from the core duplex and to melting of duplex at higher temperature. The enzymatic structure mapping of a target 27-mer RNA revealed a hairpin structure that is disrupted upon binding of tri-, dodeca-, undeca- and decamer PNAs. We show that the non-bonded nucleobase overhangs on the RNA stabilize the PNA–RNA hybrids and probably assist the PNA in overcoming the stable secondary structure of the RNA target. The great stability of PNA–RNA duplex and triplex structures allowed us to identify both 1:1 and 2:1 PNA–RNA complexes using matrix-assisted laser desorption/ionization time-of -flight mass spectrometry. Therefore, it is possible to successfully target mixed sequences in structured regions of messenger RNA with short PNA oligonucleotides that form duplex and triplex structures that can arrest elongating ribosomes.