Short pyrimidine stretches containing mixed base PNAs are versatile tools to induce translation elongation arrest and truncated protein synthesis.
Recently, we showed that antisense peptide nucleic acids (PNA) containing a short pyrimidine stretch (C(4)TC(3)) invade Ha-ras mRNA hairpin structures to form highly stable duplex and triplex complexes that contribute to the arrest of translation elongation. The antisense PNA targeted to codon 74 of Ha-ras was designed to bind in antiparallel configuration (the N-terminal of the PNA faces the 3'-end of target mRNA), as PNA/RNA duplexes are most stable in this configuration. In order to show that different sequences in the coding region could be targeted successfully with antisense PNAs, we extended our study to three other purine-rich targets. We show that the tridecamer PNA (targeted to codon 149) containing a CTC(3)T pyrimidine stretch forms with the complementary oligoribonucleotide (ORN) a stable (PNA)(2)/ORN triplex at neutral pH (T(m) = 50 degrees C) and arrests Ha-ras mRNA translation elongation. Interestingly, the thermal stability of triplexes formed with PNAs designed to bind to the complementary ORN in a parallel orientation (the N-terminal of the PNA faces the 5'-end of target) was higher than that formed with antiparallel oriented PNAs (T(m) = 58 degrees C). Because parallel and antiparallel PNAs form stable triplexes with target sequence, they act as translation elongation blockers. These duplex-forming and partly triplex-forming PNAs targeted to Ha-ras mRNA also arrested translation elongation at specific polypurine sites contained in the mRNA coding for HIV-integrase protein. Furthermore, the tridecamer PNA containing the C(3)TC(4) motif was more active than a bis-PNA in which the Hoogsteen recognizing strand was linked to the Watson-Crick recognizing strand by a flexible linker. Pyrimidine-rich, short PNAs that form very stable duplexes with target Ha-ras mRNA inhibit translation by a mechanism that does not involve ribosome elongation arrest, whereas PNAs forming duplex and triplex structures arrest ribosome elongation. The remarkable efficacy of the tridecamer PNAs in arresting translation elongation of HIV-1 integrase mRNA is explained by their ability to form stable triplexes at neutral pH with short purine sequences.
Sénamaud-Beaufort
Catherine
C
Laboratoire de Biophysique, Muséum National d'Histoire Naturelle, INSERM 5153, CNRS UMR 8646, 75231, Paris, France.
Leforestier
Erwan
E
Saison-Behmoaras
Tula Ester
TE
D000818:Animals; D001483:Base Sequence; D024202:Electrophoretic Mobility Shift Assay; D019427:HIV Integrase / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016376:Oligonucleotides, Antisense / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology*; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects* / Q000235:genetics; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology*; D011500:Protein Synthesis Inhibitors / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology*; D016283:Proto-Oncogene Proteins p21(ras) / Q000096:biosynthesis / Q000187:drug effects / Q000235:genetics; D011687:Purines / Q000737:chemistry; D011743:Pyrimidines / Q000737:chemistry; D051381:Rats; D013057:Spectrum Analysis
D016376:Oligonucleotides, Antisense; D020135:Peptide Nucleic Acids; D011500:Protein Synthesis Inhibitors; D011687:Purines; D011743:Pyrimidines; D019427:HIV Integrase; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)