Cellular antisense activity of peptide nucleic acid (PNAs) targeted to HIV-1 polypurine tract (PPT) containing RNA.
DNA and RNA oligomers that contain stretches of guanines can associate to form stable secondary structures including G-quadruplexes. Our study shows that the (UUAAAAGAAAAGGGGGGAU) RNA sequence, from the human immunodeficiency virus type 1 (HIV-1 polypurine tract or PPT sequence) forms in vitro a stable folded structure involving the G-run. We have investigated the ability of pyrimidine peptide nucleic acid (PNA) oligomers targeted to the PPT sequence to invade the folded RNA and exhibit biological activity at the translation level in vitro and in cells. We find that PNAs can form stable complexes even with the structured PPT RNA target at neutral pH. We show that T-rich PNAs, namely the tridecamer-I PNA (C4T4CT4) forms triplex structures whereas the C-rich tridecamer-II PNA (TC6T4CT) likely forms a duplex with the target RNA. Interestingly, we find that both C-rich and T-rich PNAs arrested in vitro translation elongation specifically at the PPT target site. Finally, we show that T-rich and C-rich tridecamer PNAs that have been identified as efficient and specific blockers of translation elongation in vitro, specifically inhibit translation in streptolysin-O permeabilized cells where the PPT target sequence has been introduced upstream the reporter luciferase gene.
Boutimah-Hamoudi
Fatima
F
INSERM, U565, Acides nucléiques: dynamique, ciblage et fonctions biologiques, 57 rue Cuvier, CP26, Paris Cedex 05, F-75231, France.
Leforestier
Erwan
E
Sénamaud-Beaufort
Catherine
C
Nielsen
Peter E
PE
Giovannangeli
Carine
C
Saison-Behmoaras
Tula Ester
TE
D001426:Bacterial Proteins; D001483:Base Sequence; D024202:Electrophoretic Mobility Shift Assay; D017930:Genes, Reporter; D015497:HIV-1 / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000494:pharmacology*; D010441:Peptide Chain Elongation, Translational / Q000187:drug effects*; D020135:Peptide Nucleic Acids / Q000737:chemistry / Q000494:pharmacology*; D011687:Purines / Q000032:analysis; D012367:RNA, Viral / Q000737:chemistry / Q000187:drug effects*; D013301:Streptolysins; D013696:Temperature; D014764:Viral Proteins / Q000096:biosynthesis
D001426:Bacterial Proteins; D016376:Oligonucleotides, Antisense; D020135:Peptide Nucleic Acids; D011687:Purines; D012367:RNA, Viral; D013301:Streptolysins; D014764:Viral Proteins; C025964:streptolysin O