title: A triple helix-forming oligonucleotide-intercalator conjugate acts as a transcriptional repressor via inhibition of NF kappa B binding to interleukin-2 receptor alpha-regulatory sequence.
abstract: Oligonucleotide-directed triplex formation within upstream regulatory sequences is envisioned as a potential tool for gene inhibition. However, this approach requires that triple helix-forming oligonucleotides are chemically modified, so that the triplex is stable under physiological conditions. Here, we have compared several chemical modifications of an oligonucleotide, targeted to a natural 15-base pair homopyrimidine.homopurine sequence located in the upstream regulatory region of the gene encoding the interleukin-2 receptor alpha chain (p55, IL-2 R alpha). Methylation of the cytosines strongly stabilized the triplex. Further attachment of an intercalating agent (acridine) dramatically increased the stability of the triplex, as assessed by Tm measurements or by band shift assays. Furthermore, the acridine-derivatized oligonucleotide was more efficient in competing away high affinity DNA-binding proteins, as assessed by restriction enzyme inhibition assays. Using a novel footprinting assay, we have further shown that the interaction of the methylcytosine-substituted, acridine-derivatized oligonucleotide with a plasmidic target, harboring the IL-2 R alpha regulatory region, remains highly sequence specific, occurs at physiological pH and is independent of the superhelicity of the plasmid. Acridine derivatization did not impair the exquisite target specificity of triplex formation, since the derivatized oligonucleotide inhibited the binding of nuclear proteins to the overlapping NF kappa B enhancer sequence on an IL-2 R alpha target and not on the related human immunodeficiency virus long terminal repeat target. Finally, the oligonucleotide inhibited the NF kappa B-dependent tax-induced transcriptional activation of the IL-2 R alpha chloramphenicol acetyltransferase construct in live cells, whereas it did not have any effect on a human immunodeficiency virus long terminal repeat chloramphenicol acetyltransferase construct. We conclude that this modified oligonucleotide acts as a transcriptional repressor for the IL-2 R alpha gene via triple helix formation with regulatory sequences.
authors.affiliation: Laboratoire d'Immunobiologie, Unité de Recherche Associée 1156 Centre National de la Recherche Scientifique, Institut Gustave Roussy, Villejuif, France.
authors.forename: M
authors.initials: M
authors.lastname: Grigoriev
authors.forename: D
authors.initials: D
authors.lastname: Praseuth
authors.forename: P
authors.initials: P
authors.lastname: Robin
authors.forename: A
authors.initials: A
authors.lastname: Hemar
authors.forename: T
authors.initials: T
authors.lastname: Saison-Behmoaras
authors.forename: A
authors.initials: A
authors.lastname: Dautry-Varsat
authors.forename: N T
authors.initials: NT
authors.lastname: Thuong
authors.forename: C
authors.initials: C
authors.lastname: Hélène
authors.forename: A
authors.initials: A
authors.lastname: Harel-Bellan
mesh_terms: D000166:Acridines / Q000494:pharmacology*; D001483:Base Sequence; D002460:Cell Line; D003850:Deoxyribonuclease I; D004742:Enhancer Elements, Genetic / Q000187:drug effects; D006678:HIV / Q000235:genetics; D016325:HIV Long Terminal Repeat; D006801:Humans; D007364:Intercalating Agents / Q000494:pharmacology*; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000494:pharmacology*; D010957:Plasmids; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D015375:Receptors, Interleukin-2 / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid / Q000187:drug effects; D015183:Restriction Mapping; D014158:Transcription, Genetic / Q000187:drug effects*; D015533:Transcriptional Activation; D014162:Transfection
chemical_list: D000166:Acridines; D007364:Intercalating Agents; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D015375:Receptors, Interleukin-2; D003850:Deoxyribonuclease I