title: Monitoring DNA triplex formation using multicolor fluorescence and application to insulin-like growth factor I promoter downregulation.
abstract: Inhibition of insulin-like growth factor I (IGF-I) signaling is a promising antitumor strategy and nucleic acid-based approaches have been investigated to target genes in the pathway. Here, we sought to modulate IGF-I transcriptional activity using triple helix formation. The IGF-I P1 promoter contains a purine/pyrimidine (R/Y) sequence that is pivotal for transcription as determined by deletion analysis and can be targeted with a triplex-forming oligonucleotide (TFO). We designed modified purine- and pyrimidine-rich TFOs to bind to the R/Y sequence. To monitor TFO binding, we developed a fluorescence-based gel-retardation assay that allowed independent detection of each strand in three-stranded complexes using end-labeling with Alexa 488, cyanine (Cy)3 and Cy5 fluorochromes. We characterized TFOs for their ability to inhibit restriction enzyme activity, compete with DNA-binding proteins and inhibit IGF-I transcription in reporter assays. TFOs containing modified nucleobases, 5-methyl-2'-deoxycytidine and 5-propynyl-2'-deoxyuridine, specifically inhibited restriction enzyme cleavage and formed triplexes on the P1 promoter fragment. In cells, deletion of the R/Y-rich sequence led to 48% transcriptional inhibition of a reporter gene. Transfection with TFOs inhibited reporter gene activity to a similar extent, whereas transcription from a mutant construct with an interrupted R/Y region was unaffected, strongly suggesting the involvement of triplex formation in the inhibitory mechanisms. Our results indicate that nuclease-resistant TFOs will likely inhibit endogenous IGF-I gene function in cells.
authors.affiliation: Acides nucléiques: dynamique, ciblage et fonctions biologiques, INSERM U565, Paris, France.|Département Régulations, développement et diversité moléculaire, MNHN - CNRS UMR7196, Paris, France.
authors.forename: Nadia
authors.initials: N
authors.lastname: Hégarat
authors.affiliation: Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia.
authors.forename: Darya
authors.initials: D
authors.lastname: Novopashina
authors.affiliation: Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia.
authors.forename: Alesya A
authors.initials: AA
authors.lastname: Fokina
authors.affiliation: Acides nucléiques: dynamique, ciblage et fonctions biologiques, INSERM U565, Paris, France.|Département Régulations, développement et diversité moléculaire, MNHN - CNRS UMR7196, Paris, France.
authors.forename: Alexandre S
authors.initials: AS
authors.lastname: Boutorine
authors.affiliation: Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia.
authors.forename: Alya G
authors.initials: AG
authors.lastname: Venyaminova
authors.affiliation: Acides nucléiques: dynamique, ciblage et fonctions biologiques, INSERM U565, Paris, France.|Département Régulations, développement et diversité moléculaire, MNHN - CNRS UMR7196, Paris, France.
authors.forename: Danièle
authors.initials: D
authors.lastname: Praseuth
authors.affiliation: Acides nucléiques: dynamique, ciblage et fonctions biologiques, INSERM U565, Paris, France.|Département Régulations, développement et diversité moléculaire, MNHN - CNRS UMR7196, Paris, France.|Sorbonne Universités, UPMC Univ Paris 06, UMR_S 938, CDR Saint Antoine, Paris, France.|Faculté de Médecine and Hôpital Saint Antoine, INSERM, UMR_S 938, CDR Saint Antoine, Paris, France.
authors.forename: Jean-Christophe
authors.initials: JC
authors.lastname: François
mesh_terms: D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D015536:Down-Regulation; D005453:Fluorescence; D005456:Fluorescent Dyes; D017930:Genes, Reporter; D007334:Insulin-Like Growth Factor I / Q000235:genetics* / Q000378:metabolism; D009690:Nucleic Acid Conformation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats
chemical_list: D005456:Fluorescent Dyes; C548904:insulin-like growth factor-1, rat; C051802:triplex DNA; D007334:Insulin-Like Growth Factor I; D004247:DNA
keywords: DNA-binding protein; IGF-I promoter; antigene strategy; fluorescent probes; triple helix