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Monitoring DNA triplex formation using multicolor fluorescence and application to insulin-like growth factor I promoter downregulation

Authors

  • Nadia Hégarat,

    1. Acides nucléiques: dynamique, ciblage et fonctions biologiques, INSERM U565, Paris, France
    2. Département Régulations, développement et diversité moléculaire, MNHN - CNRS UMR7196, Paris, France
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  • Darya Novopashina,

    1. Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia
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  • Alesya A. Fokina,

    1. Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia
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  • Alexandre S. Boutorine,

    1. Acides nucléiques: dynamique, ciblage et fonctions biologiques, INSERM U565, Paris, France
    2. Département Régulations, développement et diversité moléculaire, MNHN - CNRS UMR7196, Paris, France
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  • Alya G. Venyaminova,

    1. Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia
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  • Danièle Praseuth,

    1. Acides nucléiques: dynamique, ciblage et fonctions biologiques, INSERM U565, Paris, France
    2. Département Régulations, développement et diversité moléculaire, MNHN - CNRS UMR7196, Paris, France
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  • Jean-Christophe François

    Corresponding author
    1. Acides nucléiques: dynamique, ciblage et fonctions biologiques, INSERM U565, Paris, France
    2. Département Régulations, développement et diversité moléculaire, MNHN - CNRS UMR7196, Paris, France
    3. Sorbonne Universités, UPMC Univ Paris 06, UMR_S 938, CDR Saint Antoine, Paris, France
    4. Faculté de Médecine and Hôpital Saint Antoine, INSERM, UMR_S 938, CDR Saint Antoine, Paris, France
    • Correspondence

      J.-C. François, INSERM – UPMC UMRS938, CDR Saint Antoine, Faculté de Médecine Pierre et Marie Curie, Room 1107, 27 rue Chaligny F-75571 Paris 12, France

      Fax: +33 1 40 01 13 43

      Tel: +33 1 40 01 13 31

      E-mail: jean-christophe.francois@inserm.fr

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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.

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