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

Autor: Hégarat N; Acides nucléiques: dynamique, ciblage et fonctions biologiques, INSERM U565, Paris, France.; Département Régulations, développement et diversité moléculaire, MNHN - CNRS UMR7196, Paris, France., Novopashina D; Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia., Fokina AA; Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia., Boutorine AS; Acides nucléiques: dynamique, ciblage et fonctions biologiques, INSERM U565, Paris, France.; Département Régulations, développement et diversité moléculaire, MNHN - CNRS UMR7196, Paris, France., Venyaminova AG; Laboratory of RNA Chemistry, Institute of Chemical Biology and Fundamental Medicine, Siberian Division of Russian Academy of Sciences, Novosibirsk, Russia., Praseuth D; Acides nucléiques: dynamique, ciblage et fonctions biologiques, INSERM U565, Paris, France.; Département Régulations, développement et diversité moléculaire, MNHN - CNRS UMR7196, Paris, France., François JC; 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.
Jazyk: angličtina
Zdroj: The FEBS journal [FEBS J] 2014 Mar; Vol. 281 (5), pp. 1417-1431. Date of Electronic Publication: 2014 Jan 27.
DOI: 10.1111/febs.12714
Abstrakt: 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.
(© 2014 FEBS.)
Databáze: MEDLINE
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