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Monitoring DNA triplex formation using multicolor fluorescence and application to insulin‐like growth factor I promoter downregulation
Author(s) -
Hégarat Nadia,
Novopashina Darya,
Fokina Alesya A.,
Boutorine Alexandre S.,
Venyaminova Alya G.,
Praseuth Danièle,
François JeanChristophe
Publication year - 2014
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.12714
Subject(s) - microbiology and biotechnology , reporter gene , oligonucleotide , biology , dna , transfection , transcription (linguistics) , transcription factor , chemistry , promoter , gene , biochemistry , gene expression , linguistics , philosophy
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 TFO s 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 TFO s for their ability to inhibit restriction enzyme activity, compete with DNA ‐binding proteins and inhibit IGF ‐I transcription in reporter assays. TFO s 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 TFO s 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 TFO s will likely inhibit endogenous IGF ‐I gene function in cells.