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

• Published in: The FEBS journal Vol. 281; no. 5; pp. 1417 - 1431
• Main Authors: Hégarat, Nadia, Novopashina, Darya, Fokina, Alesya A., Boutorine, Alexandre S., Venyaminova, Alya G., Praseuth, Danièle, François, Jean‐Christophe
• Format: Journal Article
• Language: English
• Published: England Blackwell Publishing Ltd 01.03.2014
• Subjects: Animals; antigene strategy; Base Sequence; Bases (nucleic acids); Binding; Cell Line; Cellular biology; Clonal deletion; Deoxyribonucleic acid; DNA; DNA - chemistry; DNA - genetics; DNA - metabolism; DNA‐binding protein; Down-Regulation; Enzymatic activity; Enzyme activity; Enzymes; Fluorescence; Fluorescent Dyes; fluorescent probes; Fluorophores; Gene deletion; Gene expression; Genes, Reporter; Growth factors; IGF‐I promoter; Insulin; Insulin-Like Growth Factor I - genetics; Insulin-Like Growth Factor I - metabolism; Insulin-like growth factors; Nuclease; Nucleic Acid Conformation; Nucleic acids; Oligonucleotides; Promoter Regions, Genetic; Protein Binding; Rats; Reporter gene; Transcription; Transfection; triple helix; Tumors; triple helix; DNA-binding protein; antigene strategy; IGF-I promoter; fluorescent probes
• ISSN: 1742-464X; 1742-4658
• DOI: 10.1111/febs.12714

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. We targeted the purine/pyrimidine (R/Y) sequence in the IGF-I promoter using modified triplex‐forming oligonucleotides (TFOs). A fluorescence‐based gel retardation assay that allows simultaneous detection of all three strands was developed and used to monitor triplex formation and to demonstrate protein binding to DNA. We showed that a TFO specifically inhibited IGF-I transcription in cells using a reporter gene assay.