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 pyrimidinerich 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.

Synthesis and triplex-forming properties of oligonucleotides capable of recognizing corresponding DNA duplexes containing four base pairs

“…Triplex-forming oligonucleotides (TFOs) can recognize the major groove of the corresponding DNA duplex via Hoogsteen base pairs. ( 1 ) A TFO could be a useful molecular tool for gene regulation ( 2 – 7 ) and specific gene modification. ( 8 , 9 ) However, unmodified TFOs have two serious drawbacks: low binding affinities and high sequence-dependencies.…”

Selective Preference of Parallel DNA Triplexes Is Due to the Disruption of Hoogsteen Hydrogen Bonds Caused by the Severe Nonisostericity between the G*GC and T*AT Triplets

“…It is well documented that Watson and Crick paired DNA duplex interacts with sequence specific oligonucleotides to form triple helices stabilised by either Hoogsteen (parallel triplex) or reverse Hoogsteen (antiparallel triplex) pair of hydrogen bonds. Evidences for involvement of triple helices in biological processes come from their participation in regulation of gene expression [ 1 , 2 ], DNA damage and repair [ 3 , 4 ], RNA processing and folding [ 5 , 6 , 7 ] and chromatin organization [ 8 ]. Triplexes are also known to impair DNA polymerization and influence DNA recombination process [ 9 ].…”

Fluorescently Sensing of DNA Triplex Assembly Using an Isoquinoline Alkaloid as Selector, Stabilizer, Inducer, and Switch‐On Emitter

“…However, a TFO rich in purines can associate with the polypurine stretch of dsDNA in an inverse phosphate‐backbone polarity orientation (antiparallel) through reverse‐Hoogsteen hydrogen bonding . Because the dsDNA major groove is usually the binding field of many functional enzymes and proteins while they are exerting their physiological action, the triplex structure is believed to regulate DNA‐related bioactivities including gene expression, DNA damage/repair, and even disease . Additionally, since the TFO binds to its dsDNA counterpart by means of the much weaker Hoogsteen/reverse‐Hoogsteen hydrogen bonding relative to the typical Waterson–Crick hydrogen bonding for dsDNA, the triplex is more flexible than dsDNA in nature and thus has found broad applications in molecular switches, nanodevices, and drug release .…”