The human c-Src proto-oncogene promoter contains multiple targets for triplex-forming oligonucleotides

The overexpression and activation of the human c-Src proto-oncogene is closely associated with cancer of the colon and breast. Characterization of the 5' region of the c-Src gene revealed that the promoter is very GC rich, regulated by the Sp family of transcription factors, and contains four perfect homopolypurine/homopolypyrimidine tracts (Pu:Py tracts). These Pu:Py tracts (TC1, TC1.1, TC2, and TC3) are located near or overlap critical Spl binding sites required for full activation of the gene. Triplex-forming oligonucleotides (TFOs) can be targeted to such sequences with high affinity to form intermolecular triple-helical DNA and modulate transcriptional activity. We therefore designed a series of antiparallel purine-based TFOs and measured their ability to form triplexes with the c-Src promoter Pu:Py tracts using comigration, bandshift, and chemical footprint techniques. With one interesting exception, all of the TFOs were found to bind with specificity and high affinity (67 nM-28 nM) to their target sequences at physiologic pH. These results indicate that the c-Src gene can successfully form stable triplexes under physiologic conditions and is, therefore, an excellent candidate for triplex-mediated transcriptional downregulation.