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.
Ritchie
S
S
Department of Biochemistry, University of Saskatchewan, Saskatoon, Canada.
Bonham
K
K
D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics / Q000502:physiology; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D016391:Genes, src / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D000090063:Proto-Oncogene Mas; D016415:Sequence Alignment; D013379:Substrate Specificity
C000715470:MAS1 protein, human; D009841:Oligonucleotides; D000090063:Proto-Oncogene Mas; C051802:triplex DNA; D004247:DNA