High-resolution assessment of protein DNA binding affinity and selectivity utilizing a fluorescent intercalator...

Protein titration displacement of ethidium bromide bound to hairpin deoxyoligonucleotides containing any sequence of interest provides a well-defined titration curve (measuring the loss of fluorescence derived from the DNA bound ethidium bromide) that provides both absolute binding constants (K(a)) and stoichiometry of binding. This use of a fluorescent intercalator displacement (FID) assay for establishing protein DNA binding affinity and selectivity is demonstrated with the examination of the LEF-1 HMG domain binding to hairpin deoxyoligonucleotides containing its commonly accepted consensus sequence 5'-CTTTGWW (W=A or T) and those modified (5'-CTNTGWW) to examine sequences implicated in early studies (5'-CTNTG). The effectiveness of the FID assay coupled with its technically non-demanding experimental use makes it an attractive alternative or complement to selection screening, footprinting or affinity cleavage, and electrophoretic mobility shift assays for detecting, characterizing, and quantitating protein DNA binding affinity and selectivity.