High-resolution assessment of protein DNA binding affinity and selectivity utilizing a fluorescent intercalator displacement (FID) assay.
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.
Ham
Young-Wan
YW
Department of Chemistry and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA.
Tse
Winston C
WC
Boger
Dale L
DL
D000818:Animals; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004247:DNA / Q000737:chemistry*; D004268:DNA-Binding Proteins / Q000494:pharmacology; D004996:Ethidium; D005456:Fluorescent Dyes / Q000737:chemistry*; D007364:Intercalating Agents / Q000737:chemistry*; D051821:Lymphoid Enhancer-Binding Factor 1; D051379:Mice; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D014157:Transcription Factors / Q000494:pharmacology
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D007364:Intercalating Agents; C497574:Lef1 protein, mouse; D051821:Lymphoid Enhancer-Binding Factor 1; D011506:Proteins; D014157:Transcription Factors; D004247:DNA; D004996:Ethidium