Stable fluorescent dye-DNA complexes in high sensitivity detection of protein-DNA interactions. Application to heat...

The gel mobility-shift assay is an important tool for the study of protein-nucleic acid interactions. High detection sensitivity is typically attained by radioisotopic labeling of the target nucleic acid fragments. A novel fluorescence methodology offers significant advantages over this conventional approach. Ethidium, thiazole orange, and oxazole yellow homodimers form stable, highly fluorescent complexes with double-stranded DNA that can be detected in gels by a laser-excited, confocal, fluorescence scanning system with a sensitivity higher than that attainable with radioisotopic labeling. We describe here the use of these dyes in a gel-mobility assay to detect complexes of a truncation of the Kluyveromyces lactis heat shock transcription factor, containing the trimerization and DNA-binding domains (HSFDT), with target DNA. At an appropriate molar DNA base pair to dye ratio, the labeling of a DNA fragment with dimeric dye did not affect the binding to HSFDT. The detection of the fluorescent-dye labeled HSFDT-DNA complexes with the laser scanner achieves a spatial resolution far superior to that of conventional autoradiography and permits analysis of multimer protein-DNA complexes that are not resolved by traditional detection methods. We have used this technique to demonstrate that HSF forms multimeric complexes on DNA by addition of trimeric units. The latter conclusion is based on an analysis of the mobilities of the multiple HSFDT-DNA complexes and on a two-color mobility-shift fluorescence assay that uses a mutant of HSFDT engineered for site-specific labeling with fluorescein and target DNA labeled with an "energy transfer" dye, thiazole orange-thiazole blue heterodimer.