Determination of transcription factor binding to DNA

摘要

A method for determining the interaction between a nucleic acid and a nucleic acid binding molecule, such a transcription factor, comprises contacting a nucleic acid with a fluorescently labeled nucleic acid binding molecule and measuring the diffusion time of the nucleic acid binding molecule in solution. The fluorescent label may be a low molecular weight compound, or a fusion with a green fluorescent protein. The diffusion time in solution may be measured by single molecule detection in a confocal detection volume. Preferably, the diffusion time is measured by fluorescence correlation spectrometry (FCS) or fluorescence cross-correlation spectrometry (FCCS). For FCCS, the nucleic acid molecule carries a second, different fluorescent labeled group. The method may be used to identify binding sites for transcription factors using overlapping genomic DNA molecules. Alternatively, the effects of test compounds on interaction between a nucleic acid and a nucleic acid binding molecule are determined. The method is exemplified using fusions of GFP and the homeodomain proteins Antennapedia (Antp) and Sex combs reduced (Scr).