This article describes a method to measure transcription factor (TF)-DNA interactions using fluorescence anisotropy. The protocol involves preparing a multi-well plate with gel containing fluorophore-tagged DNA and TF, followed by analysis using high-performance fluorescence anisotropy microscopy.
Transcription factor, TF, is a protein that binds to a gene's specific DNA region and regulates transcription.
To measure TF-DNA interactions by fluorescence anisotropy, take a multi-well plate. Add molten gel mixed with a buffered solution, containing known concentrations of fluorophore-tagged reference DNA comprising a TF-binding site and a higher concentration of TF molecules, to the well and incubate.
As the labeled reference DNA distributes in the gel, a higher TF molecule concentration ensures all the DNA molecules are TF-bound. Add a buffered solution containing unlabeled competitor DNA with a single-point mutation.
Analyze the plate's gel using high-performance fluorescence anisotropy microscopy.
Polarized light excites the reference DNA-bound fluorophore, which reorients due to molecular tumbling and emits multi-directional depolarized light ― a phenomenon called fluorescence anisotropy, FA.
Upon binding to TF, the reference DNA-bound fluorophore's molecular weight increases and it tumbles slower, emitting more polarized light that increases FA.
The unlabeled competitor DNA diffuses across the gel matrix over time, forming a high-to-low concentration gradient. A higher unlabeled competitor DNA concentration displaces the labeled-reference DNA, binding itself to TF. The released labeled-reference oligomers tumble faster, decreasing FA.
Plot the fluorophore's FA and the competitor DNA's concentration gradient over time ― which decreases as more competitor DNA molecules displace the reference DNA.
To prepare the titration and calibration wells of a 96-well plate, transfer two gel stock aliquots to a 75-degree Celsius incubator shaker.
For each titration well, add 1.4 nanomoles reference DNA, transcription factor protein at a final concentration of 20 to 60 nanomoles, 0.2 millimolar DTT, and the binding buffer to 240 microliters of the melted gel.
Mix thoroughly by inverting and shaking the tube. Then, slowly pipette 200 microliters per well of the DNA-containing gel in the designated titration wells of a 96-well plate. For each calibration well, add 5 nanomoles Nile blue dye to 240 microliters of the melted gel. Avoiding air bubbles, slowly pipette 200 microliters of the dye-containing gel solution in five to six wells of the 96-well plate.
Place the gel on a perfectly horizontal surface and incubate for 10 minutes at room temperature, and another 10 minutes at 4 degrees Celsius. Use a multiwell plate reader to check the homogeneity of the gel height levels in different wells of the plate.
To prepare the competitor DNA solution, first, combine the labeled reference DNA, the protein, and the three-fold concentrated binding buffer. Then, mix 20 microliters of the solution with 40 microliters of each of the annealed competitor DNA solutions.
For each calibration well, combine the appropriate amounts of one of the annealed competitor DNA solutions and the three-fold concentrated binding buffer containing 15-millimolar Nile blue dye solution. Finally, add 50 microliters of the mixed competitor solutions on top of the gels as simultaneously as possible.
To begin image acquisition, place the 96-well plate on the microscope stage. Then, take time series of z-stack images of wells until complete unbinding of the protein from the reference DNA.
Perform the titration assay according to the manuscript. Then, use HiP-FA software to create titration curves for individual competitor sequences. Then, click the "Export" button to obtain the dissociation constant and concentration of the active protein in each titration well.
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