High Sensitivity Measurement of Transcription Factor-DNA Binding Affinities by Competitive Titration Using Fluorescence Microscopy

To improve the understanding of transcription factor-DNA binding, we developed a method to measure dissociation constants at a large-scale using for instance anisotropy measurements. This technique allows obtaining automatically full titration curves to determine the dissociation constant with high sensitivity. It works in solution at equilibrium, has moderate throughput and large dynamical range.

We applied HiP-FA to refine the binding affinity landscape of transcription factors. However, the protocol could be applied to other kinds of binding events like protein-protein or protein-drug interactions, for example. I advise to first perform multiple titrations with the same binding partners to get an estimation about the reproducibility of the measurements.

If available, use an automated system for even better reducibility. To anneal the DNA oligomers of the reference DNA, mix seven microliters of each ten millimolar di-labeled forward and unlabeled reverse strand in one tube. For the competitor DNA, mix 20 microliters of each 100 millimolar unlabeled strand into two wells of a 96 welled PCR plate.

Then, use a standard PCR machine to heat up the DNA solutions to 70 degrees Celsius for the three minutes. Then, reduce to room temperature at the rate of 0.1 degrees Celsius per second. Use a microwave oven to melt 0.5 grams agarose in 100 milliliters binding buffer.

Add double distilled water to adjust the final volume to compensate for possible evaporation. Prepare ten milliliter stock aliquots. 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 of reference DNA, transcription factor protein, at a final concentration of 20-60 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 ten minutes at room temperature, and another 10 minutes at 4 degrees Celsius.

Use a multi-well 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. Take special care when pipetting the gel solutions that are viscous. Inaccurate pipetting can lower the accuracy for the determination of the dissociation constants.

In this study, HiP-FA method is applied to determine the DNA-binding preferences of a B-zipped transcription factor. Giant, from the fly segmentation gene network. The high similarity of the PWM's for replicates that were prepared either manually or using automation techniques demonstrated the high reproducibility of the HiP-FA method.

PWM's obtained by this method and other methods are overall similar. However, significant deviations can be seen at positions two and seven of the core motif where mutations can lead to either complete loss of binding, or much stronger binding than previous measurements. We use HiP-FA to generate refinements for the bindings of tens of transcription factor and it shows that obtains in the end a better prediction of data.

We believe that its measure of high accuracy can be very useful to better understand TF-DNA binding for all of the system or for all kinds of interactions.