Fluorescence Anisotropy to Determine Transcription Factor-DNA Binding Affinity

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.