Probing Protein-DNA Interactions with Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy Utilizing...

Protein-DNA interactions are vital to cellular processes. Recently, single-molecule protein-induced fluorescence enhancement (smPIFE) has emerged as a promising tool for studying these interactions, which utilizes only simple single-dye labeling and provides sensitivity to short-range interactions. However, its application to freely diffusing molecules has been limited because it is difficult to quantify the PIFE-induced change in fluorescence intensity within a confocal observation volume. To overcome this problem, we adopted two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) to quantitatively assess PIFE through fluorescence lifetime information. Using a Cy3-labeled DNA hairpin and a restriction enzyme BamHI, we identified three fluorescence components corresponding to unbound DNA, bound DNA, and a binding-insensitive population. Based on the obtained results, the dissociation constant and the diffusion coefficients of bound and unbound DNA were determined. This work demonstrates the high potential of PIFE-2D FLCS for studying protein-DNA interactions at the single-molecule level in complex environments.