Microsecond Conformational Dynamics of Biopolymers Revealed by Dynamic-Quenching Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy with Single Dye Labeling.

The single-molecule Förster resonance energy transfer (smFRET) technique is widely used for studying conformational dynamics of biopolymers. However, smFRET requires double dye labeling and is usually utilized for detecting dynamics on slow time scales (≳ milliseconds). In this Letter, we report dynamic-quenching two-dimensional fluorescence lifetime correlation spectroscopy (DQ 2D FLCS) that can elucidate the microsecond conformational dynamics of biopolymers with only single dye labeling. In DQ 2D FLCS, the difference in solvent accessibility of the labeled dye makes the fluorescence lifetime different, which is used for distinguishing different conformers. By applying DQ 2D FLCS to a singly labeled DNA hairpin, we successfully detect microsecond interconversion dynamics between the open and closed forms and evaluate the state-specific solvent accessibility of each form with Stern-Volmer analysis. Because DQ 2D FLCS is sensitive to the local structural change, it is complementary to FRET-based 2D FLCS and thus is a new, powerful tool for studying structural dynamics of biopolymers.