Login / Signup

Scanning Two-Dimensional Fluorescence Lifetime Correlation Spectroscopy: Conformational Dynamics of DNA Holliday Junction from Microsecond to Subsecond.

Wooseok HeoKazuto HasegawaKenji OkamotoYasushi SakoKunihiko IshiiTahei Tahara
Published in: The journal of physical chemistry letters (2022)
Single-molecule Förster resonance energy transfer (smFRET) is widely utilized to investigate the structural heterogeneity and dynamics of biomolecules. However, it has been difficult to simultaneously achieve a wide observation time window, a high structure resolution, and a high time resolution with the current smFRET methods. Herein, we introduce a new method utilizing two-dimensional fluorescence lifetime correlation spectroscopy (2D FLCS) and surface immobilization techniques. This method, scanning 2D FLCS, enables us to examine the structural heterogeneity and dynamics of immobilized biomolecules on a time scale from microsecond to subsecond by slowly scanning the sample stage at the rate of ∼1 μm/s. Application to the DNA Holliday junction (HJ) complex under various [Mg 2+ ] conditions demonstrates that scanning 2D FLCS enables tracking reaction kinetics from 25 μs to 30 ms with a time resolution as high as 1 μs. Furthermore, the high structure resolution of scanning 2D FLCS allows us to unveil the ensemble nature of each isomer state and the heterogeneity of the dynamics of the HJ.
Keyphrases