Development of Lipid-Tethered Fluorescence Correlation Spectroscopy and Its Application to Conformational Dynamics of Hairpin DNA

Fluorescence correlation spectroscopy (FCS) is widely used to probe conformational dynamics of biomolecules; however, rapid diffusion in solution often obscures submillisecond to millisecond conformational transitions. Here, we introduce lipid-tethered FCS (LT-FCS), in which molecules are tethered to a cholesterol-rich supported lipid bilayer (SLB) to reduce lateral diffusion while maintaining molecular exchange in a focal region. The average residence time of molecules in the focal region is approximately 30-fold longer on the SLB than in solution, enabling clear separation of submillisecond conformational transition contributions in the autocorrelation function from the diffusion-related contributions. Furthermore, the use of fluorescence lifetime information on LT-FCS enables us to quantitatively analyze millisecond conformational transitions. We verified the capability of LT-FCS to analyze the submillisecond to millisecond conformational transition dynamics of hairpin DNAs with different stem lengths. LT-FCS thus provides a practical approach to access conformational dynamics in the submillisecond to millisecond regime, bridging the gap between conventional FCS and single-molecule FRET techniques.