Real-time submillisecond single-molecule FRET dynamics of freely diffusing molecules with liposome tethering

Abstract

Single-molecule fluorescence resonance energy transfer (smFRET) is one of the powerful techniques for deciphering the dynamics of unsynchronized biomolecules. However, smFRET is limited in its temporal resolution for observing dynamics. Here, we report a novel method for observing real-time dynamics with submillisecond resolution by tethering molecules to freely diffusing 100-nm-sized liposomes. The observation time for a diffusing molecule is extended to 100 ms with a submillisecond resolution, which allows for direct analysis of the transition states from the FRET time trace using hidden Markov modelling. We measure transition rates of up to 1,500 s–1 between two conformers of a Holliday junction. The rapid diffusional migration of Deinococcus radiodurans single-stranded DNA-binding protein (SSB) on single-stranded DNA is resolved by FRET, faster than that of Escherichia coli SSB by an order of magnitude. Our approach is a powerful method for studying the dynamics and movements of biomolecules at submillisecond resolution.

Two conformational states in D-shaped DNA: Effects of local denaturation

“…Recently, we performed single-molecule experiments in which we applied alternating laser excitation fluorescence resonance energy transfer (ALEX-FRET)1920 to D-shaped DNA composed of 30 bp ds portions and ss portions of various lengths18. The FRET signal from the two dyes attached to the ds portion near both ends (see Fig.…”

Recent advancement of light‐based single‐molecule approaches for studying biomolecules

“…In diffusion‐based smFRET, point detectors that use a single pixel such as avalanche photodiodes or photomultiplier tubes provide sub‐millisecond temporal resolution, but the observation window is limited to the ~1 ms diffusion time of molecules through a sub‐femtoliter confocal detection volume. However, tethering a biomolecule to freely diffusing liposomes increased the observation time to >10 ms (Figure b; J. Y. Kim, Kim, & Lee, ). In immobilized smFRET, the frame rate of the camera limits the temporal resolution.…”

“…However, tethering a biomolecule to freely diffusing liposomes increased the observation time to >10 ms (Figure 2b; J. Y. Kim, Kim, & Lee, 2015). In immobilized smFRET, the frame rate of the camera limits the temporal resolution.…”

A real‐time in vitro assay to evaluate the release of macromolecules from liposomes

“…FRET occurs when there is overlap of the emission spectrum of the donor and absorbance spectrum of the acceptor and for this to occur, the distance between the donor and acceptor moieties must be less that <10 nm . If the donor and acceptor moieties are conjugated onto different macromolecules, they may be separated by more than 10 nm, and FRET (quenching) will not occur.…”

“…FRET occurs when there is overlap of the emission spectrum of the donor and absorbance spectrum of the acceptor 12 and for this to occur, the distance between the donor and acceptor moieties must be less that <10 nm. 9 If the donor and acceptor moieties are conjugated onto different macromolecules, they may be separated by more than 10 nm, and FRET (quenching) will not occur. Consequently, the applicability of FRET to studies with macromolecules is postulated to depend of the molecular weight (hydrodynamic radius) of the macromolecules, the labelling density of donor or acceptor moieties, the location of the donor/acceptor moieties within the tertiary structure of the macromolecules and the proximity of the macromolecules.…”