5 years ago

Single-Molecule Photoactivation FRET: A General and Easy-to-Implement Approach to Break the Concentration Barrier

Single-Molecule Photoactivation FRET: A General and Easy-to-Implement Approach to Break the Concentration Barrier
Sijia Peng, Ruirui Sun, Wenjuan Wang, Chunlai Chen
Single-molecule fluorescence resonance energy transfer (sm-FRET) has become a widely used tool to reveal dynamic processes and molecule mechanisms hidden under ensemble measurements. However, the upper limit of fluorescent species used in sm-FRET is still orders of magnitude lower than the association affinity of many biological processes under physiological conditions. Herein, we introduce single-molecule photoactivation FRET (sm-PAFRET), a general approach to break the concentration barrier by using photoactivatable fluorophores as donors. We demonstrate sm-PAFRET by capturing transient FRET states and revealing new reaction pathways during translation using μm fluorophore labeled species, which is 2–3 orders of magnitude higher than commonly used in sm-FRET measurements. sm-PAFRET serves as an easy-to-implement tool to lift the concentration barrier and discover new molecular dynamic processes and mechanisms under physiological concentrations. Make use of the dark side: Photoactivatable fluorophores reduce the fluorescent background in FRET studies. Alternating between activation and excitation lasers leads to swift activation of the fluorophores once they bind and avoids exciting unbound activated fluorophores before they diffuse out of the excitation volume. This easy-to-implement single-molecule photoactivation FRET (sm-PAFRET) method lifts the FRET concentration barrier by 2–3 orders of magnitude.

Publisher URL: http://onlinelibrary.wiley.com/resolve/doi

DOI: 10.1002/anie.201702731

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