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

Sijia PengRuirui SunWenjuan WangChunlai Chen
Published in: Angewandte Chemie (International ed. in English) (2017)
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.
Keyphrases
  • single molecule
  • energy transfer
  • living cells
  • atomic force microscopy
  • quantum dots
  • genome wide
  • machine learning
  • brain injury
  • cerebral ischemia
  • fluorescent probe
  • high speed