Biomolecular systems,such as proteins,crucially rely on dynamic processes at the nanoscale.Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular dr...Biomolecular systems,such as proteins,crucially rely on dynamic processes at the nanoscale.Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that controlbiomolecular systems.Single-molecule fluorescence resonance energy transfer(smFRET)is a powerful technique to observe inreal-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states.Currently,this technique is fundamentally limited by irreversible photobleaching,causing the untimely end of the experiment andthus,a narrow temporal bandwidth of≤3 orders of magnitude.Here,we introduce“DyeCycling”,a measurement scheme withwhich we aim to break the photobleaching limit in smFRET.We introduce the concept of spontaneous dye replacement bysimulations,and as an experimental proof-of-concept,we demonstrate the intermittent observation of a single biomolecule forone hour with a time resolution of milliseconds.Theoretically,DyeCycling can provide>100-fold more information per singlemolecule than conventional smFRET.We discuss the experimental implementation of DyeCycling,its current and fundamentallimitations,and specific biological use cases.Given its general simplicity and versatility,DyeCycling has the potential torevolutionize the field of time-resolved smFRET,where it may serve to unravel a wealth of biomolecular dynamics by bridgingfrom milliseconds to the hour range.展开更多
文摘Biomolecular systems,such as proteins,crucially rely on dynamic processes at the nanoscale.Detecting biomolecular nanodynamics is therefore key to obtaining a mechanistic understanding of the energies and molecular driving forces that controlbiomolecular systems.Single-molecule fluorescence resonance energy transfer(smFRET)is a powerful technique to observe inreal-time how a single biomolecule proceeds through its functional cycle involving a sequence of distinct structural states.Currently,this technique is fundamentally limited by irreversible photobleaching,causing the untimely end of the experiment andthus,a narrow temporal bandwidth of≤3 orders of magnitude.Here,we introduce“DyeCycling”,a measurement scheme withwhich we aim to break the photobleaching limit in smFRET.We introduce the concept of spontaneous dye replacement bysimulations,and as an experimental proof-of-concept,we demonstrate the intermittent observation of a single biomolecule forone hour with a time resolution of milliseconds.Theoretically,DyeCycling can provide>100-fold more information per singlemolecule than conventional smFRET.We discuss the experimental implementation of DyeCycling,its current and fundamentallimitations,and specific biological use cases.Given its general simplicity and versatility,DyeCycling has the potential torevolutionize the field of time-resolved smFRET,where it may serve to unravel a wealth of biomolecular dynamics by bridgingfrom milliseconds to the hour range.
