Ghosh Srijayee, Schmid Sonja
Department of Chemistry, University of Basel, Basel, Switzerland.
QRB Discov. 2024 Dec 3;5:e8. doi: 10.1017/qrd.2024.11. eCollection 2024.
Single Molecule Förster Resonance Energy Transfer (smFRET) is a popular technique to directly observe biomolecular dynamics in real time, offering unique mechanistic insight into proteins, ribozymes, and so forth. However, inevitable photobleaching of the fluorophores puts a stringent limit on the total time a surface-tethered molecule can be monitored, fundamentally limiting the information gain through conventional smFRET measurements. DyeCycling addresses this problem by using reversibly - instead of covalently - coupled FRET fluorophores, through which it can break the photobleaching limit and theoretically provide unlimited observation time. In this perspective paper, we discuss the potential of various fluorogenic strategies to suppress the background fluorescence caused by unbound, freely diffusing fluorophores inherent to the DyeCycling approach. In comparison to nanophotonic background suppression using zero-mode waveguides, the fluorogenic approach would enable DyeCycling experiments on regular glass slides with fluorogenic FRET probes that are quenched in solution and only fluoresce upon target binding. We review a number of fluorogenic approaches and conclude, among other things, that short-range quenching appears promising for realising fluorogenic DyeCycling on regular glass slides. We anticipate that our discussion will be relevant for all single-molecule fluorescence techniques that use reversible fluorophore binding.
单分子荧光共振能量转移（smFRET）是一种用于实时直接观察生物分子动力学的常用技术，能为蛋白质、核酶等提供独特的机制见解。然而，荧光团不可避免的光漂白对表面 tethered 分子的可监测总时间施加了严格限制，从根本上限制了通过传统 smFRET 测量获得的信息。染料循环通过使用可逆而非共价偶联的 FRET 荧光团来解决这个问题，借此它可以突破光漂白限制并理论上提供无限的观察时间。在这篇观点论文中，我们讨论了各种荧光生成策略在抑制染料循环方法中固有的未结合、自由扩散荧光团所引起的背景荧光方面的潜力。与使用零模式波导的纳米光子背景抑制相比，荧光生成方法将能够使用在溶液中淬灭且仅在与靶标结合时才发荧光的荧光 FRET 探针在普通载玻片上进行染料循环实验。我们回顾了一些荧光生成方法，并得出结论，除其他外，短程淬灭对于在普通载玻片上实现荧光染料循环似乎很有前景。我们预计我们的讨论将与所有使用可逆荧光团结合的单分子荧光技术相关。
