Can DyeCycling break the photobleaching limit in single-molecule FRET?

Abstract

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 control biomolecular systems. Single-molecule fluorescence resonance energy transfer (smFRET) is a powerful technique to observe in real-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 and thus, a prohibitively narrow temporal bandwidth of ≤ 3 orders of magnitude. Here, we introduce ″DyeCycling″, a measurement scheme with which we aim to break the photobleaching limit in single-molecule FRET. We introduce the concept of spontaneous dye replacement by simulations, and as an experimental proof-of-concept, we demonstrate the intermittent observation of a single biomolecule for one hour with a time resolution of milliseconds. Theoretically, DyeCycling can provide >100-fold more information per single molecule than conventional smFRET. We discuss the experimental implementation of DyeCycling, its current and fundamental limitations, and specific biological use cases. Given its general simplicity and versatility, DyeCycling has the potential to revolutionize the field of time-resolved smFRET, where it may serve to unravel a wealth of biomolecular dynamics by bridging from milliseconds to the hour range.

Orientation-Locked DNA Origami for Stable Trapping of Small Proteins in the Nanopore Electro-Osmotic Trap

“…Consequently, the vertically locked spheres provide more observation time for dynamic processes that occur in many protein systems. 8 Furthermore, owing to the reduced thermal noise by the cholesterol-induced linkage to the pore, the events show an improved signal-to-noise ratio, and we also found a better reproducibility from experiment to experiment due to the better-defined orientation-locked configuration.…”