Bleaching‐resistant, Near‐continuous Single‐molecule Fluorescence and FRET Based on Fluorogenic and Transient DNA Binding

Abstract

Photobleaching of fluorescent probes limits the observation span of typical single‐molecule fluorescence measurements and hinders observation of dynamics at long timescales. Here, we present a general strategy to circumvent photobleaching by replenishing fluorescent probes via transient binding of fluorogenic DNAs to complementary DNA strands attached to a target molecule. Our strategy allows observation of near‐continuous single‐molecule fluorescence for more than an hour, a timescale two orders of magnitude longer than the typical photobleaching time of single fluorophores under our conditions. Using two orthogonal sequences, we show that our method is adaptable to Förster Resonance Energy Transfer (FRET) and that can be used to study the conformational dynamics of dynamic structures, such as DNA Holliday junctions, for extended periods. By adjusting the temporal resolution and observation span, our approach enables capturing the conformational dynamics of proteins and nucleic acids over a wide range of timescales.

iMAX FRET (Information Maximized FRET) for Multipoint Single-Molecule Structural Analysis

“…iMAX FRET has many advantages over established techniques. First, as we use the stochastic exchange scheme for probing all possible points in a molecule with otherwise identical probes, the imaging time can be cut down considerably compared to other DNA hybridization-based imaging techniques. ,, Probe-labeled samples can be prepared within 24 h, while weak-binder-based measurement can be completed in as little as 2 min. Second, iMAX FRET offers simplicity in sample preparation, circumventing the requirements associated with sample preparation required for other methods, such as crystallization.…”

“…Unlike hitherto reported methods that require prior structural knowledge to interpret data, iMAX FRET is the first method that enables ab initio structural analysis solely from smFRET data. The unique “one-pot measurement scheme” for stochastic multipoint sampling, i.e., no multiple repeated measurement with buffer exchange required, is realized by repurposing the probe exchange scheme, which has been utilized in recent studies to overcome photobleaching of organic dyes for long-term kinetics measurement. , Using our newly developed software pipeline, we show that iMAX FRET data can determine up to six distances from four positions in 3D space, from which the conformation of a molecule can be reconstructed through geometrical modeling. iMAX FRET provides a comprehensive understanding of structural heterogeneity within a biological sample.…”

Single-Molecule FRET at 10 MHz Count Rates

“…Single-molecule Förster resonance energy transfer (smFRET) experiments are a cornerstone in the investigation of biomolecular dynamics, as they enable the observation of the molecule of interest “in action”, specifically within its natural environment, and give a real-time movie that can span time scales from milliseconds to minutes or in some cases even hours . Crucially, although methods enabling access to faster time scales through correlation techniques exist, , time-resolving individual rare events, such as the rapid jumps across the barriers between two conformational states (e.g., protein or nucleic acid folding), require very high count rates from single molecules. , These transition paths have gained increasing interest, and smFRET experiments have been playing a major role in revealing the nature and time scales of these paths. , However, the photon count rate (PCR) required for accessing the relevant microsecond time scale mandates the use of high excitation intensities, leading to saturation and increased photobleaching, which makes the task of measuring these transition paths very challenging.…”

Single-Molecule FRET at 10 MHz Count Rates

“…Single-molecule Förster Resonance Energy Transfer (smFRET) experiments are a cornerstone in the investigation of biomolecular dynamics [1] as they enable the observation of the molecule of interest ‘in action’, specifically within its natural environment, and give a real-time movie that can span timescales from milliseconds to minutes or in some cases even hours [2]. Crucially, although methods enabling access to faster timescales through correlation techniques exist [3, 4] time-resolving individual rare events, such as the rapid jumps across the barriers between two conformational states (e.g.…”