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WIREs Syst Biol Med
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Recent advancement of light‐based single‐molecule approaches for studying biomolecules

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Recent advances in single‐molecule techniques have led to new discoveries in analytical chemistry, biophysics, and medicine. Understanding the structure and behavior of single biomolecules provides a wealth of information compared to studying large ensembles. However, developing single‐molecule techniques is challenging and requires advances in optics, engineering, biology, and chemistry. In this paper, we will review the state of the art in single‐molecule applications with a focus over the last few years of development. The advancements covered will mainly include light‐based in vitro methods, and we will discuss the fundamentals of each with a focus on the platforms themselves. We will also summarize their limitations and current and future applications to the wider biological and chemical fields. This article is categorized under: Laboratory Methods and Technologies > Imaging Laboratory Methods and Technologies > Macromolecular Interactions, Methods Analytical and Computational Methods > Analytical Methods
Single‐molecule fluorescence resonance energy transfer (smFRET). (a) Molecular mechanisms of nuclease activity in CRISPR‐Cas9 system revealed by monitoring the conformational changes by smFRET. A proposed model of HNH nuclease domain rearrangement between crystal structures of gRNA:Cas9 (PDB ID, 4ZT0) and gRNA:Cas9:DNA (PDB ID, 5F9R) (left). Schematic diagram of smFRET assay to monitor HNH rearrangement in real time (right). Green and red probes denote FRET donor and acceptor. (Reprinted with permission from Dagdas, Chen, Sternberg, Doudna, & Yildiz (). Copyright 2017 Science Advances licensed under a Creative Commons Attribution 4.0 (CC BY) and reproduced here). (b) Microinjection of smFRET labeled proteins into live cells. (c) Delivery of dye‐labeled antibodies into cells using the bacterial toxin Stepolysin O. (d) Knock‐in genome editing using CRISPR‐Cas9 for tagging endogenous proteins. (e‐g) High contrast single‐molecule imaging techniques. (e) Multi‐spot confocal microscopy. (f) Tilted light sheet microscopy. (g) Highly inclined swept tile (HIST) microscopy. Inset shows the sweeping of the tile providing an extending field‐of‐view
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Interferometric scattering mass spectrometry (iSCAMS). The interference between light scattered by individual biomolecules (dotted lines) and light reflected (IR) at the glass/water interface upon illumination (IIN) produces a destructive interference pattern on the camera as single‐molecule images (bottom). These images are simulated. Scale bar = 200 nm. The image contrast is proportional to the mass of the detected biomolecule
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Tethering‐free single‐molecule trapping techniques. (a) The anti‐Brownian electrokinetic (ABEL) trap uses electrodes to counteract the Brownian motion of a freely diffusing dye‐labeled single‐molecule whose position is monitored by a scanning laser. The fluorescent intensity, lifetime, and spectrum of the single‐molecule are measured to interpret the changes of the protein structure. (b) Geometry‐induced electrostatic (GIE) trapping confines a diffusing single‐molecule via electrostatic interactions with the walls of the well. The effective charge (qeff) of the single‐molecule can be determined via its escape time (tesc) from the well. (c) The self‐induced back‐action (SIBA) trap uses an opaque film with a double nanohole aperture to trap freely diffusing single‐molecules. Inset middle: zoomed view of double nanohole aperture. Inset right: zoomed view of a trapped biomolecule
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Pull‐down protein assays. (a) Sandwich enzyme‐linked immunosorbent assay (ELISA), where the target protein is anchored to the walls of a multiwell plate and detected via a color change upon the addition of detection antibodies labeled with horseradish peroxidase (HRP) and a substrate molecule such as o‐phenylenediamine (OPD). (b) Digital ELISA, where the target protein is immobilized on beads functionalized with antibodies and labeled with an antibody that produces a fluorescent signal upon the addition of a substrate molecule. The beads are deposited onto a microwell plate where only one bead can fit in each well, sealed with oil, and imaged. (c) Single‐molecule pull‐down (SiMPull), where proteins are immobilized by capture antibodies to a microscope slide/coverslip flow chamber, labeled with fluorescent detection antibodies, and imaged. Bar graph shows analysis of the number of detected alpha‐synuclein (α‐SYN) protein and oligomerized fraction determined via intensity analysis from healthy (CTRL) and Parkinson's disease (PD) samples
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High‐throughput and high‐content single‐molecule assays. (a) Comparison between single‐molecule images using (left) Gaussian illumination and (right) uniform flat‐field illumination. Scale bars = 10 μm. (b) Liposome tethering increases diffusion time of single‐molecules through a confocal detection volume. (c) Spectrally resolved imaging allows the distinction of fluorophores with similar emission spectra, yielding simultaneous multicolor imaging. (d) Temporally resolved imaging increases the temporal resolution beyond that of the frame rate by spreading dynamics across a spatial dimension
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Laboratory Methods and Technologies > Macromolecular Interactions, Methods
Laboratory Methods and Technologies > Imaging
Analytical and Computational Methods > Analytical Methods

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