Abstract
Recent advances in single-molecule imaging have resulted in a series of discoveries regarding characteristic behavior and dynamics of individual molecules. Among the single-molecule imaging techniques, fluorescence resonance energy transfer (FRET) measurement is relatively easy to set up, yet is a powerful method; it can visualize substrate binding and dissociation as well as intramolecular structural changes within a single molecule in real time. Here, we first review single-molecule fluorescence imaging techniques that open a way to establish single-molecule FRET (smFRET) measurement. Then, we describe two examples of the characteristic dynamics of individual molecules revealed by smFRET: antibiotic-mediated protein translation inhibition and the intramolecular structural changes in CRISPR-Cas9, a versatile genome-editing tool. Finally, we introduce some of the latest advances in smFRET technique.
Highlights
FRET techniques have been widely used for measuring the dynamics of biomolecules because of its high sensitivity as a nanoscale distance sensor
To detect single molecule fluorescent probes using optical microscopy, the techniques to reduce background light combined with an intense illumination and highly sensitive camera systems are required
In 1990, Shera et al succeeded in distinguishing fluorescent signals emitted from a single fluorescent probe molecule using pulsed light to induce photoexcitation of a low-concentration fluorescent probe solution streaming through a flow cell (Shera et al 1990)
Summary
FRET techniques have been widely used for measuring the dynamics of biomolecules because of its high sensitivity as a nanoscale distance sensor. Between two closely located fluorescent molecules, energy in an excited donor fluorescent probe is resonantly transferred to an adjacent acceptor fluorescent probe, thereby decreasing the donor’s fluorescence intensity and increasing the acceptor’s fluorescence intensity The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between the two fluorescent molecules. Based on the ratio of fluorescence intensities of two fluorescent molecules, it can achieve high signal-to-noise ratio in measurements of binding and dissociation reactions compared with measurements involving a single fluorescent molecule. These advantages have made FRET an extensively used technique for researching the dynamics of biomolecules. We first outlines the single-molecule imaging techniques, which provide the basis for establishing smFRET measurement, presents examples of protein dynamics research employing smFRET, and introduces some state- of-the-art smFRET applications
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