A practical guide to single-molecule FRET

Abstract

Despite the explosive growth in the biological applications of single molecule methods over the last decade, these techniques have thus far been practiced mostly by researchers who are biophysically oriented. This is partly because of the lack of commercial instruments in many cases and also because of the perceived steep learning curve and need for expensive equipments. We wish to provide a practical guide to using Förster (or Fluorescence) Resonance Energy Transfer (FRET) at the single molecule level, focusing on the study of immobilized molecules that allow measurements of single molecule reaction trajectories from about 1 millisecond to many minutes. An instrument can be built at a reasonable cost using various off-the-shelf components and operated reliably using current well-established protocols and freely available software.The future holds the promise of personalized DNA sequencing and high throughput screening for pathogens at affordable cost and viable time. These promises are riding high on a surge of single-molecule based technologies that enable us to manipulate and probe individual molecules. Using this approach, several important biological riddles that have intrigued scientists for a long time are coming under the microscope. As Feynman famously said "It is very easy to answer many of these fundamental biological questions; you just look at the thing!" 1 . Single-molecule methods are allowing us to do just that 2,3 . They may one day become an elementary tool for characterizing proteins, signaling pathways or any biological phenomenon. In the hopes of facilitating this objective, we provide a brief, but practical guide for single-molecule FRET 4 measurements (smFRET) [5][6][7] , one of the most general and adaptable single-molecule techniques. Since its humble beginning under nonaqueous conditions in 1996 8 , smFRET has rapidly developed to answer fundamental questions about replication, recombination, transcription, translation, RNA folding and catalysis, non-canonical DNA dynamics, protein folding and conformational changes, various motor proteins, membrane fusion proteins, ion channels, signal transduction, to name just a few, and the list keeps growing at a fast pace. Since it is not the purpose of this review to survey the vast literature on such studies, we refer the reader to reviews in the field and the references therein 6, 9-13 .Correspondence to: Taekjip Ha. Competing interests statement:The authors declare no competing financial interests. In FRET measurements, the extent of non-radiative energy transfer between two fluorescent dye molecules, termed donor and acceptor, reports the intervening distance which can be estimated from the ratio of acceptor to total emission intensity ( Fig. 1) 4,14,15 . This efficiency of energy transfer, E is given as E = [1 + (R/R 0 ) 6 ] −1 , where R is the inter-dye distance and R 0 is the Förster radius at which E = 0.5 (Fig. 1a). Conformational dynamics of single molecules can be observed in real-time by tracking FRET changes (Fig. 1b). The ad...

Single-molecule approaches to characterizing kinetics of biomolecular interactions

“…An approach that is substantially more sensitive to true association uses Fluorescence Resonance Energy Transfer (FRET), a process in which the excitation energy of a donor fluorophore is transferred to an acceptor dye via an induced dipole-dipole interaction. The strong distance dependence of this interaction causes the transfer efficiency to be close to unity in the case of a physical association and close to zero when the two binding partners are separated by more than 10 nm [26].…”

“…Similar FRET-based approaches have been used to study the kinetics of association of proteins with SNARE complexes [28,29] and the interaction of chromatin remodeling factors with nucleosomes [30]. The obvious advantage of FRET-based methods is that they also allow the study of conformational changes within complexes [4,11,26,[31][32][33].…”

Nobel Lecture: Single-molecule spectroscopy, imaging, and photocontrol: Foundations for super-resolution microscopy

“…All of this has occurred due not only to my efforts, but also due in major part to the clever and insightful research performed by many researchers around the world too numerous to mention 19 here. Beyond super-resolution microscopy, just observing single molecules and their behaviors continues to lead to tantalizing scientific advances, whether this is simply tracking single-molecule motions (184), or inferring biomolecular interactions and conformations with FRET (185,186) or extracting photodynamics from trapped single molecules (187)(188), or determining enzymatic mechanisms (189). The future of single-molecule spectroscopy and super-resolution imaging is very bright.…”

Single‐Molecule Photoactivation FRET: A General and Easy‐To‐Implement Approach To Break the Concentration Barrier

“…Total internal reflection fluorescence (TIRF) microscopy based single‐molecule fluorescence resonance energy transfer (sm‐FRET) is commonly used to examine internal conformational dynamics of surface‐attached molecules or interactions between attached and freely‐diffusing molecules 1. TIRF microscopy restricts its excitation volume to a thin layer of evanescent field, which decays within a few hundred nanometers above the microscope coverslip surface, and presents a better signal‐to‐noise ratio (SNR) than Epi‐illumination.…”