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...
Myosin V is a dimeric molecular motor that moves processively on actin, with the center of mass moving approximately 37 nanometers for each adenosine triphosphate hydrolyzed. We have labeled myosin V with a single fluorophore at different positions in the light-chain domain and measured the step size with a standard deviation of <1.5 nanometers, with 0.5-second temporal resolution, and observation times of minutes. The step size alternates between 37 + 2x nm and 37 - 2x, where x is the distance along the direction of motion between the dye and the midpoint between the two heads. These results strongly support a hand-over-hand model of motility, not an inchworm model.
The analysis of single-molecule fluorescence resonance energy transfer (FRET) trajectories has become one of significant biophysical interest. In deducing the transition rates between various states of a system for time-binned data, researchers have relied on simple, but often arbitrary methods of extracting rates from FRET trajectories. Although these methods have proven satisfactory in cases of well-separated, low-noise, two- or three-state systems, they become less reliable when applied to a system of greater complexity. We have developed an analysis scheme that casts single-molecule time-binned FRET trajectories as hidden Markov processes, allowing one to determine, based on probability alone, the most likely FRET-value distributions of states and their interconversion rates while simultaneously determining the most likely time sequence of underlying states for each trajectory. Together with a transition density plot and Bayesian information criterion we can also determine the number of different states present in a system in addition to the state-to-state transition probabilities. Here we present the algorithm and test its limitations with various simulated data and previously reported Holliday junction data. The algorithm is then applied to the analysis of the binding and dissociation of three RecA monomers on a DNA construct.
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