A Combined Single Molecule FRET/Magnetic Tweezers Instrument to Calibrate Molecular Tension - Based Fluorescence Probes (original) (raw)
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Micromachines
Single-molecule Förster resonance energy transfer (smFRET) inherits the strategy of measurement from the effective “spectroscopic ruler” FRET and can be utilized to observe molecular behaviors with relatively high throughput at nanometer scale. The simplicity in principle and configuration of smFRET make it easy to apply and couple with other technologies to comprehensively understand single-molecule dynamics in various application scenarios. Despite its widespread application, smFRET is continuously developing and novel studies based on the advanced platforms have been done. Here, we summarize some representative examples of smFRET research of recent years to exhibit the versatility and note typical strategies to further improve the performance of smFRET measurement on different biomolecules.
Toward dynamic structural biology: Two decades of single-molecule Förster resonance energy transfer
Classical structural biology can only provide static snapshots of biomacromolecules. Single-molecule Förster resonance energy transfer (smFRET) paved the way for studying dynamics in macromolecular structures under biologically relevant conditions. Since its first implementation in 1996, smFRET experiments have confirmed previously hypothesized mechanisms and provided new insights into many fundamental biological processes, such as DNA maintenance and repair, transcription, translation, and membrane transport. We review 22 years of contributions of smFRET to our understanding of basic mechanisms in biochemistry, molecular biology, and structural biology. Additionally, building on current state-of-the-art implementations of smFRET, we highlight possible future directions for smFRET in applications such as biosensing, high-throughput screening, and molecular diagnostics.
FRETBursts: An Open Source Toolkit for Analysis of Freely-Diffusing Single-Molecule FRET
Single-molecule Förster Resonance Energy Transfer (smFRET) allows probing intermolecular interactions and conformational changes in biomacromolecules, and represents an invaluable tool for studying cellular processes at the molecular scale. smFRET experiments can detect the distance between two fluorescent labels (donor and acceptor) in the 3-10 nm range. In the commonly employed confocal geometry, molecules are free to diffuse in solution. When a molecule traverses the excitation volume, it emits a burst of photons, which can be detected by single-photon avalanche diode (SPAD) detectors. The intensities of donor and acceptor fluorescence can then be related to the distance between the two fluorophores. While recent years have seen a growing number of contributions proposing improvements or new techniques in smFRET data analysis, rarely have those publications been accompanied by software implementation. In particular, despite the widespread application of smFRET, no complete softwa...
Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes
Journal of Visualized Experiments, 2021
Single-molecule Förster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. It has been used in a wide range of biophysical and molecular biological experiments, including the measurement of molecular forces, characterization of conformational dynamics of biomolecules, observation of intracellular colocalization of proteins, and determination of receptor-ligand interaction times. In a widefield microscopy configuration, experiments are typically performed using surface-immobilized probes. Here, a method combining single-molecule tracking with alternating excitation (ALEX) smFRET experiments is presented, permitting the acquisition of smFRET time traces of surface-bound, yet mobile probes in plasma membranes or glass-supported lipid bilayers. For the analysis of recorded data, an automated, open-source software collection was developed supporting (i) the localization of fluorescent signals, (ii) single-particle tracking, (iii) determination of FRET-related quantities including correction factors, (iv) stringent verification of smFRET traces, and (v) intuitive presentation of the results. The generated data can conveniently be used as input for further exploration via specialized software, e.g., for the assessment of the diffusional behavior of probes or the investigation of FRET transitions. Additionally, the FRET phenomenon has been widely used for colocalization studies of membrane-associated and intracellular proteins on a bulk level 5 , 6. In the last two decades, the method was adapted for monitoring smFRET
Quantitative single molecule FRET efficiencies using TIRF microscopy
Faraday Discuss., 2015
Förster resonance energy transfer (FRET) microscopy at the single molecule level has the potential to yield information on intra and intermolecular distances within the 2–10 nm range of molecules or molecular complexes that undergo frequent conformation changes. A pre-requirement for obtaining accurate distance information is to determine quantitative instrument independent FRET efficiency values. Here, we applied and evaluated a procedure to determine quantitative FRET efficiencies directly from individual fluorescence time traces of surface immobilized DNA molecules without the need for external calibrants. To probe the robustness of the approach over a wide range of FRET efficiencies we used a set of doubly labelled double stranded DNA samples, where the acceptor position was varied systematically. Interestingly, we found that fluorescence contributions arising from direct acceptor excitation following donor excitation are intrinsically taken into account in these conditions as o...
Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study
Nature methods, 2018
Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving re...
Advanced microscopy methods allow obtaining information on (dynamic) conformational changes in biomolecules via measuring a single molecular distance in the structure. It is, however, extremely challenging to capture the full depth of a three-dimensional biochemical state, binding-related structural changes or conformational cross-talk in multi-protein complexes using one-dimensional assays. In this paper we address this fundamental problem by extending the standard molecular ruler based on Förster resonance energy transfer (FRET) into a two-dimensional assay via its combination with protein-induced fluorescence enhancement (PIFE). We show that donor brightness (via PIFE) and energy transfer efficiency (via FRET) can simultaneously report on e.g., the conformational state of double stranded DNA (dsDNA) following its interaction with unlabelled proteins (BamHI, EcoRV, and T7 DNA polymerase gp5/trx). The PIFE-FRET assay uses established labelling protocols and single molecule fluorescence detection schemes (alternating-laser excitation, ALEX). Besides quantitative studies of PIFE and FRET ruler characteristics, we outline possible applications of ALEX-based PIFE-FRET for single-molecule studies with diffusing and immobilized molecules. Finally, we study transcription initiation and scrunching of E. coli RNA-polymerase with PIFE-FRET and provide direct evidence for the physical presence and vicinity of the polymerase that causes structural changes and scrunching of the transcriptional DNA bubble. Advanced microscopy methods have become powerful tools for structural studies of biomolecules. These methods can complement classical biochemical and biophysical techniques 1,2 , but most importantly emerged as key player in understanding structural dynamics 3,4. The underlying biophysical concept is straight forward: Construct a one-dimensional molecular ruler, in which the biochemical state of the system can be read out as a distance-related measure. Such a molecular ruler often uses a photophysical property such as fluorophore brightness or fluorescence lifetime to provide information on the structure of biomolecules in real time 5. A classic example for such a molecular ruler is Förster-type resonance energy transfer (FRET) 5 , which allows achieving structural information with a spatial resolution in the nanometre range and (sub)millisecond temporal resolution 6–10. However, other photophysical effects such as photo-induced electron transfer (PET) 11–14 or protein-induced fluorescence enhancement (PIFE) 15–32 can be used for similar purposes. Since the fluorescent signal can be read out with high time-resolution, even fast conformational changes 33–39 , as well as interactions between biomolecules, can be mapped in physiologically relevant environments in vitro 40,41 and in vivo 42,43 with a sensitivity allowing to address individual molecules. These molecular rulers suffer from limitations such as their restricted distance ranges and the need for labelling with fluorescent dyes. Most importantly, in the assessment of a three-dimensional (dynamic) structure, the largest limitation is embedded in the information accessible to these methods, which at best follow a single distance to capture a complex structural state.
Precision and accuracy of single-molecule FRET measurements - a worldwide benchmark study
arXiv (Cornell University), 2017
Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards ensuring both the reproducibility and accuracy of measuring FRET efficiencies are currently lacking. Here we report the results of a worldwide, comparative, blind study, in which 20 labs determined the FRET efficiencies of several dye-labeled DNA duplexes. Using a unified and straightforward method, we show that FRET efficiencies can be obtained with a standard deviation between ΔE = ±0.02 and ±0.05. We further suggest an experimental and computational procedure for converting FRET efficiencies into accurate distances. We discuss potential uncertainties in the experiment and the modelling. Our extensive quantitative assessment of intensity-based smFRET measurements and correction procedures serve as an essential step towards validation of distance networks with the ultimate aim to archive reliable structural models of biomolecular systems obtained by smFRETbased hybrid methods.
A practical guide to single-molecule FRET
Nature Methods, 2008
Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.