This work was supported by King Abdullah University of Science and Technology through core funding and Competitive Research Award (CRG3) to S. M. H.

The authors declare no competing financial interests.

In this study,&#160;different smFRET techniques were implemented&#160;to determine the kinetics of HJ resolution by GEN130. Similar smFRET approaches were used to follow the double-flap DNA conformational requirement and cleavage by the DNA replication and repair flap endonuclease 142,43,44. Here, critical steps in this protocol are discussed. The silanization reaction should be free from any trace of humidity. The pegylation solution should be applied rapidly to the silanized glass once PEG is dissolved to avoid hydrolysis. In the multi-channel flow cell, any trapped air in the adhesive sheet should be removed to avoid leakage between neighboring channels. The PCA solution should be freshly prepared since it oxidizes over time. The addition of 10 N NaOH should be dropwise, with vortexing in between. The fluorescence background in the coverslip should be minimal before flowing the fluorescently labeled HJ. The imaging in the flow cell should be performed in one direction to avoid imaging bleached areas. In ALEX experiments, the power of the red laser should be reduced to avoid rapid bleaching of the acceptor. In the time-lapse experiments, the cycle time has to be shorter than the fastest event.
smFRET is a sensitive technique that can provide valuable real-time insights in biomolecular reactions. However, this method has several technical challenges, among which is achieving measurable change in FRET during the biochemical reaction. This is necessary to obtain well-separated features in the histograms and distinguishable states in the time-traces. In many cases, smFRET requires careful design of the substrates, selection of the fluorophore pairs and their positions, and amplification of FRET changes in the DNA substrate because of the little structural changes in the substrate45. Another approach for performing FRET is to use labeled proteins46. The observation window in FRET is limited by the stability of the acceptor such as Cy5 or Alexa Fluor 647 which tends to bleach more rapidly than the donor (Cy3 in this case). Therefore, FRET requires a continuous search for stable fluorophores to extend the experiment duration and efforts to develop oxygen scavenging systems to prolong the fluorescence signal and maximize the signal-to-noise ratio47,48.
Among the tips for troubleshooting in smFRET is balancing the several parameters involved in imaging such as the laser power, exposure time, cycle time, and number of cycles to maximize the fluorescence emission, prolong the experiment duration, and achieve appropriate sampling intervals for the enzyme dynamics. Longer observation times and minimal effects from photobleaching are essential to obtain high fidelity dwell time distributions that represent the enzyme dynamics. ALEX generates better histograms since this method is subjected to lower contributions from photobleached particles compared to single-color FRET. However, the temporal resolution in ALEX is lower than that in single-color FRET.
Finally, smFRET&#8217;s emphasis on detecting conformational/structural changes in individual molecules in real-time bridges the gap between high resolution structural techniques (i.e., X-ray crystallography, nuclear magnetic resonance, electron microscopy), which provides atomic resolution structural details under static conditions and bulk methods that yield the ensemble average of a measurable property. In many aspects, smFRET has proven to be a powerful technique for studying biological systems in real-time.

Single-molecule methods based on fluorescence detection provide high signal-to-noise ratios1. FRET is a spectroscopic technique that can measure distances in the range of 1&#8211;10 nm, rendering this technique as a molecular ruler for measuring distances in the nanometer range2,3. The absorption spectrum of the acceptor has a partial spectral overlap with the donor&#8217;s emission spectrum at the shorter wavelength end. FRET is mediated by the radiation-less energy transfer between a donor and acceptor pair, whereas the efficiency of energy transfer is dependent on the distance and orientation of the acceptor4.
Several approaches have been implemented to minimize the background and improve the detection efficiency of the fluorescence signal5,6. One approach is confocal microscopy, in which a pinhole restricts the excitation spot to a size below the diffraction limit7. Another approach is total internal reflection fluorescence (TIRF), which is a wide-field illumination technique in which the light is directed off-axis above a critical angle8. The light is then totally internally reflected at the interface between the glass and aqueous solution, generating an evanescent wave that only illuminates the fluorophores attached to the glass surface and prevents background from the fluorophores in the rest of the solution.
In confocal microscopy, the molecules can be either freely diffusing or surface immobilized. The attained temporal resolution can be within microseconds to several milliseconds9. The confocal detection for a single molecule is performed by single-photon avalanche diode (SPAD) and point-by-point scanning of the region of interest10. In TIRF, a time-series of a few hundreds of molecules immobilized on the surface is recorded by a position-sensitive two-dimensional charge coupled detector (CCD). The CCD amplifies the fluorescence signal either by intensified phosphor screen and microchannel plate or on-chip multiplication of photoelectrons (EMCCD). The temporal resolution is dependent on the readout speed and quantum efficiency of the CCD and usually on the order of few tens of milliseconds6.
HJ is a central intermediate in DNA repair and recombination11,12,13,14. HJ has two continuous and two crossing strands that connect between the continuous strands without intersecting each other. HJ exists in solution as X-stacked conformers, which undergo continuous isomerization by the continuous strands becoming crossing and the crossing strands becoming continuous in the other conformer15. Isomer preference of the HJ is dependent on the core sequence and ionic environment and has been extensively studied by FRET16,17,18,19.
GEN120 is a monomeric protein in solution21 and requires dimerization to cleave the HJ, thus allowing proper separation of the recombined strands22,23. The stacking conformer preference of the HJ influences the outcome of genetic recombination by setting the orientation of the resolution by the HJ resolvases24. Understanding how GEN1 binds the HJ, coordinates the two incisions, and ensures its full resolution have all been under intensive study21,22,23,25,26,27,28,29,30.
In this study, an objective based TIRF set-up is used as described previously31. Two-color alternating excitation (ALEX) is applied to determine the conformational changes upon the interaction of GEN1 with fluorophore labeled HJ. ALEX produces 2D histograms based on two ratiometric parameters FRET efficiency E, which is donor-acceptor distance-dependent, and the stoichiometry parameter S, which measures the donor-acceptor stoichiometry32. ALEX enables the sorting of fluorescent species based on the stoichiometries of the fluorophores including donor-only, acceptor-only, and mixed subpopulations. ALEX can extend the use of FRET to the full range and can detect differences in fluorophore brightness and oligomerization as well as monitor macromolecule-ligand interactions33.
It is found that GEN1 consistently succeeds in resolving the HJ within the lifetime of the GEN1-HJ complex. The time-dependent conformational changes are derived from the time-traces of individual molecules, while the histograms represent the distribution of the underlying populations. Using time-lapse single-color FRET, fast on-rates and slow off-rates for the GEN1 dimer are demonstrated, which increase the affinity of the assembled GEN1 dimer at the first incision product.

<table><tbody><tr><td>(&amp;plusmn;)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox)</td><td>Sigma-Aldrich</td><td>238813</td><td></td></tr><tr><td>0.1 M sodium bicarbonate buffer</td><td>Fisher</td><td>144-55-8</td><td></td></tr><tr><td>10 % Novex Tris-Borate-EDTA gel</td><td>Thermo Fisher Scientific</td><td>EC6275BOX</td><td></td></tr><tr><td>100 X TIRF objective</td><td>Olympus</td><td>NAPO 1.49</td><td></td></tr><tr><td>3,4-dihroxybenzoic acid (PCA)</td><td>Sigma-Aldrich</td><td>P5630</td><td></td></tr><tr><td>3-aminopropyltriethoxysilane (APTES)</td><td>Sigma-Aldrich</td><td>741442</td><td></td></tr><tr><td>6% Novex Tris-Borate-EDTA gel</td><td>Thermo Fisher Scientific</td><td>EC6265BOX</td><td></td></tr><tr><td>Adhesive sheet</td><td>Grace bio-labs</td><td>SA-S-1L</td><td></td></tr><tr><td>Benchtop refrigerated centrifuge</td><td>Eppendorf</td><td>Z605212</td><td></td></tr><tr><td>Biotin-PEG</td><td>Laysan Bio</td><td>Biotin-PEG-SVA 5000</td><td></td></tr><tr><td>Bovine Serum Albumin (BSA)</td><td>New England Biolabs</td><td>B9001S</td><td></td></tr><tr><td>Calcium Chloride Dihydrate</td><td>Sigma-Aldrich</td><td>31307</td><td></td></tr><tr><td>cation exchange column</td><td>GE healthcare</td><td>MonoS (4.6/100)</td><td></td></tr><tr><td>Cell distruptor</td><td>Constant Cell Disruption System</td><td>TS5/40/CE/GA</td><td></td></tr><tr><td>Coomassie Brilliant Blue</td><td>MP Biomedicals</td><td>808274</td><td></td></tr><tr><td>Cy3 emission filter</td><td>Chroma</td><td>HQ600/40M-25</td><td></td></tr><tr><td>Cy5/Alexa Fluor 647 emission filter</td><td>Chroma</td><td>HQ700/40M-25</td><td></td></tr><tr><td>Dichroic for DV2 filter cube</td><td>Photometrics</td><td>630dcxr-18x26</td><td></td></tr><tr><td>Dithiothreitol (DTT)</td><td>Thermo Scientific</td><td>R0861</td><td></td></tr><tr><td>Drill</td><td>Dremel</td><td>200-1/21</td><td></td></tr><tr><td>Electronic cutter</td><td>Copam</td><td>CP-2500</td><td></td></tr><tr><td>EMCCD camera</td><td>Hamamatsu</td><td>C9100-13</td><td></td></tr><tr><td>Epoxy glue</td><td>Devcon</td><td>14250</td><td></td></tr><tr><td>FPLC Aktapurifier UPC 10</td><td>GE Healthcare</td><td>28406268</td><td></td></tr><tr><td>GelQuant.NET software</td><td>biochemlabsolutions.com</td><td>Version 1.8.2</td><td></td></tr><tr><td>GEN1 entry vector</td><td>Harvard plasmid repository</td><td>HSCD00399935</td><td></td></tr><tr><td>Glycerol</td><td>Sigma Life Science</td><td>G5516</td><td></td></tr><tr><td>green laser (emission 532 nm)</td><td>Coherent</td><td>Compass 315M-100</td><td></td></tr><tr><td>Heparin column</td><td>GE healthcare</td><td>HiTrap Heparin column</td><td></td></tr><tr><td>HEPES</td><td>BDH</td><td>BDH4162</td><td></td></tr><tr><td>Image splitter</td><td>Photometrics</td><td>Dualview (DV2)</td><td></td></tr><tr><td>Imidazole</td><td>Sigma-Aldrich</td><td>I2399</td><td></td></tr><tr><td>Inverted microscope</td><td>Olympus</td><td>IX81</td><td></td></tr><tr><td>Isopropyl-&amp;szlig;-D-thiogalactoside (IPTG)</td><td>Goldbio.</td><td>12481C100</td><td></td></tr><tr><td>Laser scanner</td><td>GE healthcare</td><td>Typhoon Trio</td><td></td></tr><tr><td>LB Broth</td><td>Fisher Scientific</td><td>BP1426-500</td><td></td></tr><tr><td>Long pass 532nm filter</td><td>Semrock</td><td>LPD02-532RU-25</td><td></td></tr><tr><td>Magnesium Chloride</td><td>Sigma Life Science</td><td>M8266</td><td></td></tr><tr><td>mPEG</td><td>Laysan Bio</td><td>mPEG-SVA 5000</td><td></td></tr><tr><td>Neutravidin</td><td>Pierce</td><td>31000</td><td></td></tr><tr><td>Ni-NTA column</td><td>GE healthcare</td><td>HisTrap FF</td><td></td></tr><tr><td>NuPAGE 10% Bis-Tris gels</td><td>Novex Life technologies</td><td>NP0301BOX</td><td></td></tr><tr><td>NuPAGE 10% Bis-Tris Protein Gels</td><td>Thermo Fisher Scientific</td><td>NP0302PK2</td><td></td></tr><tr><td>Origin software</td><td>OriginLab Corporation</td><td>Version 8.5</td><td></td></tr><tr><td>Phenylmethylsulfonyl fluoride (PMSF)</td><td>Alexis Biochemicals</td><td>270-184-G025</td><td></td></tr><tr><td>Phosphate-buffered saline</td><td>GIBCO</td><td>14190</td><td></td></tr><tr><td>Polyethylene Tubing (I.D. 0.76 mm O.D. 1.22mm)</td><td>Fisher (Becton Dickinson)</td><td>427416</td><td></td></tr><tr><td>Protocatechuate 3,4-dioxygenase (3,4-PCD)</td><td>Sigma-Aldrich</td><td>P8279-25UN</td><td></td></tr><tr><td>Quad-band dichroic</td><td>Chroma Inc</td><td>Z405/488/532/640rpc</td><td></td></tr><tr><td>red laser (emission 640 nm)</td><td>Coherent</td><td>Cube 640 100C</td><td></td></tr><tr><td>Sodium Chloride</td><td>Fisher Chemical</td><td>S271</td><td></td></tr><tr><td>Sorvall RC-6 plus centrifuge</td><td>Thermo Fisher Scientific</td><td>46910</td><td></td></tr><tr><td>Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>Nanodrop 2000</td><td></td></tr><tr><td>Syringe pump</td><td>Harvard Apparatus</td><td>70-3007</td><td></td></tr><tr><td>Teflon tweezers</td><td>Rubis</td><td>K35A</td><td></td></tr><tr><td>Tris Base</td><td>Promega</td><td>H5135</td><td></td></tr><tr><td>Ultracentrifuge</td><td>Beckman Coulter</td><td>Optima L-90K</td><td></td></tr><tr><td>Ultrafiltration membrane</td><td>Millipore</td><td>UFC90300</td><td></td></tr></tbody></table>

single-molecule f&ouml;rster resonance energy transfer methods for real-time investigation of the holliday junction resolution by gen1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the population. Single-molecule F&#246;rster resonance energy transfer (smFRET) provides a recording of the conformational changes taking place by individual molecules in real-time. Therefore, smFRET is powerful in measuring structural changes in the enzyme or substrate during binding and catalysis. This work presents a protocol for single-molecule imaging of the interaction of a four-way Holliday junction (HJ) and gap endonuclease I (GEN1), a cytosolic homologous recombination enzyme. Also presented are single-color and two-color alternating excitation (ALEX) smFRET experimental protocols to follow the resolution of the HJ by GEN1 in real-time. The kinetics of GEN1 dimerization are determined at the HJ, which has been suggested to play a key role in the resolution of the HJ and has remained elusive until now. The techniques described here can be widely applied to obtain valuable mechanistic insights of many enzyme-DNA systems.

Watch this Scientific Journal Video about Single-Molecule FÃ¶rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1 at JoVE.com

Single-Molecule F&#246;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Presented here is a protocol for performing single-molecule F&#246;rster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.

Single-Molecule F&ouml;rster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

Bulk methods measure the ensemble behavior of molecules, in which individual reaction rates of the underlying steps are averaged throughout the ...

single-molecule-frster-resonance-energy-transfer-methods-for-real

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

9.3K Views.  King Abdullah University of Science and Technology (KAUST). Presented here is a protocol for performing single-molecule Förster resonance energy transfer to study HJ resolution. Two-color alternating excitation is used for determining the dissociation constants. Single-color time lapse smFRET is then applied in real-time cleavage assays to obtain the dwell time distribution prior to HJ resolution.

Video: Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule level in multiparameter fluorescence detection (MFD) mode is presented here. MFD maximizes the usage of all &#34;dimensions&#34; of fluorescence to reduce photophysical and experimental artifacts and allows for the measurement of interdye distance with an accuracy up to ~1 &#197; in rigid biomolecules. This method was used to identify three conformational states of the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor to explain the activation of the receptor upon ligand binding. When comparing the known crystallographic structures with experimental measurements, they agreed within less than 3 &#197; for more dynamic biomolecules. Gathering a set of distance restraints that covers the entire dimensionality of the biomolecules would make it possible to provide a structural model of dynamic biomolecules.

A protocol for high-precision FRET experiments at the single molecule level is presented here. Additionally, this methodology can be used to identify three conformational states in the ligand-binding domain of the N-methyl-D-aspartate (NMDA) receptor. Determining precise distances is the first step towards building structural models based on FRET experiments.

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Biochemistry

Real-time Observation of the DNA Strand Exchange Reaction Mediated by Rad51

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament on single-stranded DNA (ssDNA) and captures double-stranded DNA (dsDNA) non-specifically to interrogate it for a homologous sequence. After encountering homology, Rad51 catalyzes DNA strand exchange to mediate pairing of the ssDNA with the complementary strand of the dsDNA. This reaction is highly regulated by numerous accessary proteins in vivo. Although conventional biochemical assays have been successfully employed to examine the role of such accessory protein in vitro, kinetic analysis of intermediate formation and its progression into a final product has proven challenging due to the unstable and transient nature of the reaction intermediates. To observe these reaction steps directly in solution, fluorescence resonance energy transfer (FRET)-based real-time observation systems of this reaction were established. Kinetic analysis of real-time observations shows that the DNA strand exchange reaction mediated by Rad51 obeys a three-step reaction model involving the formation of a three-strand DNA intermediate, maturation of this intermediate, and the release of ssDNA from the mature intermediate. The Swi5-Sfr1 complex, an accessary protein conserved in eukaryotes, strongly enhances the second and third steps of this reaction. The FRET-based assays presented here enable us to uncover the molecular mechanisms through which recombination accessary proteins stimulate the DNA strand exchange activity of Rad51. The primary goal of this protocol is to enhance the repertoire of techniques available to researchers in the field of homologous recombination, particularly those working with proteins from species other than Schizosaccharomyces pombe, so that the evolutionary conservation of the findings presented herein can be determined.

Fluorescence resonance energy transfer-based real-time observation systems of the DNA strand exchange reaction mediated by Rad51 were developed. Using the protocols presented here, we are able to detect the formation of reaction intermediates and their conversion into products, while also analyzing the enzymatic kinetics of the reaction.

The DNA strand exchange reaction mediated by Rad51 is a critical step of homologous recombination. In this reaction, Rad51 forms a nucleoprotein filament ...

Combining QD-FRET and Microfluidics to Monitor DNA Nanocomplex Self-Assembly in Real-Time

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically functional inside the cell, nucleic acid-based therapeutics require an efficient intracellular delivery system. One widely adopted approach is to complex DNA with a gene carrier to form nanocomplexes via electrostatic self-assembly, facilitating cellular uptake of DNA while protecting it against degradation. The challenge lies in the rational design of efficient gene carriers, since premature dissociation or overly stable binding would be detrimental to the cellular uptake and therapeutic efficacy. Nanocomplexes synthesized by bulk mixing showed a diverse range of intracellular unpacking and trafficking behavior, which was attributed to the heterogeneity in size and stability of nanocomplexes. Such heterogeneity hinders the accurate assessment of the self-assembly kinetics and adds to the difficulty in correlating their physical properties to transfection efficiencies or bioactivities. We present a novel convergence of nanophotonics (i.e. QD-FRET) and microfluidics to characterize the real-time kinetics of the nanocomplex self-assembly under laminar flow. QD-FRET provides a highly sensitive indication of the onset of molecular interactions and quantitative measure throughout the synthesis process, whereas microfluidics offers a well-controlled microenvironment to spatially analyze the process with high temporal resolution (~milliseconds). For the model system of polymeric nanocomplexes, two distinct stages in the self-assembly process were captured by this analytic platform. The kinetic aspect of the self-assembly process obtained at the microscale would be particularly valuable for microreactor-based reactions which are relevant to many micro- and nano-scale applications. Further, nanocomplexes may be customized through proper design of microfludic devices, and the resulting QD-FRET polymeric DNA nanocomplexes could be readily applied for establishing structure-function relationships. 

We present a novel and powerful integration of nanophotonics (QD-FRET) and microfluidics to investigate the formation of polyelectrolyte polyplexes, which is expected to provide better control and synthesis of uniform and customizable polyplexes for future nucleic acid-based therapeutics.

Advances in genomics continue to fuel the development of therapeutics that can target pathogenesis at the cellular and molecular level. Typically ...

Biology

Single-Molecule Dwell-Time Analysis of Restriction Endonuclease-Mediated DNA Cleavage

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for hundreds of individual restriction endonuclease (REase) molecules in one experiment. This assay does not require protein labeling and can be carried out with a single imaging channel. In addition, the results of multiple individual experiments can be pooled to generate well-populated dwell-time distributions. Analysis of the resulting dwell-time distributions can help elucidate the DNA cleavage mechanism by revealing the presence of kinetic steps that cannot be directly observed. Example data collected using this assay with the well-studied REase, EcoRV - a dimeric Type IIP restriction endonuclease that cleaves the palindromic sequence GAT&#8595;ATC (where &#8595; is the cut site) - are in agreement with prior studies. These results suggest that there are at least three steps in the pathway to DNA cleavage that is initiated by introducing magnesium after EcoRV binds DNA in its absence, with an average rate of 0.17 s-1 for each step.

Using quantum-dot-labeled DNA and total internal reflection fluorescence microscopy, we can investigate the reaction mechanism of restriction endonucleases while using unlabeled protein. This single-molecule technique allows for massively multiplexed observation of individual protein-DNA interactions, and data can be pooled to generate well-populated dwell-time distributions.

This novel total internal reflection fluorescence microscopy-based assay facilitates the simultaneous measurement of the length of the catalytic cycle for ...

Making Precise and Accurate Single-Molecule FRET Measurements using the Open-Source smfBox

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes measurements on freely diffusing biomolecules more accessible. This overview includes a step-by-step protocol for using this instrument to make measurements of precise FRET efficiencies in duplex DNA samples, including details of the sample preparation, instrument setup and alignment, data acquisition, and complete analysis routines. The presented approach, which includes how to determine all the correction factors required for accurate FRET-derived distance measurements, builds on a large body of recent collaborative work across the FRET Community, which aims to establish standard protocols and analysis approaches. This protocol, which is easily adaptable to a range of biomolecular systems, adds to the growing efforts in democratising smFRET for the wider scientific community.

This article provides step-by-step instructions for making fully-corrected accurate FRET measurements on individual, freely diffusing biomolecules using the open-source, inexpensive smfBox, from switch on, through alignment and focusing, to data collection and analysis.

The smfBox is a recently developed cost-effective, open-source instrument for single-molecule F&#246;rster Resonance Energy Transfer (smFRET), which makes ...

F&#246;rster Resonance Energy Transfer Measurements in Living Plant Cells

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser scanning microscopes have become a workhorse for biologists. Commercial systems offer high flexibility in laser power adjustment and detector sensitivity and often combine different detectors to obtain the perfect image. However, the comparison of intensity-based data from different experiments and setups is often impossible due to this flexibility. Biologist-friendly procedures are of advantage and allow for simple and reliable adjustment of laser and detector settings. 
Furthermore, as FRET experiments in living cells are affected by the variability in protein expression and donor-acceptor ratios, protein expression levels must be considered for data evaluation. Described here is a simple protocol for reliable and reproducible FRET measurements, including routines for the estimation of protein expression and adjustment of laser intensity and detector settings. Data evaluation will be performed by calibration with a fluorophore fusion of known FRET efficiency. To improve simplicity, correction factors have been compared that have been obtained in cells and by measuring recombinant fluorescent proteins.

A protocol is provided for setting up a standard confocal laser-scanning microscope for in vivo F&#246;rster resonance energy transfer measurements, followed by data evaluation.

Sensitized emission-based F&#246;rster resonance energy transfer (FRET) experiments are easily done but depend on the microscopic setup. Confocal laser ...

F&#246;rster Resonance Energy Transfer Mapping: A New Methodology to Elucidate Global Structural Features

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between biomolecules in vitro as well as within cells. In FRET, the efficiency of energy transfer, measured by changes in fluorescence intensity or lifetime, relates to the distance between two fluorescent molecules or labels. Determination of dynamics and conformational changes from the distances are just some examples of applications of this method to biological systems. Under certain conditions, this methodology can add to and enhance existing X-ray crystal structures by providing information regarding dynamics, flexibility, and adaptation to binding surfaces. We describe the use of FRET and associated distance determinations to elucidate structural properties, through the identification of a binding site or the orientations of dimer subunits. Through judicious choice of labeling sites, and often employment of multiple labeling strategies, we have successfully applied these mapping methods to determine global structural properties in a protein-DNA complex and the SecA-SecYEG protein translocation system. In the SecA-SecYEG system, we have used FRET mapping methods to identify the preprotein-binding site and determine the local conformation of the bound signal sequence region. This study outlines the steps for performing FRET mapping studies, including identification of appropriate labeling sites, discussion of possible labels including non-native amino acid residues, labeling procedures, how to perform measurements, and interpreting the data.

The study details the methodology of FRET mapping including the selection of labeling sites, choice of dyes, acquisition, and data analysis. This methodology is effective at determining binding sites, conformational changes, and dynamic motions in protein systems and is most useful if performed in conjunction with existing 3-D structural information.

F&#246;rster resonance energy transfer (FRET) is an established fluorescence-based method used to successfully measure distances in and between ...

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes

Single-molecule F&#246;rster 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.

This article presents a method for spatiotemporal analysis of mobile, single-molecule F&#246;rster resonance energy transfer (smFRET)-based probes using widefield fluorescence microscopy. The newly developed software toolkit allows the determination of smFRET time traces of moving probes, including the correct FRET efficiency and the molecular positions, as functions of time.

Single-molecule F&#246;rster resonance energy transfer (smFRET) is a versatile technique reporting on distances in the sub-nanometer to nanometer range. ...

Bioengineering

Studying DNA Looping by Single-Molecule FRET

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into nucleosomes. Thermodynamics of dsDNA bending has been studied by a method called cyclization which relies on DNA ligase to covalently join short sticky ends of a dsDNA. However, ligation efficiency can be affected by many factors that are not related to dsDNA looping such as the DNA structure surrounding the joined sticky ends, and ligase can also affect the apparent looping rate through mechanisms such as nonspecific binding. Here, we show how to measure dsDNA looping kinetics without ligase by detecting transient DNA loop formation by FRET (Fluorescence Resonance Energy Transfer). dsDNA molecules are constructed using a simple PCR-based protocol with a FRET pair and a biotin linker. The looping probability density known as the J factor is extracted from the looping rate and the annealing rate between two disconnected sticky ends. By testing two dsDNAs with different intrinsic curvatures, we show that the J factor is sensitive to the intrinsic shape of the dsDNA.

This study presents a detailed experimental procedure to measure looping dynamics of double-stranded DNA using single-molecule Fluorescence Resonance Energy Transfer (FRET). The protocol also describes how to extract the looping probability density called the J factor.

Bending of double-stranded DNA (dsDNA) is associated with many important biological processes such as DNA-protein recognition and DNA packaging into ...

F&#246;rster Resonance Energy Transfer (FRET)

F&#246;rster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent molecule can non-radiatively transfer energy to an acceptor molecule if their respective emission and absorbance spectra overlap. The amount of energy transferred&#8212;and consequently the overall emission of sample&#8212;depends on the proximity of an acceptor-donor pair of photoluminescent molecules. FRET analysis is combined with other biochemistry techniques to obtain detailed information of biomolecular structures and interactions from this &#8220;spectroscopic ruler.&#8221;
This video covers the principles and concepts of FRET analysis. The procedure focuses on preparing samples for FRET and ways to present and interpret data. Finally, the applications include monitoring conformational and cellular processes by labeling parts of a cell or protein, monitoring enzyme reactions that alter protein structures, and using FRET to monitor aggregation of monomers expressed by cells.
<div class="chapter_text" id="script_0">
F&#246;rster Resonance Energy Transfer, or FRET, is a non-radiative transfer of energy between light-emitting molecules, and is often used to investigate close-range biochemical interactions. FRET only occurs when fluorescent molecules are spaced within 10 nm of each other. FRET analysis can be combined with other techniques to obtain detailed structural information. This video will introduce the underlying principles of FRET, summarize a protocol and data presentation, and discuss some biochemical applications.
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A photoluminescent molecule such as a fluorophore is excited by absorbing electromagnetic radiation at a wavelength in its absorption spectrum. As it relaxes, it emits light at a wavelength within its emission spectrum. For more information about fluorescence, see JoVE&#39;s video on fluorescence microscopy. Different fluorophores absorb and emit light at different wavelengths, which frequently overlap. If the emission spectrum of a fluorophore overlaps significantly with the absorption spectrum of another fluorophore, the &#8220;donor&#8221; will release a virtual photon, which is absorbed by the &#8220;acceptor&#8221;. When an excited donor is within 10 nm of an acceptor, energy is transferred from donor to acceptor by dipole-dipole interactions. The release of energy by emission of light from the donor correspondingly decreases. Meanwhile, the excited acceptor emits light at its emission wavelength. The FRET response is evaluated in terms of efficiency, or the percentage of energy released from the donor by FRET rather than by fluorescence or other radiative processes. The efficiency depends strongly on the distance between the donor and acceptor, which allows FRET to act as a &#39;molecular&#39; or &#39;spectroscopic&#39; ruler.
In biochemistry, FRET is often used qualitatively to observe conformational changes in molecules by monitoring fluorophores as they move in and out of FRET range of each other. Similarly, cellular functions can be studied with molecules containing a FRET pair. If the labeled molecule is cleaved by enzyme activity, FRET stops and the observed fluorescence wavelength changes.
Now that you understand the principles behind FRET, let&#39;s look at an overview of a protocol and a few ways to present and interpret the data.
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Prior to the experiment, the biomolecules of interest, typically DNA or proteins, are engineered with fluorescent tags, using molecular biology techniques.&#160;Common ways to introduce the modified genetic material into the cells include transfection and electroporation.
Then, the cells are prepared for FRET visualization on a fluorescence microscope.&#160;For instance, the molecules may be immobilized on a slide for single-molecule FRET, or samples are loaded into wells for high-throughput screening.
Then, the excitation lasers, microscope, and associated equipment are prepared. (A) FRET experiments often involve powerful lasers; (B) so appropriate PPE and safety procedures should be used.&#160;The sample is then placed in the instrument and illuminated with the excitation laser.
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For experiments monitoring cell behavior, color images showing differences or changes in emission intensity are used. Donor and acceptor emission intensities are plotted together to track FRET response over time.
FRET data can also be fitted to various functions for more complex analyses. Depending on the experiment, data may be presented in multiple ways to best represent the results, making FRET a flexible experimental tool.
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Now that you&#39;re familiar with the basics of running and analyzing a FRET experiment, let&#39;s look at some applications of FRET in biochemistry research.
FRET can be used to study conformational changes or cellular processes by labeling parts of the protein or cell predicted to move within 10 nm of each other with a FRET pair. For example, protein sensors are prepared by labeling receptors with a pair of fluorophores. The FRET response is monitored live by confocal microscopy. Variation of emission wavelength and intensity indicate conformational changes.
FRET can also be used by preparing molecules with an active FRET pair and observing changes in the response. When the substrate is cleaved, FRET is disrupted, causing an increase in donor emission and a decrease in acceptor emission. The emissions are analyzed to determine contributions by donor, acceptor, and FRET. Once the direct emission factors are calculated for the cyan and yellow fluorescent proteins, the concentration and kinetic parameters of the substrate can be determined.
Cells designed to express monomers containing either of a FRET pair function as &#39;sensors&#39; for interactions between those monomers. If aggregation of those monomers is induced, a FRET response is observed. This can be used to investigate protein aggregation triggered by &#39;seeding&#39; of misfolded proteins. Here, cells were transduced with aggregates of the protein of interest, incubated, and analyzed with flow cytometry.
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You&#39;ve just watched JoVE&#39;s video on F&#246;rster Resonance Energy Transfer, or FRET. This video contained the underlying principles of FRET, preparation and analysis of a FRET experiment, and a few biochemical applications.
Thanks for watching!
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F&amp;#246;rster Resonance Energy Transfer (FRET)

F&#246;rster resonance energy transfer (FRET) is a phenomenon used to investigate close-range biochemical interactions. In FRET, a donor photoluminescent ...

Single-Molecule Förster Resonance Energy Transfer Methods for Real-Time Investigation of the Holliday Junction Resolution by GEN1

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