Conventional Fluorescence Resonance Energy Transfer Research Articles

Single-Molecule FRET-Based Multiplexed Detection.

Single-molecule multiplexed detection is a high-promise toolkit for the expanding field ofbiosensing and molecular diagnostics. Among many single-molecule techniques available today for biomarker sensing including fluorescence, force, electrochemical, spectroscopic, barcoding, and other techniques, fluorescence-based approaches are arguably the most widely used methods due to their high sensitivity, selectivity, and readily available fluorophore-labeling schemes for a wide variety of biomolecules. However, multiplexed imaging using fluorescence techniques has proven to be challenging due to the sophisticated labeling schemes often requiring multiple FRET (fluorescence resonance energy transfer) pairs and/or excitation sources, which lead to overlapping signals and complicate data analysis. Here, we describe a single-molecule FRET method that enables multiplexed analysis while still using only one FRET pair, and thus the described approach is a significant step forward from conventional FRET methods.

Single-Molecule FRET X.

Fluorescence resonance energy transfer (FRET) is a photophysical phenomenon that has been repurposed as a biophysical tool to measure nanometer distances. With FRET by DNA eXchange, or FRET X, many points of interest (POIs) in a single object can be probed, overcoming a major limitation of conventional single-molecule FRET. In FRET X, short fluorescently labeled DNA imager strands specifically and transiently bind their complementary docking strands on a target molecule, such that at most a single FRET pair is formed at each point in time and multiple POIs on a single molecule can be readily probed. Here, we describe the sample preparation, image acquisition, and data analysis for structural analysis of DNA nanostructures with FRET X.

Engineering highly efficient NIR-II FRET platform for Background-Free homogeneous detection of SARS-CoV-2 neutralizing antibodies in whole blood

Förster or fluorescence resonance energy transfer (FRET) enables to probe biomolecular interactions, thus playing a vital role in bioassays. However, conventional FRET platforms suffer from limited sensitivity due to the low FRET efficiency and poor anti-interference of existing FRET pairs. Here we report a NIR-II (1000–1700 nm) FRET platform with extremely high FRET efficiency and exceptional anti-interference capability. This NIR-II FRET platform is established based on a pair of lanthanides downshifting nanoparticles (DSNPs) by employing Nd3+ doped DSNPs as an energy donor and Yb3+ doped DSNPs as an energy acceptor. The maximum FRET efficiency of this well-engineered NIR-II FRET platform reaches up to 92.2%, which is much higher than most commonly used ones. Owing to the all-NIR advantage (λex = 808 nm, λem = 1064 nm), this highly efficient NIR-II FRET platform exhibits extraordinary anti-interference in whole blood, and thus enabling background-free homogeneous detection of SARS-CoV-2 neutralizing antibodies in clinical whole blood sample with high sensitivity (limit of detection = 0.5 μg/mL) and specificity. This work opens up new opportunities for realizing highly sensitive detection of various biomarkers in biological samples with severe background interference.

A comparative study of conventional FRET and light harvesting properties of Rh-110/Rh-6G and Rh-19/Rh-B organic dye pairs impregnated in sol-gel glasses

Two different pairs of laser dyes, Rhodamine-110 (Rh-110)/Rhodamine-6G (Rh-6G) and Rhodamine-19 (Rh-19)/Rhodamine-B (Rh-B) (the first dye in each pair as a donor and the second as an acceptor) were impregnated in silica samples prepared by the sol-gel method and spectroscopically studied using absorption and steady-state fluorescence techniques. The critical transfer distance (R0), actual distance (r) between the donor and acceptor, overlap integral [J()], FRET (fluorescence resonance energy transfer) efficiency (E), and antenna effect efficiency (AE) were investigated in detail based on the variation in acceptor concentration. The FRET efficiency, antenna effect efficiency, and actual donor-acceptor distance for Rh-110/Rh-6G and Rh-19/Rh-B dye pairs corresponding to acceptor concentration ranges (3.83 to 7.65) × 10−5 M l−1 and (3.71 to 8.34) × 10−5 M l−1, respectively, were found to be in the ranges of 57.38% to 74.89%, 36.97% to 24.13%, 5.44 nm to 4.77 nm, and 77.01%. Furthermore, maximum FRET efficiencies of 85.68% and 87.63% and antenna effect efficiencies of 36.97% and 40.95% for Rh-110/Rh-6G and Rh-19/Rh-B, respectively, were also reported. Our results demonstrate the superior FRET efficiency of Rh-19/Rh-B over Rh-110/Rh-6G dye pair in sol-gel glasses, while the antenna effect efficiency of Rh-110/Rh-6G is higher than that of Rh-19/Rh-B for the same donor to acceptor (D/A) ratio. Finally, Rh-110/Rh-6G is a better energy harvester than the Rh-19/Rh-B dye pair at the common D/A ratio. These results are explained in terms of molecular structure similarity, polarity, and rigidity of donor and acceptor.

Wash-Free Detection of Nucleic Acids with Photoswitch-Mediated Fluorescence Resonance Energy Transfer against Optical Background Interference.

The optical background such as autofluorescence and light scattering poses a big challenge to quantify nucleic acids with conventional fluorescence-based methods. We report here high-contrast nucleic acid detection with photoswitch-mediated fluorescence resonance energy transfer (FRET), which strongly occurs between the open forms of the photoswitch (a naphthopyran) and the signal fluorophores brought to the surface of the nanoprobes (≲15 nm). The fluorescence change (ΔF) upon UV irradiation is highly sensitive and more robust to quantify the target DNAs than traditional intensity measurements. Therefore, the method works in samples with strong background fluorescence from the unbound fluorophores. The photoswitchable nanoprobes could be easily prepared and interrogated in capillaries for high-throughput measurements. The method was evaluated in both sandwich-like hybridization and DNA label-free detection with a nucleic stain SG. Without DNA amplification and sample pretreatment of blood serum, the photoswitchable nanoprobes provided a limit of detection of 0.5 nM, which is ∼6 to 20 times lower than conventional FRET.

Twin-FRET: A New Molecular Ruler for Biomolecules

Conformational changes in biomolecules underpin all biological processes and being able to quantify these structural changes in solution is crucial to understand biological function. By carefully positioning two fluorophores within the biomolecule, it is possible to use fluorescence resonance energy transfer (FRET) as a molecular ruler to measure the desired distance. In current FRET assays, the biomolecule needs to be labelled with two different chemical fluorophores acting as a donor-acceptor FRET pair. Incorporation of these two chemically different species at specific positions within the biomolecule is challenging due to limited chemical labelling strategies. Here, we present a radically different strategy for measuring distances in biomolecules. We have developed the concept of TWIN-FRET which removes the need for two different fluorophores attached to the biomolecule by chemically encoding the FRET pair within the structure of the fluorophore itself. We have designed and synthesised a fluorescent molecule to prove this concept. The fluorophore (FH) has an acid-base equilibrium with a ground state pKa∼8.9. We have derivatized the fluorophore to its succinimide ester, and used this derivative, to label a duplex DNA with two molecules of the same fluorophore at specific positions. Our results demonstrate the transfer of non-radiative energy from the neutral (FH) to the anionic (F−) state of the fluorescent molecule. We further demonstrate the use of TWIN-FRET to measure nanometer-size distances within the DNA duplex, and we obtained distance values similar to those obtained using a conventional FRET pair (Alexa488-Cy3). By removing the need to introduce two different chemical structures within the biomolecule, we greatly simplified the methodology to measure nanometer-size distances in biomolecules. We expect this technique to be widely used in structural and biophysical studies of nucleic acids, proteins and interactions between them.

Role of quantum dot in designing FRET based sensors

Nowadays Fluorescence Resonance Energy Transfer (FRET), which involves the nonradiative transfer of excitation energy from an excited state donor to a proximal ground-state acceptor, may be regarded as a useful tool in the design of ratiometric sensors. Recently, quantum dots (QDs), a kind of semiconductor nanoparticles, have been employed to modulate FRET. Compared with conventional FRET, quantum dot mediated FRET (QD-FRET) is advantageous because of their unique photophysical properties such as size tunable, narrow emission spectra but broad excitation spectra, strong signal intensity, high photostability, and good biocompatibility. Owing to these superior properties QDs have obtained huge development in many fields, such as drug testing, cell imaging, virus detection, molecular biology, medical diagnosis, electron transfer detection and so on. In view of above-mentioned achievements, QDs is a kind of promising material which can definitely be used as donors or acceptors in the FRET system. This review highlights the modern development of sensitive and selective ratiometric FRET based sensors where QDs has been used as fluorophores.

Optofluidic FRET Lasers and Their Applications in Novel Photonic Devices and Biochemical Sensing

Incorporating fluorescence resonance energy transfer (FRET) into a laser cavity can increase the sensitivity of FRET-based biochemical sensors due to the nonlinear dependence of the lasing output on the FRET parameters. Here, we carry out a comprehensive theoretical analysis of optofluidic FRET lasers based on a Fabry–Perot microcavity using a rate equation model. We compare conceptually distinct cases of donor and acceptor molecules diffusing freely in a bulk solution versus molecules connected by a fixed-length linker and show that the latter arrangement is especially well suited for sensing of low-concentration analytes. By comparing FRET lasing-based sensors with conventional FRET sensors, we show that for optimal pump fluence and FRET-pair concentration, FRET lasing can lead to more than 100-fold enhancement in detection sensitivities of conformational changes in the Forster radius range. We also show that for optimal experimental conditions, donor and acceptor emission intensities become over 20-fold more sensitive to FRET-pair concentration changes in the presence of FRET lasing. We study the dependence of the sensitivity enhancement on the cavity Q-factor. We show that the highest enhancements can be obtained for Q-factors between $10^4\text{--}10^6$ , and enhancement values decrease for Q-factors above $10^6$ due to the radiative energy transfer in the cavity.

Multi step FRET among three laser dyes Pyrene, Acriflavine and Rhodamine B

Fluorescence Resonance Energy Transfer (FRET) system using three dyes has been demonstrated. It has been observed that multi step energy transfer occurred from Pyrene to Rhodamine B via Acriflavine. Here Acriflavine acts as an antenna to receive energy from Pyrene and transfer the same to Rhodamine B. This multi step FRET system is advantageous compared to the conventional FRET as this can be used to study molecular level interaction beyond conventional FRET distance (1–10nm) as well as studying multi-branched macromolecules. The introduction of clay enhances the FRET efficiencies among the dye pair, which is an advantage to make the multi step system more useful. Similar approach can be used for increasing FRET efficiencies by using other dyes.

TripleFRET measurements in flow cytometry

A frequently used method for viewing protein interactions and conformation, Förster (fluorescence) resonance energy transfer (FRET), has traditionally been restricted to two fluorophores. Lately, several methods have been introduced to expand FRET methods to three species. We present a method that allows the determination of FRET efficiency in three-dye systems on a flow cytometer. TripleFRET accurately reproduces energy transfer efficiency values measured in two-dye systems, and it can indicate the presence of trimeric complexes, which is not possible with conventional FRET methods. We also discuss the interpretation of energy transfer values obtained with tripleFRET in relation to spatial distribution of labeled molecules, specifically addressing the limitations of using total energy transfer to determine molecular distance.

First Realization of Single-Molecule Four-Color FRET

Single-molecule FRET (Fluorescence Resonance Energy Transfer) has provided unprecedented details on fundamental processes in biology. However, information of single inter-fluorophore distances in conventional two-color FRET is insufficient to completely capture the intrinsic complexity of many biological systems. Despite recent developments of single-molecule three-color FRET techniques, there is an ever increasing demand for more advanced FRET techniques, four-color FRET, as single-molecule approaches are being expanded to include biological systems with multiple components.Here, we report single-molecule four-color FRET technique both in confocal and in total-internal-reflection fluorescence microscopies. Real-time determination of six inter-fluorophore FRET efficiencies allowed us to probe the correlated motion of four arms of the Holliday junction. The technique was also applied to assess the correlation of RecA-mediated strand exchange events at both ends of a synaptic complex. We expect that the technique will have broad applications in measuring the correlated dynamics of complex biological systems.

OS-FRET: A New One-Sample Method for Improved FRET Measurements

Fluorescence resonance energy transfer (FRET) is a powerful tool for studying macromolecular assemblies in vitro under near-physiological conditions. Here we present a new type of one-sample FRET (OS-FRET) method employing a novel, nonfluorescent methanethiosulfonate-linked acceptor that can be reversibly coupled to a target sulfhydryl residue via a disulfide bond. After the quenched donor emission is quantitated, the acceptor is removed by reduction, allowing measurement of unquenched donor emission in the same sample. Previous one-sample methods provide distinct advantages in specific FRET applications. The new OS-FRET method is a generalizable spectrochemical approach that can be applied to macromolecular systems lacking essential disulfide bonds and eliminates the potential systematic errors of some earlier one-sample methods. In addition, OS-FRET enables quantitative FRET measurements in virtually any fluorescence spectrometer or detection device. Compared to conventional multisample FRET methods, OS-FRET conserves sample, increases the precision of data, and shortens the time per measurement. The utility of the method is illustrated by its application to a protein complex of known structure formed by CheW and the P4-P5 fragment of CheA, both from Thermotoga maritima. The findings confirm the practicality and advantages of OS-FRET. Anticipated applications of OS-FRET include analysis of macromolecular structure, binding and conformational dynamics, and high-throughput screening for interactions and inhibitors.

Carbon Nanotubes as a Low Background Signal Platform for a Molecular Aptamer Beacon on the Basis of Long-Range Resonance Energy Transfer

Although holding the advantages of both an aptamer and a molecular beacon (MB), a molecular aptamer beacon (MAB) needs complicated and expensive modifications at both of its ends and usually has a high background signal because of the low energy transfer efficiency between the donor and the acceptor. To overcome these shortcomings, in this study, we develop a long-range resonance energy transfer (LrRET) system by separating the donor from the acceptor, wherein only one end of the MAB is fluorescently labeled and acts as the energy donor and multiwalled carbon nanotubes (MWCNTs) are introduced as the energy acceptor. To test the feasibility of the newly designed MAB system, adenosine triphosphate (ATP) has been employed as a proof-of-concept target. It is found that the fluorescence of the designed MAB is completely quenched by MWCNTs, supplying a very low background signal. Then the quenched fluorescence is recovered significantly with the addition of ATP, so that ATP can be detected in the range of 0.8-80 μM with a limit of detection of 0.5 μM (3σ). Compared with the conventional fluorescence resonance energy transfer, the efficiency of LrRET between the dye and MWCNTs is much higher. Since only one end of the MAB needs the modification, the present strategy is simple and cost-effective. Furthermore, the use of MWCNTs can greatly reduce the fluorescence background of the MAB and supply a high sensitivity, showing its generality for detection of a variety of targets.

Proximity or no proximity: That is the question—But the answer is more complex

THE significance of molecular interactions has been recognized since the dawn of molecular biology, and the real importance of the vast amount of information hidden in the human genome can only be understood if the interactome is also defined. Conventionally, molecular interactions are investigated by chemical crosslinking and coprecipitation. Although these approaches are widely used, they do not provide quantitative information, suffer from the lack of cell-by-cell resolution, and potential artifacts arising from high protein expression and membrane solubilization in the case of membrane proteins. Cytometry offers several alternative methods to assess protein–protein interactions. Fluorescence resonance energy transfer (FRET) has made its way to become the golden standard in quantitative analysis of protein interactions (1). In FRET, a fluorescent donor dye residing on a protein of interest transfers its excitation energy to a nearby acceptor molecule which labels another protein. The rate of this radiationless interaction decreases with the sixth power of the separation distance making FRET a sensitive tool for molecular interactions. In conventional FRET, the pairwise interaction between two spectroscopically distinct molecules is analyzed, and the principle has recently been applied for the simultaneous investigation of two pairwise interactions (2) and for the investigation of the three-way interaction among three molecules (3,4). Although FRET usually takes place between spectroscopically distinct molecules, the same process can also occur between molecules of the same type. This flavor of the interaction is called homo-FRET, and it has been used for the detection of molecular trimers (5) and even larger clusters (6,7). The combination of total internal reflection microscopy with FRET allowed the high-throughput screening of molecular interactions (8). A multitude of correlation techniques are available for the analysis of protein clustering. The mother of all such approaches is fluorescence correlation spectroscopy (FCS) in which the autocorrelation of fluorescence signals is measured in a femtoliter volume to determine the diffusion constant (9,10). FCS has been applied in confocal microscopy for the accurate and quantitative measurement of association states and concentrations in the intracellular space (11,12) and for the monitoring of dynamic properties of membrane proteins (13,14). Fluorescence cross-correlation spectroscopy (FCCS) has been used for the detection of the association of two fluorescently labeled proteins. Dynamic and transient interactions are more conveniently detected by FCCS than by FRET (15). However useful the aforementioned correlation techniques are, they require dedicated instrumentation. The realization that a temporal confocal image series taken by a conventional confocal microscope contains all the necessary information to calculate the diffusion constant and the association state of the fluorescently labeled substance has lead to the development of raster image correlation spectroscopy (RICS) (16) and number and brightness analysis (N&B) (17). Both of these techniques

Simultaneous Live Cell Imaging Using Dual FRET Sensors with a Single Excitation Light

Fluorescence resonance energy transfer (FRET) between fluorescent proteins is a powerful tool for visualization of signal transduction in living cells, and recently, some strategies for imaging of dual FRET pairs in a single cell have been reported. However, these necessitate alteration of excitation light between two different wavelengths to avoid the spectral overlap, resulting in sequential detection with a lag time. Thus, to follow fast signal dynamics or signal changes in highly motile cells, a single-excitation dual-FRET method should be required. Here we reported this by using four-color imaging with a single excitation light and subsequent linear unmixing to distinguish fluorescent proteins. We constructed new FRET sensors with Sapphire/RFP to combine with CFP/YFP, and accomplished simultaneous imaging of cAMP and cGMP in single cells. We confirmed that signal amplitude of our dual FRET measurement is comparable to of conventional single FRET measurement. Finally, we demonstrated to monitor both intracellular Ca2+ and cAMP in highly motile cardiac myocytes. To cancel out artifacts caused by the movement of the cell, this method expands the applicability of the combined use of dual FRET sensors for cell samples with high motility.

Two-Photon Excitation Fluorescence Resonance Energy Transfer with Small Organic Molecule as Energy Donor for Bioassay

A fluorescence resonance energy transfer (FRET) model using two-photon excitable small organic molecule DMAHAS as energy donor has been constructed and tried in an assay for avidin. In the FRET model, biotin was conjugated to the FRET donor, and avidin was labeled with a dark quencher DABS-Cl. Binding of DABS-Cl labeled avidin to biotinylated DMAHAS resulted in the quenching of fluorescence emission of the donor, based on which a competitive assay for free avidin was established. With using such donors that are excited in IR region, it is capable of overcoming some primary shortcomings of conventional one-photon FRET methods, especially in bioassays, such as the interference from background fluorescence or scattering light, the coexcitation of the energy acceptor with the donor. And such small molecules also show advantages over inorganic up-converting particles that also give anti-Stokes photoluminescence and have been applied as FRET donor recently. The results of this work suggest that two-photon excitable small molecules could be a promising energy donor for FRET-based bioassays.

Three-Color Alternating-Laser Excitation of Single Molecules: Monitoring Multiple Interactions and Distances

We introduce three-color alternating-laser excitation (3c-ALEX), a fluorescence resonance energy transfer (FRET) method that measures up to three intramolecular distances and complex interaction stoichiometries of single molecules in solution. This tool extends substantially the capabilities of two-color ALEX, which employs two alternating lasers to study molecular interactions (through probe stoichiometry S) and intramolecular distances (through FRET efficiency E), and sorts fluorescent molecules in multi-dimensional probe-stoichiometry and FRET-efficiency histograms. Probe-stoichiometry histograms allowed analytical sorting, identification, and selection of diffusing species; selected molecules were subsequently represented in FRET-efficiency histograms, generating up to three intramolecular distances. Using triply labeled DNAs, we established that 3c-ALEX enables 1), FRET-independent analysis of three-component interactions; 2), observation and sorting of singly, doubly, and triply labeled molecules simultaneously present in solution; 3), measurements of three intramolecular distances within single molecules from a single measurement; and 4), dissection of conformational heterogeneity with improved resolution compared to conventional single-molecule FRET. We also used 3c-ALEX to study large biomolecules such as RNA polymerase-DNA transcription complexes, and monitor the downstream translocation of RNA polymerase on DNA from two perspectives within the complex. This study paves the way for advanced single-molecule analysis of complex mixtures and biomolecular machinery.

Resonance energy transfer from a fluorescent dye to a metal nanoparticle

A quantum mechanical theory of the rate of excitation energy transfer from a fluorescent dye molecule to the surface plasmonic modes of a spherical metal nanoparticle is presented. The theory predicts the distance dependence of the transfer rate to vary as 1/d(sigma), with sigma=3-4 at intermediate distances, in partial agreement with the recent experimental results. Förster's 1/d(6) dependence is recovered at large separations. The predicted rate exhibits nontrivial nanoparticle size dependence, ultimately going over to an asymptotic, a(3) size dependence. Unlike in conventional fluorescence resonance energy transfer, the orientational factor is found to vary between 1 and 4.

GPCR dimerisation

The concept that GPCRs exist and potentially function as dimers and/or higher oligomers has progressed recently from hypothesis to being widely accepted. A range of techniques has contributed to this understanding, including co-immunoprecipitation and various forms of fluorescence and bioluminescence resonance energy transfer. Although co-immunoprecipitation studies indicate the capacity of a wide range of GPCRs to form hetero-dimers as well as homo-dimers, this approach is not well suited to examine selectivity of interactions. Both bioluminescence resonance energy transfer (BRET) and fluorescence resonance energy transfer (FRET) have been applied to the detection of GPCR dimerisation in intact cells and BRET and FRET have been used to attempt to quantitate the fraction of GPCRs present as dimers. Following heterologous expression, a considerable fraction of many GPCRs is not fully processed and is trafficked to the proteasome or lysosome for destruction. A distinct limitation of both BRET and conventional FRET approaches is that both the energy donor and energy acceptor tags are inside the cell. Time-resolved FRET employing N-terminally epitope-tagged GPCRs has been used to allow detection only of dimers trafficked successfully to the cell surface. Reports indicating the appearance of distinct pharmacology and function following co-expression of two GPCRs are fascinating. Much remains to be examined, however, on the specificity and mechanisms of these interactions and to develop techniques to monitor the function only of hetero-dimers when the corresponding homo-dimers must also be present.

Photodestruction intermediates probed by an adjacent reporter molecule.

We used a fluorescence resonance energy transfer donor molecule to probe the multiple intermediates in the photoinduced destruction of an acceptor molecule. These intermediates are nonemitting but are still able to quench the fluorescence of the donor at a distance scale shorter than conventional fluorescence resonance energy transfer, suggesting novel biophysical applications.