Förster Resonance Energy Transfer Pair Research Articles

Development of mKate3/HaloTag7 (JFX650) and CFP/YFP Dual-Fluorescence (or Förster) Resonance Energy Transfer Pairs for Visualizing Dual-Molecular Activity.

Although several imaging strategies for dual fluorescence (or Förster) resonance energy transfer (FRET) biosensors have been reported, their implementation is challenging because of the limited performance of fluorescent proteins and the spectral overlap of FRET biosensors. These processes often require additional data calibration to eliminate artifacts. Many CFP/YFP FRET biosensors have been developed. In this study, we introduced the mKate3/HT7(JFX650) FRET pair, which effectively formed two pairs of FRET pairs for dual-FRET imaging when combined with the CFP/YFP FRET pair. The FRET donor mKate3 exhibited higher brightness than its predecessor mKate. The FRET acceptor, HT7(JFX650), is a HaloTag7 protein covalently conjugated with a far-red JFX650-THL ligand. The pair comprising mKate3 and HT7(JFX650) represents an excellent FRET dyad, exhibiting a high FRET efficiency ratio. To use the FRET pair for dual FRET biosensor imaging, we constructed PKA and K+ biosensors based on the mKate3/HT7(JFX650) FRET pair. These biosensors can be used along with CFP/YFP biosensors to simultaneously detect the responses of intracellular PKA/Src, PKA/Ca2+, and K+/Ca2+ under different stimuli. The findings revealed that dual FRET biosensors, which are based on the combination of CFP/YFP and mKate3/HT7 (JFX650), exhibit adequate compatibility and can be used to visualize multiple molecular activities in a live cell.

Portable Near-Infrared to Near-Infrared Platform for Homogeneous Quantification of Biomarkers in Complex Biological Samples.

Förster resonance energy transfer (FRET)-based homogeneous immunoassay obviates tedious washing steps and thus is a promising approach for immunoassays. However, a conventional FRET-based homogeneous immunoassay operating in the visible region is not able to overcome the interference of complex biological samples, thus resulting in insufficient detection sensitivity and poor accuracy. Here, we develop a near-infrared (NIR)-to-NIR FRET platform (Ex = 808 nm, Em = 980 nm) that enables background-free high-throughput homogeneous quantification of various biomarkers in complex biological samples. This NIR-to-NIR FRET platform is portable and easy to operate and is mainly composed of a high-performance NIR-to-NIR FRET pair based on lanthanide-doped nanoparticles (LnNPs) and a custom-made microplate reader for readout of NIR luminescence signals. We demonstrate that this NIR-to-NIR FRET platform is versatile and robust, capable of realizing highly sensitive and accurate detection of various critical biomarkers, including small molecules (morphine and 1,25-dihydroxyvitamin D), proteins (human chorionic gonadotropin), and viral particles (adenovirus) in unprocessed complex biological samples (urine, whole blood, and feces) within 5-10 min. We expect this NIR-to-NIR FRET platform to provide low-cost healthcare for populations living in resource-limited areas and be widely used in many other fields, such as food safety and environmental monitoring.

Effect of Hybridization on the Photoluminescence Properties of Atomically Precise Silver Nanoclusters

AbstractThe Förster Resonance Energy Transfer (FRET) is a widely used mechanism harnessed for sensing applications. For temporal stability of the energy transfer between donor and acceptor molecules their photostability and maintenance of the coupling geometry are important. A donor‐acceptor system was created from silicon nanoclusters (Si NCs) and silver nanoclusters (dihydrolopic acid (DHLA)‐protected Ag29 NCs). Optical spectroscopy and lifetime measurements helped to understand the energy transfer process between the Si NCs and Ag29 NCs. Compared with donor‐acceptor pairs based on gold nanocluster and organic dyes, the FRET pair formed between Si NCs and Ag29 (DHLA)12 NCs reaches a coequal efficiency of 60 %. A fine‐tuning of the system‘s parameters opens the door to investigating the sensitivity of the two nanocluster FRET system to various species and incorporating highly photostable moieties in a process relying on photoluminescence.

A wearable microneedle patch incorporating reversible FRET-based hydrogel sensors for continuous glucose monitoring

Continuous glucose monitors are crucial for diabetes management, but invasive sampling, signal drift and frequent calibrations restrict their widespread usage. Microneedle sensors are emerging as a minimally-invasive platform for real-time monitoring of clinical parameters in interstitial fluid. Herein, a painless and flexible microneedle sensing patch is constructed by a mechanically-strong microneedle base and a thin layer of fluorescent hydrogel sensor for on-site, accurate, and continuous glucose monitoring. The Förster resonance energy transfer (FRET)-based hydrogel sensors are fabricated by facile photopolymerizations of acryloylated FRET pairs and glucose-specific phenylboronic acid. The optimized hydrogel sensor enables quantification of glucose with reversibility, high selectivity, and signal stability against photobleaching. Poly (ethylene glycol diacrylate)-co-polyacrylamide hydrogel is utilized as the microneedle base, facilitating effective skin piercing and biofluid extraction. The integrated microneedle sensor patch displays a sensitivity of 0.029 mM−1 in the (patho)physiological range, a low detection limit of 0.193 mM, and a response time of 7.7 min in human serum. Hypoglycemia, euglycemia and hyperglycemia are continuously monitored over 6 h simulated meal and rest activities in a porcine skin model. This microneedle sensor with high transdermal analytical performance offers a powerful tool for continuous diabetes monitoring at point-of-care settings.

Resonant energy transfer among naturally available bio materials for white light emission

In this work we have studied the fluorescence of natural dyes and generated nearly pure white light with chromaticity intensity (CIE) coordinate (0.35,0.35). The colour rendering index (CRI) and colour temperature corresponding to the CIE coordinate are calculated and these dyes are ideal for cool white light emission. It was observed that a broadband and simultaneous emission involving anthocyanin and polyphenol ellergic acid from jamun, curcumin and chlorophyll from spinach leaves played a vital role in obtaining a CIE index close to that of pure white light. The white light emission is due to the Förster resonance energy transfer (FRET) from curcumin to anthocyanin and ellergic acid to curcumin. Efficiency of FRET is calculated and different possibilities studied. For the polyphenol ellergic acid, curcumin FRET pair the spectral overlap integral and the efficiency are 3.29 × 10−24 m2, 99.97% and for the curcumin, anthocyanin pair, they are 4.03 × 10–24 m2, 76%, respectively.

Surfactant-Assisted Förster Resonance Energy Transfer from a Quantum Dot Complex for Highly Stable White Light Emission.

Herein we report the fabrication of a surfactant modified quantum dot complex (S-QDC, having λem = 485 nm) nanocomposite (composed of cetyltrimethylammonium bromide surfactants and a zinc-quinolate complex attached ZnS quantum dot), the donor capability of S-QDC in Förster resonance energy transfer (FRET) with an acceptor organic molecule (λem = 573 nm), and finally their utilization in the FRET-based white light emission having features near to mid-day sunlight. The Förster distance, energy transfer efficiency, donor-acceptor distance, number of binding sites, and binding constant are evaluated to be 3.48 nm, 85.74%, 2.58 nm, 0.94, and 1.87 × 104 M-1, respectively, for the current electrostatically driven FRET pair. The solid polymer coated FRET pair composite emits white light having chromaticity color coordinates of (0.33, 0.33) and correlated color temperature of 5350 K and also shows long-term atmospheric white luminescence stability up to 30 days, photostability, and thermal stability with preservation of their pristine morphology.

Improvement of the Green-Red Förster Resonance Energy Transfer-Based Ca2+ Indicator by Using the Green Fluorescent Protein, Gamillus, with a Trans Chromophore as the Donor.

To monitor the Ca2+ dynamics in cells, various genetically encoded Ca2+ indicators (GECIs) based on Förster resonance energy transfer (FRET) between fluorescent proteins are widely used for live imaging. Conventionally, cyan and yellow fluorescent proteins have been often used as FRET pairs. Meanwhile, bathochromically shifted indicators with green and red fluorescent protein pairs have various advantages, such as low toxicity and autofluorescence in cells. However, it remains difficult to develop them with a similar level of dynamic range as cyan and yellow fluorescent protein pairs. To improve this, we used Gamillus, which has a unique trans-configuration chromophore, as a green fluorescent protein. Based on one of the best high-dynamic-range GECIs, Twitch-NR, we developed a GECI with 1.5-times higher dynamic range (253%), Twitch-GmRR, using RRvT as a red fluorescent protein. Twitch-GmRR had high brightness and photostability and was successfully applied for imaging the Ca2+ dynamics in live cells. Our results suggest that Gamillus with trans-type chromophores contributes to improving the dynamic range of GECIs. Therefore, selection of the cis-trans isomer of the chromophore may be a fundamental approach to improve the dynamic range of green-red FRET indicators, unlimited by GECIs.

Förster resonance energy transfer within single chain nanoparticles.

Single chain nanoparticles (SCNPs) are a highly versatile polymer architecture consisting of single polymer chains that are intramolecularly crosslinked. Currently, SCNPs are discussed as powerful macromolecular architectures for catalysis, delivery and sensors. Herein, we introduce a methodology based on Förster Resonance Energy Transfer (FRET) to evidence the folding of single polymer chains into SCNPs via fluorescence readout. We initially introduce a molecular FRET pair based on a bimane and nitrobenzoxadiazole (NBD) moiety and study its fluorescence properties in different solvents. We subsequently construct a low dispersity polymer chain carrying NBD units, while exploiting the bimane units for intramolecular chain collapse. Upon chain collapse and SCNP formation - thus bringing bimane and NBD units into close proximity - the SCNPs report their folded state by a strong and unambiguous FRET fluorescence signal. The herein introduced reporting of the folding state of SCNPs solely relies on an optical readout, opening avenues to monitoring SCNP folding without recourse to complex analytical methodologies.

Cascade Energy Transfer and White‐Light Emission in Chirality‐Controlled Crystallization‐Driven Two‐Dimensional Co‐assemblies from Donor and Acceptor Dye‐Conjugated Polylactides

AbstractAchieving predictable and programmable two‐dimensional (2D) structures with specific functions from exclusively organic soft materials remains a scientific challenge. This article unravels stereocomplex crystallization‐driven self‐assembly as a facile method for producing thermally robust discrete 2D‐platelets of diamond shape from biodegradable semicrystalline polylactide (PLA) scaffolds. The method involves co‐assembling two PLA stereoisomers, namely, PY‐PDLA and NMI‐PLLA, which form stereocomplex (SC)‐crystals in isopropanol. By conjugating a well‐known Förster resonance energy transfer (FRET) donor and acceptor dye, namely, pyrene (PY) and naphthalene monoimide (NMI), respectively, to the chain termini of these two interacting stereoisomers, a thermally robust FRET process can be stimulated from the 2D array of the co‐assembled dyes on the thermally resilient SC‐PLA crystal surfaces. Uniquely, by decorating the surface of the SC‐PLA crystals with an externally immobilized guest dye, Rhodamine‐B, similar diamond‐shaped structures could be produced that exhibit pure white‐light emission through a surface‐induced two‐step cascade energy transfer process. The FRET response in these systems displays remarkable dependence on the intrinsic crystalline packing, which could be modulated by the chirality of the co‐assembling PLA chains. This is supported by comparing the properties of similar 2D platelets generated from two homochiral PLLAs (PY‐PLLA and NMI‐PLLA) labeled with the same FRET pair.

Cascade Energy Transfer and White-Light Emission in Chirality-Controlled Crystallization-Driven Two-Dimensional Co-assemblies from Donor and Acceptor Dye-Conjugated Polylactides.

Achieving predictable and programmable two-dimensional (2D) structures with specific functions from exclusively organic soft materials remains a scientific challenge. This article unravels stereocomplex crystallization-driven self-assembly as a facile method for producing thermally robust discrete 2D-platelets of diamond shape from biodegradable semicrystalline polylactide (PLA) scaffolds. The method involves co-assembling two PLA stereoisomers, namely, PY-PDLA and NMI-PLLA, which form stereocomplex (SC)-crystals in isopropanol. By conjugating a well-known Förster resonance energy transfer (FRET) donor and acceptor dye, namely, pyrene (PY) and naphthalene monoimide (NMI), respectively, to the chain termini of these two interacting stereoisomers, a thermally robust FRET process can be stimulated from the 2D array of the co-assembled dyes on the thermally resilient SC-PLA crystal surfaces. Uniquely, by decorating the surface of the SC-PLA crystals with an externally immobilized guest dye, Rhodamine-B, similar diamond-shaped structures could be produced that exhibit pure white-light emission through a surface-induced two-step cascade energy transfer process. The FRET response in these systems displays remarkable dependence on the intrinsic crystalline packing, which could be modulated by the chirality of the co-assembling PLA chains. This is supported by comparing the properties of similar 2D platelets generated from two homochiral PLLAs (PY-PLLA and NMI-PLLA) labeled with the same FRET pair.

A novel fluorescent protein pair facilitates FLIM-FRET analysis of plant immune receptor interaction under native conditions.

Elucidating protein-protein interactions is crucial for our understanding of molecular processes within living organisms. Microscopy-based techniques can detect protein-protein interactions in vivo at the single-cell level and provide information on their subcellular location. Fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) is one of the most robust imaging approaches, but it is still very challenging to apply this method to proteins which are expressed under native conditions. Here we describe a novel combination of fluorescence proteins (FPs), mCitrine and mScarlet-I, which is ideally suited for FLIM-FRET studies of low abundance proteins expressed from their native promoters in stably transformed plants. The donor mCitrine displays excellent brightness in planta, near-mono-exponential fluorescence decay, and a comparatively long fluorescence lifetime. Moreover, the FRET pair has a good spectral overlap and a large Förster radius. This allowed us to detect constitutive as well as ligand-induced interaction of the Arabidopsis chitin receptor components CERK1 and LYK5 in a set of proof-of-principle experiments. Due to the good brightness of the acceptor mScarlet-I, the FP combination can be readily utilized for co-localization studies. The FP pair is also suitable for co-immunoprecipitation experiments and western blotting, facilitating a multi-method approach for studying and confirming protein-protein interactions.

One pattern analysis (OPA) for the quantitative determination of protein interactions in plant cells

BackgroundA commonly used approach to study the interaction of two proteins of interest (POIs) in vivo is measuring Förster Resonance Energy Transfer (FRET). This requires the expression of the two POIs fused to two fluorescent proteins that function as a FRET pair. A precise way to record FRET is Fluorescence Lifetime IMaging (FLIM) which generates quantitative data that, in principle, can be used to resolve both complex structure and protein affinities. However, this potential resolution is often lost in many experimental approaches. Here we introduce a novel tool for FLIM data analysis of multiexponential decaying donor fluorophores, one pattern analysis (OPA), which allows to obtain information about protein affinity and complex arrangement by extracting the relative amplitude of the FRET component and the FRET transfer efficiency from other FRET parameters.ResultsAs a proof of concept for OPA, we used FLIM-FRET, or FLIM-FRET in combination with BiFC to reassess the dimerization and tetramerization properties of known interacting MADS-domain transcription factors in Nicotiana benthamiana leaf cells and Arabidopsis thaliana flowers. Using the OPA tool and by extracting protein BINDING efficiencies from FRET parameters to dissect MADS-domain protein interactions in vivo in transient N. benthamiana experiments, we could show that MADS-domain proteins display similar proximities within dimeric or tetrameric complexes but bind with variable affinities. By combining FLIM with BiFC, we were able to identify SEPALLATA3 as a mediator for tetramerization between the other MADS-domain factors. OPA also revealed that in vivo expression from native promoters at low levels in Arabidopsis flower meristems, makes in situ complex formation of MADS-domain proteins barely detectable.ConclusionsWe conclude that MADS-domain protein interactions are transient in situ and may involve additional, so far unknown interaction mediators. We conclude that OPA can be used to separate protein binding from information about proximity and orientation of the interacting proteins in their complexes. Visualization of individual protein interactions within the underlying interaction networks in the native environment is still restrained if expression levels are low and will require continuous improvements in fluorophore labelling, instrumentation set-ups and analysis tools.

A general method for the development of multicolor biosensors with large dynamic ranges

Fluorescent biosensors enable the study of cell physiology with spatiotemporal resolution; yet, most biosensors suffer from relatively low dynamic ranges. Here, we introduce a family of designed Förster resonance energy transfer (FRET) pairs with near-quantitative FRET efficiencies based on the reversible interaction of fluorescent proteins with a fluorescently labeled HaloTag. These FRET pairs enabled the straightforward design of biosensors for calcium, ATP and NAD+ with unprecedented dynamic ranges. The color of each of these biosensors can be readily tuned by changing either the fluorescent protein or the synthetic fluorophore, which enables simultaneous monitoring of free NAD+ in different subcellular compartments following genotoxic stress. Minimal modifications of these biosensors furthermore allow their readout to be switched to fluorescence intensity, fluorescence lifetime or bioluminescence. These FRET pairs thus establish a new concept for the development of highly sensitive and tunable biosensors.

FRET Imaging of Nonuniformly Distributed DNA SAMs on Gold Reveals the Role Played by the Donor/Acceptor Ratio and the Local Environment in Measuring the Rate of Hybridization.

Mixed DNA SAMs labeled with a fluorophore (either AlexaFluor488 or AlexaFluor647) were prepared on a single crystal gold bead electrode using potential-assisted thiol exchange and studied using Förster resonance energy transfer (FRET). A measure of the local environment of the DNA SAM (e.g., crowding) was possible using FRET imaging on these surfaces since electrodes prepared this way have a range of surface densities (ΓDNA). The FRET signal was strongly dependent on ΓDNA and on the ratio of AlexaFluor488 to AlexaFluor647 used to make the DNA SAM, which were consistent with a model of FRET in 2D systems. FRET was shown to provide a direct measure of the local DNA SAM arrangement on each crystallographic region of interest providing a direct assessment of the probe environment and its influence on the rate of hybridization. The kinetics of duplex formation for these DNA SAMs was also studied using FRET imaging over a range of coverages and DNA SAM compositions. Hybridization of the surface-bound DNA increased the average distance between the fluorophore label and the gold electrode surface and decreased the distance between the donor (D) and acceptor (A), both of which result in an increase in FRET intensity. This increase in FRET was modeled using a second order Langmuir adsorption rate equation, reflecting the fact that both D and A labeled DNA are required to become hybridized to observe a FRET signal. The self-consistent analysis of the hybridization rates on low and high coverage regions on the same electrode showed that the low coverage regions achieved full hybridization 5× faster than the higher coverage regions, approaching rates typically found in solution. The relative increase in FRET intensity from each region of interest was controlled by manipulating the donor to acceptor composition of the DNA SAM without changing the rate of hybridization. The FRET response can be optimized by controlling the coverage and the composition of the DNA SAM sensor surface and could be further improved with the use of a FRET pair with a larger (e.g., > 5 nm) Förster radius.

In vivo quantitative FRET small animal imaging: Intensity versus lifetime-based FRET

Förster resonance energy transfer (FRET) microscopy is used in numerous biophysical and biomedical applications to monitor inter- and intramolecular interactions and conformational changes in the 2–10 nm range. FRET is currently being extended to in vivo optical imaging, its main application being in quantifying drug-target engagement or drug release in animal models of cancer using organic dye or nanoparticle-labeled probes. Herein, we compared FRET quantification using intensity-based FRET (sensitized emission FRET analysis with the three-cube approach using an IVIS imager) and macroscopic fluorescence lifetime (MFLI) FRET using a custom system using a time-gated-intensified charge-coupled device, for small animal optical in vivo imaging. The analytical expressions and experimental protocols required to quantify the product fDE of the FRET efficiency E and the fraction of donor molecules involved in FRET, fD, are described in detail for both methodologies. Dynamic in vivo FRET quantification of transferrin receptor-transferrin binding was acquired in live intact nude mice upon intravenous injection of a near-infrared-labeled transferrin FRET pair and benchmarked against in vitro FRET using hybridized oligonucleotides. Even though both in vivo imaging techniques provided similar dynamic trends for receptor-ligand engagement, we demonstrate that MFLI-FRET has significant advantages. Whereas the sensitized emission FRET approach using the IVIS imager required nine measurements (six of which are used for calibration) acquired from three mice, MFLI-FRET needed only one measurement collected from a single mouse, although a control mouse might be needed in a more general situation. Based on our study, MFLI therefore represents the method of choice for longitudinal preclinical FRET studies such as that of targeted drug delivery in intact, live mice.

Direct Measurement of Polymer-Chain-End-to-End Distances by Using RAFT Chain Transfer Agent as the FRET Acceptor.

Förster resonance energy transfer (FRET) is a powerful tool for measuring distances between two molecules (donor and acceptor) in close proximity (1-10 nm), which can be employed for determining polymer end-to-end distances (Ree). However, previous works for labeling FRET pairs on chain-ends often involve relatively complex steps for materials preparation, potentially limiting their broad use in synthetic polymer systems. In this work, we introduce an anthracene-functionalized chain-transfer agent for reversible addition-fragmentation chain-transfer (RAFT) polymerizations, which can directly yield polymers containing FRET donor and acceptor molecules on respective chain-ends. This approach enables the direct use of FRET for characterizing the averaged Ree of polymers. Building on this platform, we investigate the averaged Ree of polystyrene (PS) and poly(methyl methacrylate) (PMMA) in a good solvent as a function of their molecular weight. Notably, the FRET results show good agreement with simulation results obtained from all-atom molecular dynamics, confirming its measurement accuracy. Overall, this work provides a facile and broadly applicable platform to directly determine the Ree of low molecular weight polymers by using FRET-based methods.

Evaluation by Experimentation and Simulation of a FRET Pair Comprising Fluorescent Nucleobase Analogs in Nucleosomes.

Förster resonance energy transfer (FRET) is an attractive tool for understanding biomolecular dynamics. FRET-based analysis of nucleosomes has the potential to fill the knowledge gaps between static structures and dynamic cellular behaviors. Compared with typical FRET pairs using bulky fluorophores introduced by flexible linkers, fluorescent nucleoside-based FRET pair has great potential since it can be fitted within the helical structures of nucleic acids. Herein we report on the construction of nucleosomes containing a nucleobase FRET pair and the investigation of experimental and theoretical FRET efficiencies through steady-state fluorescence spectroscopy and calculation based on molecular dynamics simulations, respectively. Distinguishable experimental FRET efficiencies were observed depending on the positions of FRET pairs in nucleosomal DNA. The tendency could be supported by theoretical study. This work suggests the possibility of our approach to analyze structural changes of nucleosomes by epigenetic modifications or internucleosomal interactions.

Biosensor Optimization Using a Förster Resonance Energy Transfer Pair Based on mScarlet Red Fluorescent Protein and an mScarlet-Derived Green Fluorescent Protein.

Genetically encoded biosensors based on Förster resonance energy transfer (FRET) are indispensable tools for monitoring biochemical changes in cells. Green and red fluorescent protein-based FRET pairs offer advantages over the classically employed cyan and yellow fluorescent protein pairs, such as better spectral separation, lower phototoxicity, and less autofluorescence. Here, we describe the development of an mScarlet-derived green fluorescent protein (designated as mWatermelon) and its use as a FRET donor to the red fluorescent protein mScarlet-I as a FRET acceptor. We tested the functionality of this FRET pair by engineering biosensors for the detection of protease activity, Ca2+, and K+. Furthermore, we described a strategy to enhance the FRET efficiency of these biosensors by modulating the intramolecular association between mWatermelon and mScarlet-I.

Ruthenium(II) Polypyridyl Complexes as FRET Donors: Structure- and Sequence-Selective DNA-Binding and Anticancer Properties.

Ruthenium(II) polypyridyl complexes (RPCs) that emit from metal-to-ligand charge transfer (MLCT) states have been developed as DNA probes and are being examined as potential anticancer agents. Here, we report that MLCT-emissive RPCs that bind DNA undergo Förster resonance energy transfer (FRET) with Cy5.5-labeled DNA, forming mega-Stokes shift FRET pairs. Based on this discovery, we developed a simple and rapid FRET binding assay to examine DNA-binding interactions of RPCs with diverse photophysical properties, including non-"light switch" complexes [Ru(dppz)2(5,5'dmb)]2+ and [Ru(PIP)2(5,5'dmb)]2+ (dppz = dipyridophenazine, 5,5'dmb = 5,5'-dimethyl-2,2'-bipyridine, PIP = 2-phenyl-imidazo[4,5-f][1,10]phenanthroline). Binding affinities toward duplex, G-quadruplex, three-way junction, and mismatch DNA were determined, and derived FRET donor-acceptor proximities provide information on potential binding sites. Molecules characterized by this method demonstrate encouraging anticancer properties, including synergy with the PARP inhibitor Olaparib, and mechanistic studies indicate that [Ru(PIP)2(5,5'dmb)]2+ acts to block DNA replication fork progression.

Advancements in a FRET Biosensor for Live-Cell Fluorescence-Lifetime High-Throughput Screening of Alpha-Synuclein.

There is a critical need for small molecules capable of rescuing pathophysiological phenotypes induced by alpha-synuclein (aSyn) misfolding and oligomerization. Building upon our previous aSyn cellular fluorescence lifetime (FLT)-Förster resonance energy transfer (FRET) biosensors, we have developed an inducible cell model incorporating the red-shifted mCyRFP1/mMaroon1 (OFP/MFP) FRET pair. This new aSyn FRET biosensor improves the signal-to-noise ratio, reduces nonspecific background FRET, and results in a 4-fold increase (transient transfection) and 2-fold increase (stable, inducible cell lines) in FRET signal relative to our previous GFP/RFP aSyn biosensors. The inducible system institutes greater temporal control and scalability, allowing for fine-tuning of biosensor expression and minimizes cellular cytotoxicity due to overexpression of aSyn. Using these inducible aSyn-OFP/MFP biosensors, we screened the Selleck library of 2684 commercially available, FDA-approved compounds and identified proanthocyanidins and casanthranol as novel hits. Secondary assays validated the ability of these compounds to modulate aSyn FLT-FRET. Functional assays probing cellular cytotoxicity and aSyn fibrillization demonstrated their capability to inhibit seeded aSyn fibrillization. Proanthocyanidins completely rescued aSyn fibril-induced cellular toxicity with EC50 of 200 nM and casanthranol supported a 85.5% rescue with a projected EC50 of 34.2 μM. Furthermore, proanthocyanidins provide a valuable tool compound to validate our aSyn biosensor performance in future high-throughput screening campaigns of industrial-scale (million-compound) chemical libraries.