Intramolecular Fluorescence Resonance Energy Transfer Research Articles

The Dual Function of the Polybasic Juxtamembrane Region of Syntaxin 1A in Clamping Spontaneous Release and Stimulating Ca2+-Triggered Release in Neuroendocrine Cells.

The exact function of the polybasic juxtamembrane region (5RK) of the plasma membrane neuronal SNARE, syntaxin 1A (Syx), in vesicle exocytosis, although widely studied, is currently not clear. Here, we addressed the role of 5RK in Ca2+-triggered release, using our Syx-based intramolecular fluorescence resonance energy transfer (FRET) probe, which previously allowed us to resolve a depolarization-induced Ca2+-dependent close-to-open transition (CDO) of Syx that occurs concomitant with evoked release, both in PC12 cells and hippocampal neurons and was abolished upon charge neutralization of 5RK. First, using dynamic FRET analysis in PC12 cells, we show that CDO occurs following assembly of SNARE complexes that include the vesicular SNARE, synaptobrevin 2, and that the participation of 5RK in CDO goes beyond its participation in the final zippering of the complex, because mutations of residues adjacent to 5RK, believed to be crucial for final zippering, do not abolish this transition. In addition, we show that CDO is contingent on membrane phosphatidylinositol 4,5-bisphosphate (PIP2), which is fundamental for maintaining regulated exocytosis, as depletion of membranal PIP2 abolishes CDO. Prompted by these results, which underscore a potentially significant role of 5RK in exocytosis, we next amperometrically analyzed catecholamine release from PC12 cells, revealing that charge neutralization of 5RK promotes spontaneous and inhibits Ca2+-triggered release events. Namely, 5RK acts as a fusion clamp, making release dependent on stimulation by Ca2+SIGNIFICANCE STATEMENT Syntaxin 1A (Syx) is a central protein component of the SNARE complex, which underlies neurotransmitter release. Although widely studied in relation to its participation in SNARE complex formation and its interaction with phosphoinositides, the function of Syx's polybasic juxtamembrane region (5RK) remains unclear. Previously, we showed that a conformational transition of Syx, related to calcium-triggered release, reported by a Syx-based FRET probe, is abolished upon charge neutralization of 5RK (5RK/A). Here we show that this conformational transition is dependent on phosphatidylinositol 4,5-bisphosphate (PIP2) and is related to SNARE complex formation. Subsequently, we show that the 5RK/A mutation enhances spontaneous release and inhibits calcium-triggered release in neuroendocrine cells, indicating a previously unrecognized role of 5RK in neurotransmitter release.

Conformational transition of polystyrene in good solvents studied by fluorescence spectroscopy

In this paper, the conformational transition of polystyrene (PS) in good solvents (i.e., dichloroethane and decalin) is studied by intramolecular fluorescence resonance energy transfer (FRET, NRET). The characteristics of FRET and PS excimer fluorescence spectroscopy in the analysis of polymer solution are systematically described. We find that FRET is prior to PS excimer fluorescence spectroscopy due to the superior sensitivity and better spectroscopic signal resolution. Based on the FRET results, it can be concluded that the chain conformation almost keeps unchanged with the increase of polymer concentration before the concentration reaches the critical overlap concentration ( C *). This spectroscopic observation excludes the existence of dynamic contact transition. In the expected C * regime, a sharp break point is observed for the fluorescence intensity versus the increase of the concentration, which is in exactly agreement with the theoretical value of C *. Additionally, the polymer chain undergoes severe collapse when the concentration reaches the semidilute regime and increases continuously. This work demonstrates that intramolecular FRET is a robust technique to sensitively characterize C *. C * could be used as a threshold to describe the conformational transition of polymer in good solvents.

Electrochemiluminescent resonance energy transfer of polymer dots for aptasensing

This work designed a three-component polymer for the preparation of polymer dots (Pdots). The polymer contained 9-(diphenylmethylene)-9H-fluorene (DPF), 9,9-dioctyl-9H-fluorene (DOF) and 1,1′-binaphthyl moieties, and was synthesized via Pd-catalyzed Suzuki reaction. It exhibited obvious yellow-colored aggregation-induced emission (AIE) for fluorescence enhancement at 543nm via an intramolecular fluorescence resonance energy transfer from DOF moiety to DPF moiety. The Pdots prepared by nanoprecipitation could be conveniently cast on electrode surface and showed a stable anodic electrochemiluminescence (ECL) emission in the presence of triethylamine as a co-reactant. The ECL emission could be effectively quenched by rhodamine B via resonance energy transfer, which led to an “off-on” switch for the design of ECL sensing methodology. Using Pb2+ as a target model, an ECL aptasensor for the detection of trace Pb2+ was proposed, which showed a linear range of 100pM to 1.0μM with a detection limit down to 38.0pM This work demonstrated the first Pdots prepared with AIE-active polymer for highly efficient ECL sensing.

Selective visualization of endogenous hypochlorous acid in zebrafish during lipopolysaccharide-induced acute liver injury using a polymer micelles-based ratiometric fluorescent probe

The development of effective method for monitoring of endogenous hypochlorous acid (HClO) in vivo is of great significance for early diagnosis of lipopolysaccharide (LPS) induced acute liver injury. Herein, we report a polymer micelles-based ratiometric fluorescent probe (PM) based on the combination of intramolecular charge transfer (ICT) mechanism and fluorescence resonance energy transfer (FRET) principle for selective visualization of endogenous HClO in vivo. Upon the reaction of PM with HClO, the electron-donating thiourea moiety is oxidized and transformed into imidazoline moiety (electron-withdrawing group), resulting in a dramatic blue shift (˃100nm) in the fluorescence emission. The as-prepared PM shows good water dispersibility (100% aqueous media), fast response (<40s), high sensitivity (a detection limit of 1.75nM), and outstanding selectivity toward HClO over other ROS/RNS (50 equiv.). In addition, the vivo imaging experiments demonstrate that PM facilitates the visualization of endogenous HClO generation with LPS induced acute liver injury in zebrafish model.

Exploring Drug-Receptor Interaction Kinetics: Lessons from a Sigma-1 Receptor Transmembrane Biosensor.

An important field of study in pharmacology comprises the investigation of drug-target interaction kinetics. Thus, assessing both the lifetime of a drug on its receptor (i.e., drug-target residence time; Copeland, 2016) and the magnitude of drug-mediated receptor activation (i.e., drug efficacy) across the time are critical to understand in vivo pharmacological activity of small-molecule drugs. Of note, while classical in vitro methods view drug-receptor interaction in terms of equilibrium affinity, the residence time model considers the dynamics of receptor conformational rearrangements, which affect drug association and dissociation. Although, classical binding experiments can also address kinetics questions, they are tedious and very time consuming. Accordingly, monitoring drug-receptor interaction dynamics by means of receptor biosensors has become fundamental for understanding how drugs trigger receptor activity over the time. Precisely, in the last years, a number of Fluorescence Resonance Energy Transfer (FRET)-based assays have been developed to accurately display drug-receptor interaction in real time (Lohse et al., 2012). Indeed, one of the most outstanding methods consists of assessing intramolecular conformational rearrangements upon receptor challenge by monitoring intramolecular FRET changes (Vilardaga et al., 2009). Thus, a FRET-based receptor biosensor is built by fusing both donor and acceptor fluorophores to the receptor sequence (Vilardaga et al., 2009). Importantly, a general consensus has prompted to basically attach these molecules (i.e., cyan and yellow fluorescent proteins, CFP and YFP, respectively) intracellularly, this is, in the cytosolic side of the receptor's structure (Figure ​(Figure1A).1A). Accordingly, when the receptor is activated and a conformational rearrangement occurs the distance and/or orientation of the fluorophores within the receptor biosensor changes and it is possible to monitor FRET changes in real time, thus permitting to finely characterize receptor's activation. Needless to say, although precision is higher than that obtained in classical binding assays, the present biosensors cannot discern between receptors expressed at the cell surface or intracellularly, thus much effort is needed in order to exactly elucidate ligand-receptor kinetics constants.

A highly selective fluorescent chemosensor for Cu2+ : synthesis and properties of a rhodamine B-containing diarylethene.

A diarylethene bearing a triazole-linked rhodamine B unit was synthesized. Its fluorescent emission was significantly enhanced in the presence of protons or Cu2+ due to transformation from the pirocyclic form to open-ring form. The fluorescence was quenched sequentially upon irradiation with 297nm light based on the intramolecular fluorescence resonance energy transfer mechanism. In an acetonitrile: water binary solvent (1: 1v/v), the compound showed significant fluorescent enhancement for Cu2+ compared with a wide range of tested metal ions with a fast response and a limit of detection of 2.86×10-8 molL-1 . Using Cu2+ and UV light as the chemical inputs, and fluorescence intensity at 597nm as the output, a logic gate was developed at the molecular level. Moreover, the compound can be used with a high accuracy to detect Cu2+ in a natural water sample.

A novel naphthalimide-rhodamine dye: Intramolecular fluorescence resonance energy transfer and ratiometric chemodosimeter for Hg2+ and Fe3+

A novel ratiometric fluorescent chemodosimeter REPN based on 1,8-naphthalimide-rhodamine was successfully synthesized. Two different strategies of fluorescence resonance energy transfer (FRET) and photo-induced electron transfer (PET) for ratiometric detection of Hg2+/Fe3+ were designed into one sensing system. REPN exhibits typical FRET signal from pyridine-naphthalimide moiety to rhodamine moiety which was induced by Hg2+ with a detection limit of 2.72 × 10−6 M in EtOH/PBS buffer (1:1, v/v, pH = 7.4) solution. However, the probe REPN performed ratiometric detection behavior via PET process combined with the classical ring-opening sensor system in the presence of Fe3+, the detection limit was 5.7 × 10−7 M. REPN emitted bright yellow fluorescence in solid state and the maximum emission wavelength was 538 nm. Besides, the silica nanoparticles loaded AIE dye REPN maintained and further reinforced the AIE effect with higher water solubility.

Light harvesting systems composed of carbazole based subphthalocyanine-BODIPY enhanced with intramolecular fluorescence resonance energy transfer (FRET)

A new series of subphthalocyanine-borondipyrromethene scaffolds containing N-ethylcarbazole moiety have been successfully designed and synthesized. The identities of synthesized compounds were confirmed by using 1H, 13C NMR, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The photophysical properties of the newly synthesized subphthalocyanine-borondipyrromethene conjugates were investigated via absorption and fluorescence spectroscopies in dichloromethane, tetrahydrofuran, benzene, and toluene solution (1 × 10−5 M). The targeted compounds exhibit a highly efficient energy transfer process, either from the excited borondipyrromethene to the subphthalocyanine core or from the excited subphthalocyanine to the mono or distyryl borondipyrromethene unit. The time resolved fluorescence studies of the subphthalocyanine- borondipyrromethene conjugates were also examined.

Click synthesis, Hg2+ sensor and Intramolecular fluorescence resonance energy transfer in novel BODIPY dendrons

Two novel intramolecular fluorescence resonance energy transfer (FRET) dendritic compounds RhB-BODIPY and NPI-BODIPY have been synthesized utilizing click reaction and characterized. The fluorescence intensity of RhB-BODIPY at 580nm was amplified with 10.7-fold on addition of Hg2+ and the fluorescence resonance energy transfer efficiency between the energy donor (BODIPY) and the energy acceptor (rhodamine) is 67%. In compound NPI-BODIPY, the naphthalimide serves as donor and BODIPY serves as acceptor. The energy transfer efficiency of NPI-BODIPY is 72%. The comparative sensing abilities of RhB-BODIPY toward a range of metal ions have been investigated and it exhibited appreciable selectivity towards Hg2+. Job’s plot clearly indicated that the binding ratio of RhB-BODIPY with Hg2+ is 1:2. The 1H NMR titration of RhB-BODIPY with Hg2+ confirmed that tertiary amines and triazoles were involved in the binding event. The detection limit estimated from the titration results were 3.34×10−7M for RhB-BODIPY. The fluorescence quantum yield of RhB-BODIPY and NPI-BODIPY at 510nm were 53% and 88% respectively. In addition, both two compounds were successfully applied to living cell imaging.

Photocontrolled Reversible Luminescent Lanthanide Molecular Switch Based on a Diarylethene-Europium Dyad.

A new europium complex coordinated between a Eu(III) ion and an unsymmetrical diarylperfluorocyclopentene yields a light-controlled diarylethene-europium dyad, DAE@TpyEu(tta)3, whose photophysical properties can be reversibly switched by optical stimuli. When DAE@TpyEu(tta)3 is exposed to 365 nm UV light, an efficient intramolecular photochromic fluorescence resonance energy transfer (pc-FRET) occurs between the emission of the Eu(3+) donor (D) and the absorption of the diarylethene acceptor (A) in closed-form DAE@TpyEu(tta)3 accompanied by luminescence quenching. However, the pc-FRET process could be effectively inhibited by visible light (λ > 600 nm) irradiation, and the lanthanide emission of DAE@TpyEu(tta)3 is rapidly recovered. Furthermore, this luminescent lanthanide molecular switch could serve as a highly reliable and sensitive "turn on" fluorescent marker in living cells irradiated by red light without any optical interference.

Synthesis and aggregation-induced emission enhancement of naphthalimide-rhodamine dye

Abstract A series of naphthalimide-rhodamine dye L1–4 with the different terminal structures were designed and synthesized. The rhodamine was used as bulky rigid substituent of naphthalimide to enhance the AIE property. All the naphthalimide-rhodamine dye exhibited the significant aggregation-induced emission enhancement. The strong fluorescence emissions in solid state were observed with emission peaks ranging from 549 to 608 nm. In high fW mixture solution, the fluorescence intensity enhanced with the increase of water content, which can be attributed to two types of aggregation-induced morphology. The AIE dye-doped silica nanoparticles (Si-NPs) were prepared and exhibited good solubility in pure water. In addition, dye L1 and L2 exhibited the selective recognition toward Hg2+ ions and were successfully applied to the bioimages in living cells. The intramolecular fluorescence resonance energy transfer mechanism from the naphthalimide moiety to the rhodamine moiety was investigated.

Intramolecular fluorescence resonance energy transfer and living cell imaging of novel pyridyltriphenylamine dye

A novel pyridyltriphenylamine-rhodamine dye PTRh and a pyridyltriphenylamine derivative PTO were synthesized and characterized by 1H NMR and HRMS-MALDI-TOF. PTRh performed typical fluorescence resonance energy transfer (FRET) signal from pyridyltriphenylamine to rhodamine along with notable color change from green to rose when interacting with Hg2+ in EtOH/H2O. And PTRh as a ratiometric probe for Hg2+ based on FRET could achieve a very low detection limit of 32 nM and energy transfer efficiency of 83.7% in aqueous organic system. On the other hand, spectra properties of PTO in its aggregates, THF/H2O mixed solution and silica nanoparticles (Si-NPs) dispersed in water were investigated. And the results indicated PTO exhibited bright green fluorescence in solid state, and PTO was successfully encapsulated in silica matrix (30–40 nm), emitting bright blue fluorescence with 11.7% quantum yield. Additionally, living cell imaging experiments demonstrated that PTRh could effectively response to intracellular Hg2+ and PTO-doped Si-NPs were well uptaken by MCF-7 breast cancer cells. It could be concluded that the chromophores are promising materials used as biosensors.

FRET-Based Ratiometric Detection of Metal Ions

Fluorescent sensors for detecting transition- and heavy-metal ions in environmental and biological systems have received considerable attention. In comparison with single emission intensity-based “turn-off” or “turn-on” sensors, ratiometric sensors can afford self-calibration by dual-channel detection and thus perform precise analyses against various environmental and instrumental factors. One of the most frequently applied mechanisms for ratiometric sensing is the binding-induced modulating of intramolecular fluorescence resonance energy transfer (FRET), which is highly dependent on the distance between the donor and acceptor, the overlap integral between the donor emission and acceptor absorption, and the relative orientation of the transition dipoles. Hence the binding-induced changes of the spatial and electronic structure can effectively modulate the FRET efficiency within a donor-acceptor system, consequently leading to change in the fluorescence intensity of the donor and acceptor.In order to develop heavy-metal ion sensors, we have proposed a new strategy for ratiometric fluorescent detection that is based on the rational integration of two sensing mechanisms of a binding-induced modulating of FRET efficiency coupled with a metal-chelating quenching of fluorescence. Synergistic coupling of these two functions leads to exceptionally large change in the emission ratio, thus allowing for ratiometric detecting of metal ions with improved sensitivity. As proof-of-concepts, a BODIPY-porphyrin dyad (1) and a series of phthalocyanine-porphyrin(Zn) hetero-oligomers (2-4) have been designed for ratiometric fluorescent detection, which show excellent selectivity and sensitivity toward Ag+ and Pb2+ respectively. Figure 1. Ratiometric fluorescent detection of Ag+ by FRET-based BODIPY-porphyrin dyad (1).

Rhodamine–naphthalene conjugate as a novel ratiometric fluorescent probe for recognition of Al3+

In this study, a novel chemosensor (HL) for Al3+ which contains rhodamine and naphthalene moieties has been designed and synthesized. In the presence of Al3+, the receptor exhibited a strong, increasing fluorescent emission centered at 550nm at the expense of the fluorescent emission of HL centered at 520nm. We proposed that the addition of Al3+ triggered intramolecular fluorescence resonance energy transfer (FRET) from naphthalene to Rhodamine 6G.

Quantitative analysis of recombination between YFP and CFP genes of FRET biosensors introduced by lentiviral or retroviral gene transfer.

Biosensors based on the principle of Förster (or fluorescence) resonance energy transfer (FRET) have been developed to visualize spatio-temporal dynamics of signalling molecules in living cells. Many of them adopt a backbone of intramolecular FRET biosensor with a cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) as donor and acceptor, respectively. However, there remains the difficulty of establishing cells stably expressing FRET biosensors with a YFP and CFP pair by lentiviral or retroviral gene transfer, due to the high incidence of recombination between YFP and CFP genes. To address this, we examined the effects of codon-diversification of YFP on the recombination of FRET biosensors introduced by lentivirus or retrovirus. The YFP gene that was fully codon-optimized to E.coli evaded the recombination in lentiviral or retroviral gene transfer, but the partially codon-diversified YFP did not. Further, the length of spacer between YFP and CFP genes clearly affected recombination efficiency, suggesting that the intramolecular template switching occurred in the reverse-transcription process. The simple mathematical model reproduced the experimental data sufficiently, yielding a recombination rate of 0.002–0.005 per base. Together, these results show that the codon-diversified YFP is a useful tool for expressing FRET biosensors by lentiviral or retroviral gene transfer.

FRET-based rhodamine–coumarin conjugate as a Fe3+ selective ratiometric fluorescent sensor in aqueous media

In this study, a novel ratiometric fluorescent sensor (HL) for Fe3+ based on the conjugation of rhodamine and coumarin has been designed and synthesized. The free sensor displays fluorescence emission at 475nm, on the addition of Fe3+ to an aqueous solution of HL, the sensor shows significant fluorescence enhancement at 550nm which should be attributed to an intramolecular fluorescence resonance energy transfer (FRET) mechanism from coumarin to Rhodamine 6G.

Intramolecular ex vivo Fluorescence Resonance Energy Transfer (FRET) of Dihydropyridine Receptor (DHPR) β1a Subunit Reveals Conformational Change Induced by RYR1 in Mouse Skeletal Myotubes

The dihydropyridine receptor (DHPR) β1a subunit is essential for skeletal muscle excitation-contraction coupling, but the structural organization of β1a as part of the macromolecular DHPR-ryanodine receptor type I (RyR1) complex is still debatable. We used fluorescence resonance energy transfer (FRET) to probe proximity relationships within the β1a subunit in cultured skeletal myotubes lacking or expressing RyR1. The fluorescein biarsenical reagent FlAsH was used as the FRET acceptor, which exhibits fluorescence upon binding to specific tetracysteine motifs, and enhanced cyan fluorescent protein (CFP) was used as the FRET donor. Ten β1a reporter constructs were generated by inserting the CCPGCC FlAsH binding motif into five positions probing the five domains of β1a with either carboxyl or amino terminal fused CFP. FRET efficiency was largest when CCPGCC was positioned next to CFP, and significant intramolecular FRET was observed for all constructs suggesting that in situ the β1a subunit has a relatively compact conformation in which the carboxyl and amino termini are not extended. Comparison of the FRET efficiency in wild type to that in dyspedic (lacking RyR1) myotubes revealed that in only one construct (H458 CCPGCC β1a -CFP) FRET efficiency was specifically altered by the presence of RyR1. The present study reveals that the C-terminal of the β1a subunit changes conformation in the presence of RyR1 consistent with an interaction between the C-terminal of β1a and RyR1 in resting myotubes.

Intramolecular fluorescence resonance energy transfer in a novel PDI–BODIPY dendritic structure: Synthesis, Hg2+ sensor and living cell imaging

A novel intramolecular fluorescence resonance energy transfer (FRET) and photo-induced electron transfer (PET) based dendritic compound PDI–BODIPY has been developed: Tetra-boron-dipyrrin (BODIPY) attached perylene bisimide by click chemistry. Compound PDI–BODIPY as a fluorescence probe for rapid, selective, and sensitive determination of Hg(II) ions has 98% of high energy transfer efficiency. The dissociation constant (Kd) of the probe PDI–BODIPY with Hg2+ is 6.67×10−12M and the detection limit estimated from the titration results was 2×10−8M. Moreover, the imaging of Hg(II) ions in Hela cells further certified the potential value of the probe.

Judgment of pure fermented soy sauce by fluorescence resonance energy transfer of OPA-tryptophan adduct

Tryptophan was detected with a flow-injection manifold after reacting with mM order of fluorogenic o-phthalaldehyde (OPA)/thiol reagent (pH 10.0) in the carrier stream (0.63mL/min). Based on the intra-molecular fluorescence resonance energy transfer of OPA-tryptophan adduct, the difference in fluorescence intensity obtained at 280 and 300nm excitation was used to detect tryptophan content with satisfactory precision (CV<6.5% for concentration higher than 0.5μM), linearity (0.1–10μM, R2=0.9893) and sensitivity (≈10nM). Since tryptophan will decompose during manufacturing non-fermented soy sauce by acid-hydrolysis procedure, the method was used to discriminate pure fermented soy sauces, adulterated soy sauces and chemical soy sauces in less than 5min. The ratio of tryptophan to total amino acid content served as the index for the judgment, and the results were validated by capillary electrophoresis.

2-Aminotryptanthrin Derivative with Pyrene as a FRET-based Fluorescent Chemosensor for Metal Ions

1-(2-tryptanthrinylaminoacetoxy)-14-(1-pyrenecarboxy)-3,6,9,12-tetraoxatetradecane (T2NH-P5P) was synthesized as a fluorescent chemosensor for metal ions. We investigated the metal-ion recognition of T2NH-P5P by separately adding Mg(2+), Ca(2+), Ba(2+), Fe(2+), Fe(3+), Co(2+), Ni(2+), Cu(2+), Ag(+), Zn(2+), Cd(2+), Hg(2+), Al(3+), and Pb(2+) in an acetonitrile solution. When using excitation at 325 nm, which corresponds to the absorption of the pyrene unit of T2NH-P5P, emission at 600 nm, from the 2-aminotryptanthrin unit, was observed, indicating that intramolecular fluorescence resonance energy transfer (FRET) occurs in T2NH-P5P (FRET-on). However, when Fe(2+), Fe(3+), Ni(2+), Cu(2+), Cd(2+), Hg(2+), and Al(3+) were added to an acetonitrile solution of T2NH-P5P, the behavior changed from FRET-on to FRET-off, which means that the fluorescence of 2-aminotryptanthrin was quenched, whereas that of the pyrene group was revived (FRET-off). Especially, this behavior was remarkable for Fe(2+), Fe(3+), Cu(2+), and Hg(2+). T2NH-P5P is suitable for use as a fluorescent chemosensor for Fe(2+), Fe(3+), Cu(2+), and Hg(2+).