Concept: Förster resonance energy transfer
C.RF-Tshr(hyt/hyt) mice have a mutated thyroid stimulating hormone receptor (P556L-TSHR) and these mice develop severe hypothyroidism. We found that C.RF-Tshr(hyt/wild) heterozygous mice are also in a hypothyroid state. Thyroid glands from C.RF-Tshr(hyt/wild) mice are smaller than those from wild-type mice, and (125)I uptake activities of the former are significantly lower than those in the latter. When TSHR (TSHR(W)) and P556L-TSHR (TSHR(M)) cDNAs were cloned and co-transfected into HEK 293 cells, the cells retained (125)I-TSH binding activity, but cAMP response to TSH was decreased to about 20% of HEK 293 cells transfected with TSHR(W) cDNA. When TSHR(W) and TSHR(M) were tagged with eCFP or eYFP, we observed fluorescence resonance energy transfer (FRET) in HEK 293 cells expressing TSHR(W)-eCFP and TSHR(W)-eYFP in the absence of TSH, but not in the presence of TSH. In contrast, we obtained FRET in HEK 293 cells expressing TSHR(W)-eCFP and TSHR (M)-eYFP, regardless of the presence or absence of TSH. These results suggest that P556L TSHR has a dominant negative effect on TSHR(W) by impairing polymer to monomer dissociation, which decreases TSH responsiveness and induces hypothyroidism in C.RF-Tshr(hyt/wild) mice.
Many genetically encoded biosensors use Förster resonance energy transfer (FRET) to dynamically report biomolecular activities. While pairs of cyan and yellow fluorescent proteins (FPs) are most commonly used as FRET partner fluorophores, respectively, green and red FPs offer distinct advantages for FRET, such as greater spectral separation, less phototoxicity, and lower autofluorescence. We previously developed the green-red FRET pair Clover and mRuby2, which improves responsiveness in intramolecular FRET reporters with different designs. Here we report the engineering of brighter and more photostable variants, mClover3 and mRuby3. mClover3 improves photostability by 60% and mRuby3 by 200% over the previous generation of fluorophores. Notably, mRuby3 is also 35% brighter than mRuby2, making it both the brightest and most photostable monomeric red FP yet characterized. Furthermore, we developed a standardized methodology for assessing FP performance in mammalian cells as stand-alone markers and as FRET partners. We found that mClover3 or mRuby3 expression in mammalian cells provides the highest fluorescence signals of all jellyfish GFP or coral RFP derivatives, respectively. Finally, using mClover3 and mRuby3, we engineered an improved version of the CaMKIIα reporter Camuiα with a larger response amplitude.
Fluorescent resonance energy transfer (FRET) with naturally exceptional selectivity is a powerful technique and widely used in chemical and biomedical analysis. However, it is still challenging for conventional FRET to perform as a high sensitivity compact sensor. Here we propose a novel ‘FRET on Fiber’ concept, in which a partially reduced graphene oxide (prGO) film is deposited on a fiber-optic modal interferometer, acting as both the fluorescent quencher for the FRET and the sensitive cladding for optical phase measurement due to refractive index changes in biochemical detection. The target analytes induced fluorescence recovery with good selectivity and optical phase shift with high sensitivity are measured simultaneously. The functionalized prGO film coated on the fiber-optic interferometer shows high sensitivities for the detections of metal ion, dopamine and single-stranded DNA (ssDNA), with detection limits of 1.2 nM, 1.3 μM and 1 pM, respectively. Such a prGO based ‘FRET on fiber’ configuration, bridging the FRET and the fiber-optic sensing technology, may serve as a platform for the realization of series of integrated ‘FRET on Fiber’ sensors for on-line environmental, chemical, and biomedical detection, with excellent compactness, high sensitivity, good selectivity and fast response.
With growing concerns about health issues worldwide, elegant sensors with high sensitivity and specificity for virus/antigens (Ag) detection are urgent to be developed. Homogeneous immunoassays (HIA) are an important technique with the advantages of small sample volumes requirement and pretreatment-free process. HIA are becoming more favorable for the medical diagnosis and disease surveillance than heterogeneous immunoassays. An important subset of HIA relies on the effect of fluorescence resonance energy transfer (FRET) via a donor-acceptor (D-A) platform, e.g., quantum dots (QDs) donor based FRET system. Being an excellent plasmonic material, silver triangular nanoplates (STNPs) have unique advantages in displaying surface plasmon resonance in the visible to near infrared spectral region, which make them a better acceptor for pairing with QDs in a FRET-based sensing system. However, the reported STNPs generally exhibited broad size distributions, which would greatly restrict their application as HIA acceptor for high detection sensitivity and specificity purpose. In this work, uniform STNPs and red-emitting QDs are firstly applied to construct FRET nanoplatform in the advanced HIA and further be exploited for analyzing virus Ag. The uniform STNPs/QDs nanoplatform based medical sensor provides a straightforward and highly sensitive method for Ag analysis in homogeneous form.
Upconverting nanoparticles (UCNPs) have attracted considerable attention as potential photosensitizer carriers for photodynamic therapy (PDT) in deep tissues. In this work, a new and efficient NIR photosensitizing nanoplatform for PDT based on red-emitting UCNPs is designed. The red emission band matches well with the efficient absorption bands of the widely used commercially available photosensitizers (Ps), benefiting the fluorescence resonance energy transfer (FRET) from UCNPs to the attached photosensitizers and thus efficiently activating them to generate cytotoxic singlet oxygen. Three commonly used photosensitizers, including chlorine e6 (Ce6), zinc phthalocyanine (ZnPc) and methylene blue (MB), are loaded onto the alpha-cyclodextrin-modified UCNPs to form Ps@UCNPs complexes that efficiently produce singlet oxygen to kill cancer cells under 980 nm near-infrared excitation. Moreover, two different kinds of drugs are co-loaded onto these nanoparticles: chemotherapy drug doxorubicin and PDT agent Ce6. The combinational therapy based on doxorubicin (DOX)-induced chemotherapy and Ce6-triggered PDT exhibits higher therapeutic efficacy relative to the individual means for cancer therapy in vitro.
Organic solar cells have the potential to become a low-cost sustainable energy source. Understanding the photoconversion mechanism is key to the design of efficient organic solar cells. In this review, we discuss the processes involved in the photo-electron conversion mechanism, which may be subdivided into exciton harvesting, exciton transport, exciton dissociation, charge transport and extraction stages. In particular, we focus on the role of energy transfer as described by F¨orster resonance energy transfer (FRET) theory in the photoconversion mechanism. FRET plays a major role in exciton transport, harvesting and dissociation. The spectral absorption range of organic solar cells may be extended using sensitizers that efficiently transfer absorbed energy to the photoactive materials. The limitations of F¨orster theory to accurately calculate energy transfer rates are discussed. Energy transfer is the first step of an efficient two-step exciton dissociation process and may also be used to preferentially transport excitons to the heterointerface, where efficient exciton dissociation may occur. However, FRET also competes with charge transfer at the heterointerface turning it in a potential loss mechanism. An energy cascade comprising both energy transfer and charge transfer may aid in separating charges and is briefly discussed. Considering the extent to which the photo-electron conversion efficiency is governed by energy transfer, optimisation of this process offers the prospect of improved organic photovoltaic performance and thus aids in realising the potential of organic solar cells.
We report a fully reversible off-on fluorescence probe for hypoxia. The design employed QSY-21 as a Förster resonance energy transfer (FRET) acceptor and cyanine dye Cy5 as a FRET donor, based on our finding that QSY-21 undergoes one-electron bioreduction to the radical under hypoxia, with an absorbance decrease at 660 nm. Then, FRET can no longer occur and the dye becomes strongly fluorescent. Upon recovery of normoxia, the radical is immediately re-oxidized to QSY-21, with loss of fluorescence due to restoration of FRET. We show that this probe, RHyCy5, can monitor repeated hypoxia-normoxia cycles in live cells.
Theory of the energy transfer efficiency and fluorescence lifetime distribution in single-molecule FRET.
- Proceedings of the National Academy of Sciences of the United States of America
- Published over 5 years ago
In single-molecule FRET experiments with pulsed lasers, not only the colors of the photons but also the fluorescence lifetimes can be monitored. Although these quantities appear to be random, they are modulated by conformational dynamics. In order to extract information about such dynamics, we develop the theory of the joint distribution of FRET efficiencies and fluorescence lifetimes determined from bins (or bursts) of photons. Our starting point is a rigorous formal expression for the distribution of the numbers of donor and acceptor photons and donor lifetimes in a bin that treats the influence of conformational dynamics on all timescales. This formula leads to an analytic result for a two-state system interconverting on a timescale slower than the interphoton time and to an efficient simulation algorithm for multistate dynamics. The shape of the joint distribution contains more information about conformational dynamics than the FRET efficiency histogram alone. In favorable cases, the connectivity of the underlying conformational states can be determined directly by simple inspection of the projection of the joint distribution on the efficiency-lifetime plane.
- The Journal of neuroscience : the official journal of the Society for Neuroscience
- Published over 4 years ago
Soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptors (SNAREs) mediate vesicle fusion with the plasma membrane on activation by calcium binding to synaptotagmin. In the present study, we used fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy between fluorescently labeled SNARE proteins expressed in cultured rat hippocampal neurons to detect resting SNARE complexes, their conformational rearrangement on exocytosis, their disassembly before endocytosis of vesicular proteins, and SNARE assembly at newly docked vesicles. Assembled SNAREs are not only present in docked vesicles; unexpected residual “orphan SNARE complexes” also reside in para-active zone regions. Real-time changes in FRET between N-terminally labeled SNAP-25 and VAMP reported a reorientation of the SNARE motif upon exocytosis, SNARE disassembly in the active zone periphery, and SNARE reassembly in newly docked vesicles. With VAMP labeled C-terminally, decreased fluorescence in C-terminally labeled syntaxin (extracellular) reported trans-cis-conformational changes in SNAREs on vesicle fusion. After fusion SNAP-25 and syntaxin disperse along with VAMP, as well as the FRET signal itself, indicating diffusion of intact SNAREs after vesicle fusion but before their peripheral disassembly. Our measurements of spatiotemporal dynamics of SNARE conformational changes and movements refine models of SNARE function. Technical advances required to detect tiny changes in fluorescence in small fractions of labeled proteins in presynaptic boutons on a time scale of seconds permit the detection of rapid intermolecular interactions between small proportions of protein partners in cellular subcompartments.
Fluorescent ligands for GPCRs (G-protein-coupled receptors) have been synthesized for a long time but their use was usually restricted to receptor localization in the cell by fluorescent imaging microscopy. During the last two decades, the emergence of new fluorescence-based strategies and the concomitant development of fluorescent measurement apparatus have dramatically widened the use of fluorescent ligands. Among the various strategies, TR (time-resolved)-FRET (fluorescence resonance energy transfer) approaches exhibit an interesting potential to study GPCR interactions with various partners. We have derived various sets of ligands that target different GPCRs with fluorophores, which are compatible with TR-FRET strategies. Fluorescent ligands labelled either with a fluorescent donor (such as europium or terbium cryptate) or with a fluorescent acceptor (such as fluorescein, dy647 or Alexa Fluor® 647), for example, kept high affinities for their cognate receptors. These ligands turn out to be interesting tools to develop FRET-based binding assays. We also used these fluorescent ligands to analyse GPCR oligomerization by measuring FRET between ligands bound to receptor dimers. In contrast with FRET strategies, on the basis of receptor labelling, the ligand-based approach we developed is fully compatible with the study of wild-type receptors and therefore with receptors expressed in native tissues. Therefore, by using fluorescent analogues of oxytocin, we demonstrated the existence of oxytocin receptor dimers in the mammary gland of lactating rats.