Bioluminescence Resonance Energy Transfer (BRET)-based Biosensing Probes Using Novel Luminescent and Fluorescent Protein Pairs
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Bioluminescence resonance energy transfer (BRET) is a useful technique for visualizing cellular functions and responses to stimuli.To construct efficient biosensing protein probes using BRET, novel luminescent and red fluorescent protein pairs, which have separate peaks of luminescence and fluorescence and can cause energy transfer efficiently, were screened.The red fluorescent protein, mScarletI, used as an acceptor, induced a highly efficient BRET signal from green or blue luminescent proteins [Emerald Luc (ELuc) or NanoLuc (NLuc)].Novel pairs of luminescent and red fluorescent protein (mScarletI) could be applied to the analysis of calcium ions (Ca 2+ ).The BRET-based biosensing protein pair of mScarletI and NLuc showed an increased intensity of the BRET signal, depending on the concentration of Ca 2+ (0-4 μM).Intracellular Ca 2+ influx was monitored in HEK293A cells stimulated with 50 mM KCl and 15 mM arginine using the BRET-based biosensing protein probe with the novel protein pair.This pair of proteins was particularly suited to cellular imaging in vitro and even in vivo.Therefore, it could be useful for various BRET-based analyses of cell and tissue samples.Keywords:
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Abstract Genetically encoded biosensors based on the principle of Förster resonance energy transfer comprise two major classes: biosensors based on fluorescence resonance energy transfer (FRET) and those based on bioluminescence energy transfer (BRET). The FRET biosensors visualize signaling-molecule activity in cells or tissues with high resolution. Meanwhile, due to the low background signal, the BRET biosensors are primarily used in drug screening. Here, we report a protocol to transform intramolecular FRET biosensors to BRET-FRET hybrid biosensors called hyBRET biosensors. The hyBRET biosensors retain all properties of the prototype FRET biosensors and also work as BRET biosensors with dynamic ranges comparable to the prototype FRET biosensors. The hyBRET biosensors are compatible with optogenetics, luminescence microplate reader assays, and non-invasive whole-body imaging of xenograft and transgenic mice. This simple protocol will expand the use of FRET biosensors and enable visualization of the multiscale dynamics of cell signaling in live animals.
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Rare-earth-doped upconversion nanoparticles (UCNPs) have often been used in combination with fluorescent dyes for sensing applications. In these systems, sensing can be achieved through the modulation of Förster resonant energy transfer (FRET) between the dye and the UCNP. The effects of FRET in such cases are complex, as the extent to which FRET is experienced by the rare-earth ions is dependent on their position within the nanoparticle. Here, we develop an analytical model to accurately describe the effects of FRET for such a system. As a proof of principle, we verify our model by considering the case of a pH sensor comprised of fluorescein isothiocyanate and Tm3+-doped UCNPs. We extend our model to the case of core–shell UCNPs and discuss the design of an optimal FRET-based biosensor using UCNPs.
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Abstract Fluorescent biosensors enable to study 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 either changing the fluorescent protein or the synthetic fluorophore, which enabled to monitor simultaneously free NAD + in different subcellular compartments upon genotoxic stress. Minimal modifications furthermore allow the readout of these biosensors to be switched to fluorescence intensity, lifetime or bioluminescence. These FRET pairs thus establish a new concept for the development of highly sensitive and tunable biosensors. Graphical abstract
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FRET plays an important role in light-induced processes in life sciences, e.g. energy transfer in light harvesting complexes. We present a method to tune the energy transfer from donor to acceptor in an optical microresonator.
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Bioluminescence resonance energy transfer (BRET) is a useful technique for visualizing cellular functions and responses to stimuli.To construct efficient biosensing protein probes using BRET, novel luminescent and red fluorescent protein pairs, which have separate peaks of luminescence and fluorescence and can cause energy transfer efficiently, were screened.The red fluorescent protein, mScarletI, used as an acceptor, induced a highly efficient BRET signal from green or blue luminescent proteins [Emerald Luc (ELuc) or NanoLuc (NLuc)].Novel pairs of luminescent and red fluorescent protein (mScarletI) could be applied to the analysis of calcium ions (Ca 2+ ).The BRET-based biosensing protein pair of mScarletI and NLuc showed an increased intensity of the BRET signal, depending on the concentration of Ca 2+ (0-4 μM).Intracellular Ca 2+ influx was monitored in HEK293A cells stimulated with 50 mM KCl and 15 mM arginine using the BRET-based biosensing protein probe with the novel protein pair.This pair of proteins was particularly suited to cellular imaging in vitro and even in vivo.Therefore, it could be useful for various BRET-based analyses of cell and tissue samples.
Resonant inductive coupling
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