Quantification of FRET-induced angular displacement by monitoring sensitized acceptor anisotropy using a dim fluorescent donor

Abstract

Förster resonance energy transfer (FRET) between fluorescent proteins has become a common platform for designing genetically encoded biosensors. For live cell imaging, the acceptor-to-donor intensity ratio is most commonly used to readout FRET efficiency, which largely depends on the proximity between donor and acceptor. Here, we introduce an anisotropy-based mode of FRET detection (FADED: FRET-induced Angular Displacement Evaluation via Dim donor), which probes for relative orientation rather than proximity alteration. A key element in this technique is suppression of donor bleed-through, which allows measuring purer sensitized acceptor anisotropy. This is achieved by developing Geuda Sapphire, a low-quantum-yield FRET-competent fluorescent protein donor. As a proof of principle, Ca2+ sensors were designed using calmodulin as a sensing domain, showing sigmoidal dose response to Ca2+. By monitoring the anisotropy, a Ca2+ rise in living HeLa cells is observed upon histamine challenging. We conclude that FADED provides a method for quantifying the angular displacement via FRET.

Förster resonance energy transfer within the neomycin aptamer

“…This would enable measurements of the angle between donor and acceptor using anisotropy measurements. 51 With the procedure described here, in particular using transient absorption experiments, the determination of the angle between donor and acceptor is also possible without fluorescence. This requires a different acceptor than tC nitro , whose excited state lives longer and is spectrally separated from the signal of the excited state of the donor.…”

Smart genetically-encoded biosensors for the chemical monitoring of living systems

“…30,50 Another approach is to use identical reporters for both donor and acceptor, thus relying on energy transfer based on homo-FRET. Such a strategy can be used to generate sensors that are read out by detecting the fluorescence anisotropy, 51,52 which is likewise quantitative and does not require the use of two different spectral bands (Fig. 5d).…”

Monitoring the in vivo siRNA release from lipid nanoparticles based on the fluorescence resonance energy transfer principle

“…Non-radioactive energy transfer occurs when the emission spectrum of the fluorescent donor molecule overlaps with the absorption spectrum of the acceptor fluorescent molecule and the distance between the two molecules is within 10 nm [19] . The fluorescence intensity of the donor is greatly reduced, while the fluorescence emitted by the acceptor is greatly enhanced [20] . Based on the FRET principle, nanogold (Au) is often used as fluorescent quencher of most fluorophores because of its wide quenching range, high quenching efficiency, good stability, sensitivity, low toxicity, and good biocompatibility [21 , 22] .…”