Journal
NATURE COMMUNICATIONS
Volume 12, Issue 1, Pages -Publisher
NATURE PORTFOLIO
DOI: 10.1038/s41467-021-22816-7
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Funding
- Interdisciplinaire Onderzoeksprogramma (IDO) KU Leuven [IDO/12/020]
- Category 1 research grant from KU Leuven [C14/16/053]
- KU Leuven Facultaire Luik Onderzoeksfonds (FLOF)
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FADED is a novel method for measuring FRET based on anisotropy rather than proximity, allowing for quantification of the relative angle between donor and acceptor. By suppressing donor bleed-through, purer sensitized acceptor anisotropy measurements can be obtained, making it suitable for live cell imaging.
Forster 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. The FRET efficiency usually predominantly depends on the proximity of donor and acceptor. Here the authors report an anisotropy-based mode of FRET detection, FRET-induced Angular Displacement Evaluation via Dim donor (FADED), to allow quantification of the relative angle between donor and acceptor.
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