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  • Title: Nontrivial Effect of the Color-Exchange of a Donor/Acceptor Pair in the Engineering of Förster Resonance Energy Transfer (FRET)-Based Indicators.
    Author: Ohta Y, Kamagata T, Mukai A, Takada S, Nagai T, Horikawa K.
    Journal: ACS Chem Biol; 2016 Jul 15; 11(7):1816-22. PubMed ID: 27232891.
    Abstract:
    Genetically encoded indicators driven by the Förster resonance energy transfer (FRET) mechanism are reliable tools for live imaging. While the properties of FRET-based indicators have been improved over the years, they often suffer from a poor dynamic range due to the lack of comprehensive understanding about how to apply an appropriate strategy to optimize the FRET parameters. One of the most successful optimizations is the incorporation of circularly permuted fluorescent proteins (cpFPs). To better understand the effects of this strategy, we systematically investigated the properties of the indicators by utilizing a set of FRET backbones consisting of native or one of the most effective cp variants (cp173FPs) with considerations of their order. As a result, the ordering of donor and acceptor FPs, which has been ignored in previous studies, was found to significantly affect the dynamic range of indicators. By utilizing these backbones, we succeeded in improving a cGMP indicator with 3.6-fold increased dynamic range and in generating an ultrasensitive cAMP indicator capable of environmental imaging, demonstrating the practical importance of the ordering of donors and acceptors in the engineering of FRET-based indicators.
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