title: Nontrivial Effect of the Color-Exchange of a Donor/Acceptor Pair in the Engineering of Förster Resonance Energy Transfer (FRET)-Based Indicators.
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.
authors.affiliation: Division of Bioimaging, Institute of Biomedical Sciences, Tokushima University Graduate School , 3-18-15 Kuramoto-cho, Tokushima City, Tokushima 770-8503, Japan.
authors.forename: Yusaku
authors.initials: Y
authors.lastname: Ohta
authors.affiliation: Division of Bioimaging, Institute of Biomedical Sciences, Tokushima University Graduate School , 3-18-15 Kuramoto-cho, Tokushima City, Tokushima 770-8503, Japan.|Okazaki Institute for Integrative Bioscience and National Institute for Basic Biology, National Institutes of Natural Sciences , Okazaki, Aichi 444-8787, Japan.|SOKENDAI (The Graduate University for Advanced Studies) , Okazaki, Aichi 444-8787, Japan.
authors.forename: Takanori
authors.initials: T
authors.lastname: Kamagata
authors.affiliation: Division of Bioimaging, Institute of Biomedical Sciences, Tokushima University Graduate School , 3-18-15 Kuramoto-cho, Tokushima City, Tokushima 770-8503, Japan.
authors.forename: Asuka
authors.initials: A
authors.lastname: Mukai
authors.affiliation: Okazaki Institute for Integrative Bioscience and National Institute for Basic Biology, National Institutes of Natural Sciences , Okazaki, Aichi 444-8787, Japan.|SOKENDAI (The Graduate University for Advanced Studies) , Okazaki, Aichi 444-8787, Japan.
authors.forename: Shinji
authors.initials: S
authors.lastname: Takada
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University , Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Takeharu
authors.initials: T
authors.lastname: Nagai
authors.affiliation: Division of Bioimaging, Institute of Biomedical Sciences, Tokushima University Graduate School , 3-18-15 Kuramoto-cho, Tokushima City, Tokushima 770-8503, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Horikawa
mesh_terms: D003116:Color; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes