Naoki Komatsu, Kenta Terai, Ayako Imanishi, Yuji Kamioka, Kenta Sumiyama, Takashi Jin, Yasushi Okada, Takeharu Nagai, Michiyuki Matsuda (2018) A platform of BRET-FRET hybrid biosensors for optogenetics, chemical screening, and in vivo imaging., Scientific reports, Volume 8, Number 1, pp. 8984
Published in 2018 Jun 12 (Electronic publication in June 12, 2018, midnight )
(Abstract) Genetically encoded biosensors based on the principle of Forster 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.