PT  - JOURNAL ARTICLE
AU  - Lars Hellweg
AU  - Anna Edenhofer
AU  - Lucas Barck
AU  - Magnus-Carsten Huppertz
AU  - Michelle. S. Frei
AU  - Miroslaw Tarnawski
AU  - Andrea Bergner
AU  - Birgit Koch
AU  - Kai Johnsson
AU  - Julien Hiblot
TI  - A general method for the development of multicolor biosensors with large dynamic ranges
AID  - 10.1101/2022.11.29.518186
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.11.29.518186
4099  - http://biorxiv.org/content/early/2022/11/29/2022.11.29.518186.short
4100  - http://biorxiv.org/content/early/2022/11/29/2022.11.29.518186.full
AB  - Fluorescent biosensors enable to study cell physiology with spatiotemporal resolution, yet most biosensors suffer from relatively low dynamic ranges. Here, we introduce a family of designed Förster Resonance Energy Transfer (FRET) pairs with near quantitative FRET efficiencies based on the reversible interaction of fluorescent proteins with a fluorescently labeled HaloTag. These FRET pairs enabled the straightforward design of biosensors for calcium, ATP and NAD+ with unprecedented dynamic ranges. The color of each of these biosensors can be readily tuned by either changing the fluorescent protein or the synthetic fluorophore, which enabled to monitor simultaneously free NAD+ in different subcellular compartments upon genotoxic stress. Minimal modifications furthermore allow the readout of these biosensors to be switched to fluorescence intensity, lifetime or bioluminescence. These FRET pairs thus establish a new concept for the development of highly sensitive and tunable biosensors.Competing Interest StatementK.J. is listed as inventor for patents related to labeling technologies filed by the Max Planck Society or the Ecole Polytechnique Federale de Lausanne (EPFL).