Robust red FRET sensors using self-associating fluorescent domains

Elucidation of subcellular signaling networks by multiparameter imaging is hindered by a lack of sensitive FRET pairs spectrally compatible with the classic CFP/YFP pair. Here, we present a generic strategy to enhance the traditionally poor sensitivity of red FRET sensors by developing self-associating variants of mOrange and mCherry that allow sensors to switch between well-defined on- and off states. Requiring just a single mutation of the mFruit domain, this new FRET pair improved the dynamic range of protease sensors up to 10-fold and was essential to generate functional red variants of CFP-YFP-based Zn2+ sensors. The large dynamic range afforded by the new red FRET pair allowed simultaneous use of differently colored Zn2+ FRET sensors to image Zn2+ over a broad concentration range in the same cellular compartment.