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ACS Chem Biol. 2013 Oct 18;8(10):2133-9. doi: 10.1021/cb400427b. Epub 2013 Aug 30.

Robust red FRET sensors using self-associating fluorescent domains.

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Laboratory of Chemical Biology, Department of Biomedical Engineering, Eindhoven University of Technology , P.O. Box 513, 5600MB Eindhoven, The Netherlands.

Erratum in

  • ACS Chem Biol. 2014 Jun 20;9(6):1387.


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 Zn(2+) sensors. The large dynamic range afforded by the new red FRET pair allowed simultaneous use of differently colored Zn(2+) FRET sensors to image Zn(2+) over a broad concentration range in the same cellular compartment.

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