Matryoshka: Ratiometric biosensors from a nested 1 cassette of green- and orange-emitting fluorescent proteins

Ast, C.; Foret, J.; Oltrogge, L.; De Michele, R.; Kleist, T.; Ho, C.-H.; Frommer, W.

doi:10.1101/143107

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bioRxiv · 10.1101/143107

Matryoshka: Ratiometric biosensors from a nested 1 cassette of green- and orange-emitting fluorescent proteins

Ast, C.·Foret, J.·Oltrogge, L.·De Michele, R.·Kleist, T.·Ho, C.-H.·Frommer, W.

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Abstract

Sensitivity, dynamic and detection range as well as exclusion of expression and instrumental artifacts are critical for the quantitation of data obtained with fluorescent protein (FP)-based biosensors in vivo. Current biosensors designs are, in general, unable to simultaneously meet all these criteria. Here, we describe a generalizable platform to create dual-FP biosensors with large dynamic ranges by employing a single FP-cassette, named GO-(Green-Orange) Matryoshka. The cassette nests a stable reference FP (large Stokes shift LSSmOrange) within a reporter FP (circularly permuted green FP). GO-Matryoshka yields green and orange fluorescence upon blue excitation. As proof of concept, we converted existing, single-emission biosensors into a series of ratiometric calcium sensors (MatryoshCaMP6s) and ammonium transport activity sensors (AmTryoshka1;3). We additionally identified the internal acid-base equilibrium as a key determinant of the GCaMP dynamic range. Matryoshka technology promises flexibility in the design of a wide spectrum of ratiometric biosensors and expanded in vivo applications.

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Ast, C., Foret, J., Oltrogge, L., De Michele, R., Kleist, T., Ho, C.-H., Frommer, W.. 2017-06-06. Matryoshka: Ratiometric biosensors from a nested 1 cassette of green- and orange-emitting fluorescent proteins. https://doi.org/10.1101/143107

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