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

Cindy Ast, Jessica Foret, Luke M. Oltrogge, Roberto De Michele, Thomas J. Kleist, Cheng-Hsun Ho and Wolf B. Frommer ()
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Cindy Ast: Carnegie Science
Jessica Foret: Carnegie Science
Luke M. Oltrogge: Stanford University
Roberto De Michele: Institute of Biosciences and Bioresources (IBBR), Italian National Research Council (CNR)
Thomas J. Kleist: Carnegie Science
Cheng-Hsun Ho: Carnegie Science
Wolf B. Frommer: Carnegie Science

Nature Communications, 2017, vol. 8, issue 1, 1-13

Abstract: 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.

Date: 2017
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DOI: 10.1038/s41467-017-00400-2

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