TY - JOUR T1 - Matryoshka: Ratiometric biosensors from a nested cassette of green- and orange-emitting fluorescent proteins JF - bioRxiv DO - 10.1101/143107 SP - 143107 AU - Cindy Ast AU - Jessica Foret AU - Luke M. Oltrogge AU - Roberto De Michele AU - Thomas J. Kleist AU - Cheng-Hsun Ho AU - Wolf B. Frommer Y1 - 2017/01/01 UR - http://biorxiv.org/content/early/2017/06/06/143107.abstract N2 - 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. ER -