Journal
NATURE COMMUNICATIONS
Volume 8, Issue -, Pages -Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/s41467-017-00400-2
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Funding
- National Science Foundation [MCB-1413254]
- NSF Graduate Research Program Fellowship
- NIH [GM27738]
- Div Of Molecular and Cellular Bioscience
- Direct For Biological Sciences [1413254] Funding Source: National Science Foundation
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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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