Ast Cindy, Foret Jessica, Oltrogge Luke M, De Michele Roberto, Kleist Thomas J, Ho Cheng-Hsun, Frommer Wolf B
Department of Plant Biology, Carnegie Science, Stanford, California, 94305, USA.
Department of Chemistry, Stanford University, Stanford, California, 94305, USA.
Nat Commun. 2017 Sep 5;8(1):431. doi: 10.1038/s41467-017-00400-2.
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.Single fluorescent protein biosensors are susceptible to expression and instrumental artifacts. Here Ast et al. describe a dual fluorescent protein design whereby a reference fluorescent protein is nested within a reporter fluorescent protein to control for such artifacts while preserving sensitivity and dynamic range.
灵敏度、动态范围和检测范围以及排除表达和仪器假象对于体内基于荧光蛋白（FP）的生物传感器所获得数据的定量分析至关重要。目前的生物传感器设计通常无法同时满足所有这些标准。在此，我们描述了一个通用平台，通过采用单个FP盒（名为GO-（绿色-橙色）套娃）来创建具有大动态范围的双FP生物传感器。该盒在一个报告荧光蛋白（环状排列的绿色荧光蛋白）内嵌套了一个稳定的参考荧光蛋白（大斯托克斯位移的LSSmOrange）。GO-套娃在蓝光激发下产生绿色和橙色荧光。作为概念验证，我们将现有的单发射生物传感器转化为一系列比率型钙传感器（MatryoshCaMP6s）和铵转运活性传感器（AmTryoshka1;3）。我们还确定内部酸碱平衡是GCaMP动态范围的关键决定因素。套娃技术有望在设计广泛的比率型生物传感器方面具有灵活性，并扩展体内应用。单荧光蛋白生物传感器易受表达和仪器假象的影响。在此，阿斯特等人描述了一种双荧光蛋白设计，即一个参考荧光蛋白嵌套在一个报告荧光蛋白内，以控制此类假象，同时保持灵敏度和动态范围。
