Sanchez Mateo I, Nguyen Quynh-Anh, Wang Wenjing, Soltesz Ivan, Ting Alice Y
Department of Genetics, Stanford University, Stanford, CA 94305.
Department of Biology, Stanford University, Stanford, CA 94305.
Proc Natl Acad Sci U S A. 2020 Dec 29;117(52):33186-33196. doi: 10.1073/pnas.2006521117. Epub 2020 Dec 15.
Molecular integrators, in contrast to real-time indicators, convert transient cellular events into stable signals that can be exploited for imaging, selection, molecular characterization, or cellular manipulation. Many integrators, however, are designed as complex multicomponent circuits that have limited robustness, especially at high, low, or nonstoichiometric protein expression levels. Here, we report a simplified design of the calcium and light dual integrator FLARE. Single-chain FLARE (scFLARE) is a single polypeptide chain that incorporates a transcription factor, a LOV domain-caged protease cleavage site, and a calcium-activated TEV protease that we designed through structure-guided mutagenesis and screening. We show that scFLARE has greater dynamic range and robustness than first-generation FLARE and can be used in culture as well as in vivo to record patterns of neuronal activation with 10-min temporal resolution.
与实时指标不同，分子整合器将瞬时细胞事件转化为稳定信号，可用于成像、筛选、分子表征或细胞操作。然而，许多整合器被设计为复杂的多组分电路，其稳健性有限，尤其是在高、低或非化学计量的蛋白质表达水平下。在这里，我们报告了钙和光双整合器FLARE的简化设计。单链FLARE（scFLARE）是一条单一多肽链，包含一个转录因子、一个LOV结构域笼化蛋白酶切割位点和一个通过结构导向诱变和筛选设计的钙激活TEV蛋白酶。我们表明，scFLARE比第一代FLARE具有更大的动态范围和稳健性，可用于体外培养以及体内，以10分钟的时间分辨率记录神经元激活模式。
