Adaptable, Turn-On Monobody (ATOM) Fluorescent Biosensors for Multiplexed Detection in Cells.
Adaptable, Turn-On Monobody (ATOM) Fluorescent Biosensors for Multiplexed Detection in Cells.
复制标题
用于细胞多重检测的适应性强的开启单体 (ATOM) 荧光生物传感器。
DOI：
10.1101/2023.03.28.534597
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Loh,StewartN
中科院分区：
文献类型：
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作者：
Sekhon,Harsimranjit;Ha,Jeung-Hoi;Presti,MariaF;Procopio,SpencerB;Mirsky,PaigeO;John,AnnaM;Loh,StewartN
生物传感器设计中的一个巨大挑战是开发一个单分子，基于荧光蛋白的平台，可以很容易地适应识别选择的目标。从概念上讲，这可以通过将小的抗体样结合结构域与荧光蛋白融合来实现，其方式为靶结合激活荧光。虽然这种设计很容易想象，但它的执行并不明显。在这里，我们创建了一个家庭的适应性，打开单体（原子）生物传感器组成的单体，循环排列在两个位置之一，插入荧光蛋白在三个表面环之一。共表达青色、黄色和红色ATOM传感器的活人类细胞的多重成像检测分别定位于细胞核、细胞质和质膜的生物传感器靶标（WDR 5、SH 2和hRAS蛋白），具有高特异性。ER-和ESTA-本地化的ATOM传感器也检测到针对这些细胞器的配体。荧光激活涉及配体依赖性发色团成熟，在细胞中荧光开启率>20倍，在体外高达100倍。的传感机制进行了验证与三个任意选择的单体插入到水母以及海葵谱系的荧光蛋白，这表明原子传感器具有不同的结合特异性和额外的颜色可以相对较快地产生。
A grand challenge in biosensor design is to develop a single molecule, fluorescent protein-based platform that can be easily adapted to recognize targets of choice. Conceptually, this can be achieved by fusing a small, antibody-like binding domain to a fluorescent protein in such a way that target binding activates fluorescence. Although this design is simple to envision, its execution is not obvious. Here, we created a family of adaptable, turn-on monobody (ATOM) biosensors consisting of a monobody, circularly permuted at one of two positions, inserted into a fluorescent protein at one of three surface loops. Multiplexed imaging of live human cells co-expressing cyan, yellow, and red ATOM sensors detected the biosensor targets (WDR5, SH2, and hRAS proteins) that were localized to the nucleus, cytoplasm, and plasma membrane, respectively, with high specificity. ER- and mitochondria-localized ATOM sensors also detected ligands that were targeted to those organelles. Fluorescence activation involved ligand-dependent chromophore maturation with fluorescence turn-on ratios of >20-fold in cells and up to 100-foldin vitro. The sensing mechanism was validated with three arbitrarily chosen monobodies inserted into jellyfish as well as anemone lineages of fluorescent proteins, suggesting that ATOM sensors with different binding specificities and additional colors can be generated relatively quickly.