Red-Shifted Fluorogenic Substrate for Detection of lacZ-Positive Cells in Living Tissue with Single-Cell Resolution
Red-Shifted Fluorogenic Substrate for Detection of lacZ-Positive Cells in Living Tissue with Single-Cell Resolution
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DOI：
10.1002/anie.201808670
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发表时间：
2018-11-26
影响因子：
16.6
通讯作者：
Urano, Yasuteru
中科院分区：
文献类型：
--
作者：
Ito, Hiroki;Kawamata, Yu;Urano, Yasuteru
编码β-半乳糖苷酶的大肠杆菌lacZ基因是一种广泛使用的报告基因，但很少有合成底物可用于在活样品中以单细胞分辨率检测其活性。我们最近报道的荧光底物SPiDER-β Gal适用于此目的，但其水解产物显示绿色荧光发射，因此需要红移类似物与绿色荧光蛋白（GFP）标记物组合使用。在这里，我们描述了一个红移的荧光底物β-半乳糖苷酶，SPiDER红-β Gal，基于硅罗丹酚支架和羧基作为分子内亲核试剂的发展。LacZ阳性细胞在活样本中以单细胞分辨率成功地用SPiDER-Red-beta Gal标记，这使我们能够将不同的细胞类型与GFP标记物结合起来可视化。
The Escherichia coli lacZ gene encoding beta-galactosidase is a widely used reporter, but few synthetic substrates are available for detecting its activity with single-cell resolution in living samples. Our recently reported fluorogenic substrate SPiDER-beta Gal is suitable for this purpose, but its hydrolysis product shows green fluorescence emission, and a red-shifted analogue is therefore required for use in combination with green fluorescent protein (GFP) markers. Herein, we describe the development of a red-shifted fluorogenic substrate for beta-galactosidase, SPiDER-Red-beta Gal, based on a silicon rhodol scaffold and a carboxylic group as the intramolecular nucleophile. LacZ-positive cells were successfully labeled with SPiDER-Red-beta Gal at single-cell resolution in living samples, which enabled us to visualize different cell types in combination with GFP markers.