Department of Chemistry, Graduate School of Science, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Department of Biotechnology, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.
Nat Chem Biol. 2023 Jan;19(1):38-44. doi: 10.1038/s41589-022-01134-z. Epub 2022 Sep 22.
Molecular fluorescent indicators are versatile tools for dynamic imaging of biological systems. We now report a class of indicators that are based on the chemigenetic combination of a synthetic ion-recognition motif and a protein-based fluorophore. Specifically, we have developed a calcium ion (Ca) indicator that is based on genetic insertion of circularly permuted green fluorescent protein into HaloTag protein self-labeled with a ligand containing the Ca chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. We have demonstrated the versatility of this design by also developing a sodium ion (Na) indicator using a crown-ether-containing ligand. This approach affords bright and sensitive ion indicators that can be applicable to cell imaging. This design can enable the development of chemigenetic indicators with ion or molecular specificities that have not been realized with fully protein-based indicators.
分子荧光指示剂是用于生物系统动态成像的多功能工具。我们现在报告一类基于合成离子识别基序与基于蛋白质的荧光团的化学遗传组合的指示剂。具体来说，我们开发了一种基于将环状排列的绿色荧光蛋白插入到带有包含钙螯合剂 1,2-双(2-氨基苯氧基)乙烷-N,N,N',N'-四乙酸的配体的 HaloTag 蛋白中的钙离子 (Ca) 指示剂。我们通过使用含冠醚的配体开发钠离子 (Na) 指示剂，证明了这种设计的多功能性。该方法提供了明亮且灵敏的离子指示剂，可适用于细胞成像。这种设计可以使具有离子或分子特异性的化学遗传指示剂的开发成为可能，而这些特异性是完全基于蛋白质的指示剂无法实现的。
