Zheng Qinsi, Ayala Anthony X, Chung Inhee, Weigel Aubrey V, Ranjan Anand, Falco Natalie, Grimm Jonathan B, Tkachuk Ariana N, Wu Carl, Lippincott-Schwartz Jennifer, Singer Robert H, Lavis Luke D
Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, United States.
Department of Biology and Department of Molecular Biology and Genetics, Johns Hopkins University, Baltimore, Maryland 21218, United States.
ACS Cent Sci. 2019 Sep 25;5(9):1602-1613. doi: 10.1021/acscentsci.9b00676. Epub 2019 Sep 5.
Rhodamine dyes exist in equilibrium between a fluorescent zwitterion and a nonfluorescent lactone. Tuning this equilibrium toward the nonfluorescent lactone form can improve cell-permeability and allow creation of "fluorogenic" compounds-ligands that shift to the fluorescent zwitterion upon binding a biomolecular target. An archetype fluorogenic dye is the far-red tetramethyl-Si-rhodamine (SiR), which has been used to create exceptionally useful labels for advanced microscopy. Here, we develop a quantitative framework for the development of new fluorogenic dyes, determining that the lactone-zwitterion equilibrium constant ( ) is sufficient to predict fluorogenicity. This rubric emerged from our analysis of known fluorophores and yielded new fluorescent and fluorogenic labels with improved performance in cellular imaging experiments. We then designed a novel fluorophore-Janelia Fluor 526 (JF)-with SiR-like properties but shorter fluorescence excitation and emission wavelengths. JF is a versatile scaffold for fluorogenic probes including ligands for self-labeling tags, stains for endogenous structures, and spontaneously blinking labels for super-resolution immunofluorescence. JF constitutes a new label for advanced microscopy experiments, and our quantitative framework will enable the rational design of other fluorogenic probes for bioimaging.
罗丹明染料在荧光两性离子和非荧光内酯之间存在平衡。将这种平衡向非荧光内酯形式调整可以提高细胞通透性，并允许创建“荧光生成”化合物——即与生物分子靶标结合后转变为荧光两性离子的配体。一种典型的荧光生成染料是远红四甲基硅罗丹明（SiR），它已被用于为先进显微镜创建极其有用的标记。在这里，我们开发了一个用于开发新型荧光生成染料的定量框架，确定内酯 - 两性离子平衡常数（ ）足以预测荧光生成能力。这个准则源于我们对已知荧光团的分析，并在细胞成像实验中产生了具有改进性能的新型荧光和荧光生成标记。然后，我们设计了一种具有类似SiR性质但荧光激发和发射波长更短的新型荧光团——Janelia Fluor 526（JF）。JF是用于荧光生成探针的通用支架，包括用于自标记标签的配体、用于内源性结构的染色剂以及用于超分辨率免疫荧光的自发闪烁标签。JF构成了用于先进显微镜实验的新标记，我们的定量框架将有助于合理设计用于生物成像的其他荧光生成探针。
