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Bioorthogonal red and far-red fluorogenic probes for wash-free live-cell and super-resolution microscopy
bioRxiv - Cell Biology Pub Date : 2020-08-07 , DOI: 10.1101/2020.08.07.241687
Philipp Werther , Klaus Yserentant , Felix Braun , Kristin Grussmayer , Vytautas Navikas , Miao Yu , Zhibin Zhang , Michael J. Ziegler , Christoph Mayer , Antoni J. Gralak , Marvin Busch , Weijie Chi , Frank Rominger , Aleksandra Radenovic , Xiaogang Liu , Edward A. Lemke , Tiago Buckup , Dirk-Peter Herten , Richard Wombacher

Small-molecule fluorophores enable the observation of biomolecules in their native context with fluorescence microscopy. Specific labelling via bioorthogonal tetrazine chemistry confers minimal label size and rapid labelling kinetics. At the same time, fluorogenic tetrazine-dye conjugates exhibit efficient quenching of dyes prior to target binding. However, live-cell compatible long-wavelength fluorophores with strong fluorogenicity have been difficult to realize. Here, we report close proximity tetrazine-dye conjugates with minimal distance between tetrazine and fluorophore. Two synthetic routes give access to a series of cell permeable and impermeable dyes including highly fluorogenic far-red emitting derivatives with electron exchange as dominant excited state quenching mechanism. We demonstrate their potential for live-cell imaging in combination with unnatural amino acids, wash-free multi-colour and super-resolution STED and SOFI imaging. These dyes pave the way for advanced fluorescence imaging of biomolecules with minimal label size.

中文翻译:

生物正交的红色和远红色荧光探针,用于免洗活细胞和超分辨率显微镜

小分子荧光团可以通过荧光显微镜观察其自然环境中的生物分子。通过生物正交四嗪化学进行的特异性标记赋予最小的标记尺寸和快速的标记动力学。同时,发荧光的四嗪-染料缀合物在靶结合之前表现出染料的有效猝灭。然而,具有强荧光性的活细胞相容的长波长荧光团已经难以实现。在这里,我们报告紧密接近的四嗪染料偶联物,四嗪和荧光团之间的距离最小。两种合成途径可获取一系列细胞可渗透和不可渗透的染料,包括高度荧光的远红色发射衍生物,其中电子交换是主要的激发态猝灭机制。我们证明了其结合非天然氨基酸,免洗多色和超分辨率STED和SOFI成像在活细胞成像中的潜力。这些染料为标记最小的生物分子的高级荧光成像铺平了道路。
更新日期:2020-08-10
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