Linker molecules convert commercial fluorophores into tailored functional probes during biolabeling: Synthesis, spectroscopic characterization and biophysical applications

商业上可用的合成荧光团已经成为成像、生化分析、DNA测序和医疗技术的标准工具。然而，它们受到各种限制和缺点的影响，这些限制和缺点对上述技术和分析产生了负面影响。典型的光物理问题是暗淡的信号、快速的信号损失或不需要的信号波动。对于许多应用，荧光团还需要功能特性，如闪烁发射、金属传感能力或高光稳定性。最后，商业荧光团的使用通常限于特定的应用，例如，用于蛋白质标记、细胞器标记、DNA测序、单分子检测等，并且如果没有重大的合成努力，就不能改变荧光团的性质或生物结合化学。在Thorben Cordes(物理和合成生物学，LMU München)和Andreas Herrmann(高分子材料和系统，RWTH Aachen)团队之间的这个合作项目中，我们提出了这些基本问题的解决方案。我们介绍了一种新的化学生物学工具，它以连接物的形式存在，允许用商业荧光团选择性地标记体外和体内的生物靶标，通过连接物使其性质变得可调。连接体分子包含允许生物结合到靶的不同分子部分、促进荧光团连接的点击单元和功能元件。这种方法是基于这样一个想法，即商业荧光团的性质在生物标记过程中被连接物改变。我们通过一锅UGI反应成功地建立了这类连接体化合物的合成路线，并可以证明连接体确实可以作为不同商业荧光团对生物靶标进行功能修饰的基础。这个DFG项目的目标是通过开发和基准在不同生物环境中扩展的连接子文库来充分探索该方法的可能性。这些功能特性将被探索，包括高光稳定性，受控的单分子闪烁，光激活和染料对二价金属离子的感知能力。同时，化学性质(极性和染料类)、亲和标签的存在和生物连接选项(蛋白质、寡核苷酸、抗体-体内和体外)将被修改。这项工作将由两名博士生分享，每人在两个相关实验室中的一个工作，以探索所有相关方面。拟议的项目包括化学合成和优化具有不同荧光团类别的不同目标上的生物标记(Herrmann组)、连接物-荧光团组合的功能和光谱表征(Cordes&Herrmann组)以及最成功的结构在成像和生物物理分析中的应用(Cordes组)。

Commercially-available synthetic fluorophores have become standard tools for imaging, biochemical assays, DNA-sequencing and medical technologies. They suffer, however, from various limitations and shortcomings that negatively impact the above-mentioned techniques and assays. Typical photophysical problems are dim signals, fast signal loss or unwanted signal fluctuations. For many applications, fluorophores additionally require functional properties such as blinking emission, metal sensing capabilities or high photostability. Finally, the use of a commercial fluorophore is often limited to a specific application, e.g., for protein labeling, organelle marking, DNA sequencing, single-molecule detection etc., and fluorophore properties or bioconjugation chemistry cannot be modified without major synthetic efforts. In this collaborative project between the groups of Thorben Cordes (Physical and Synthetic Biology, LMU München) and Andreas Herrmann (Macromolecular Materials and Systems, RWTH Aachen), we propose a solution to these fundamental problems. We introduce a novel chemical biology tool in the form of linker compounds that allow selective labeling of biological targets in vitro and in vivo with a commercial fluorophore, which becomes tunable in its properties via the linker. The linker molecule contains distinct molecular parts allowing bioconjugation to a target, a click-unit to facilitate fluorophore linkage and a functional element. The approach is based on the idea that the properties of a commercial fluorophore are modified by the linker during biolabeling. We have successfully established a synthesis route for such linker compounds via the one-pot Ugi reaction and could demonstrate that linkers can indeed serve as a basis for functional modification of different commercial fluorophores on biological targets. The goal of this DFG-project is to fully explore the possibilities of the approach by developing and benchmarking an extended library of linkers in different biological contexts. The functional properties, which will be explored, are high photostability, controlled single-molecule blinking, photoactivation and the ability of the dye to sense divalent metal cations. Simultaneously, the chemical properties (polarity and dye-class), presence of affinity-tags and bioconjugation options (proteins, oligonucleotides, antibodies - in vivo and in vitro) will be modified. The work will be shared by two PhD students, each working in one of the two involved labs, to explore all the relevant aspects. The proposed project covers chemical synthesis and optimization of biolabeling on different targets with distinct fluorophore classes (Herrmann group), functional and spectroscopic characterization of linker-fluorophore combinations (Cordes & Herrmann group) and applications of the most successful constructs in imaging and biophysical assays (Cordes group).