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

连接分子在生物标记过程中将商业荧光团转化为定制的功能探针：合成、光谱表征和生物物理应用

• 批准号: 518284393
• 负责人: Professor Dr. Thorben Cordes
• 金额: --
• 依托单位: DWI - Leibniz-Institut für Interaktive Materialien e.V.
• 依托单位国家: 德国
• 项目类别: Research Grants
• 资助国家: 德国
• 项目状态: 未结题
• 来源: https://gepris.dfg.de/gepris/projekt/518284393?language=en
• 关键词: Linker; molecules; convert; commercial; fluorophores

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).

商用合成荧光团已成为成像、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信号暗淡、信号快速丢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仅限于特定应用，例如用于蛋白质标记、细胞器标记、DNA测序、单分子检测等，并且如果不进行重大的合成努力，就无法修改荧光团的性质或生物偶联化学。在Thorben Cordes（德国慕尼黑大学物理与合成生物学）和Andreas Herrmann（德国亚琛工业大学大分子材料与系统）团队的合作项目中，我们提出了解决这些基本问题的方法。我们以连接化合物的形式引入了一种新的化学生物学工具，它允许在体外和体内用商业荧光团选择性标记生物靶标，其特性通过连接物可调节。连接分子包含不同的分子部分，允许生物偶联到目标，一个点击单元，以促进荧光团连接和功能元件。该方法是基于这样的想法，即商业荧光团的性质在生物标记过程中被连接剂修饰。我们已经通过一锅Ugi反应成功地建立了这种连接化合物的合成路线，并可以证明连接物确实可以作为生物靶标上不同商业荧光团功能修饰的基础。这个dfg项目的目标是通过在不同的生物学环境中开发和测试一个扩展的连接器库来充分探索这种方法的可能性。该染料的功能特性包括高光稳定性、可控的单分子闪烁、光活化和感应二价金属阳离子的能力。同时，化学性质（极性和染料类别），亲和标签的存在和生物偶联选择（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共同完成，他们分别在两个相关实验室中的一个工作，以探索所有相关方面。拟议的项目包括具有不同荧光团类别（Herrmann组）的不同靶标的化学合成和生物标记优化，连接-荧光团组合的功能和光谱表征（Cordes & Herrmann组）以及最成功的构建在成像和生物物理分析中的应用（Cordes组）。