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

• Applicants: Professor Dr. Thorben Cordes; Professor Dr. Andreas Herrmann
• Subject Area: Physical Chemistry of Molecules, Liquids and Interfaces, Biophysical Chemistry ; Biophysics
• Term: since 2023
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 518284393

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).

• DFG Programme: Research Grants
• International Connection: China
• Cooperation Partner: Dr. Lei Zhang