Photoactivated Click Chemistry for Labelling of DNA and Chemotherapeutics in Cells

In the last years, fluorescence-based labelling of biomolecules in living cells has gained significant importance for providing deeper insights into complex biological processes. A particularly powerful approach for this purpose are the bioorthogonal reactions, which have been developed over the past two decades to enable selective, interference-free labelling of biomolecules such as proteins and nucleic acids. Combining these bioorthogonal labelling techniques with chemotherapeutics allows for precise tracking of their interactions and mechanisms of action at the molecular level. This is especially valuable for studying cancer drugs that inhibit DNA replication, thereby disrupting cell division and ultimately leading to cell death. Using bioorthogonal labelling, the interactions of these substances with DNA can be visualized in real time, providing new insights into their effects on cellular processes.
This work is divided into three parts. In the first part, a light-controlled bioorthogonal labelling method - the so-called “photoclick reaction” - is investigated and further developed. Tetrazole-based reactants are modified so that the reaction can be triggered by exposure to visible light (>405 nm). ... mehrIn addition to the bathochromic shift of the excitation wavelength, these modifications improve the water solubility of the reactants, further optimizing the visualization of the desired biomolecule in living biological systems. The reaction occurs between tetrazoles as dyes and 2’-deoxynucleosides, which are incorporated into cellular DNA. The reactivity of the tetrazoles was characterized using UV/Vis spectroscopy and HPLC analysis and successfully applied to HeLa cells.
The second part of the work focuses on the development and visualization of photoactivatable chemotherapeutics that are non-toxic in their inactive (dark) state. Upon light activation, a part of the molecule is cleaved, activating the cancer drug and making it toxic. Since these molecules are non-fluorescent, they are first bioorthogonally labeled using copper-catalyzed azide-alkyne cycloaddition for cellular studies. This enables real-time visualization and monitoring of their effects. The activated cancer drugs are studied under different activation and incubation conditions, and their cellular target organelles are analyzed using immunofluorescence.
In the third part of this work, the method is extended to chemotherapeutics that intercalate into DNA and thereby affect replication. By combining the approaches developed in the first and second part, DNA replication in the cell is observed, and the point at which the synthesis of new DNA is inhibited is identified. For this purpose, a 2’-deoxynucleoside is co-incubated with the chemotherapeutic in cells, and bioorthogonal reactions are used to visualize newly replicated DNA. This allows detailed analysis of the therapeutic’s spectrum of action and its effects on cellular systems.
This work thus makes a significant contribution to the development of innovative labelling techniques and the precise study of chemotherapeutics that influence DNA replication in cancer cells, offering important perspectives for new therapeutic approaches.