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Abstract

In my thesis, I worked on two labeling strategies for cellular DNA in fluorescence microscopy. DNA can be studied by fluorescence microscopy with the aid of stains, that can label the total cellular DNA content in live cells. But for some applications, it is desirable that DNA labeling should only occur in specific regions. To this end, Hoechst33342 was photocaged and its in cell activation with light was investigated in a first project. The activatable DNA stain was successfully applied to label specific regions of DNA in the nucleus of living cells and in clusters of cells in zebrafish larva. Metabolic DNA labeling is frequently applied to label newly synthesized DNA. In metabolic DNA labeling, the cell activates nucleotide precursors and incorporates them into the nascent DNA chain. In a subsequent labeling reaction, the precursors can be marked with a fluorophore. Until now, the visualization of incorporated reporter nucleosides was only amenable in fixed cells. In a second project, different approaches for metabolic DNA labeling were employed and further improved for applications in living cells. Initially, a reported nucleoside building block, 5-vinyl-2’-deoxyuridine (VdU), which is marked in an inverse-electron demand Diels–Alder (iEDDA) reaction was explored. The building block had previously been shown to be effective for labeling of DNA in fixed cells, but not in living cells. I describe the development of a probe for a proximity-enhanced iEDDA labeling of VdU-tagged DNA in living cells. To overcome the slow labeling observed for vinyl-modified DNA, the screening of new metabolic precursors was undertaken, in hope of finding a building block with higher reactivity. Therefore, 7-deaza-2’-deoxyadenosine derivatives were modified with more reactive dienophiles and were investigated. Their incorporation into the DNA was verified by mass spectrometry. In fluorescence imaging, they provided the first signals in the nucleus of proliferating cells. Despite faster labeling kinetics, the overall sample preparation takes long since the reporter is introduced first and visualization with a fluorophore is only possible in the second step. Successful live cell imaging demands labeling methods with high labeling efficiency and speed. Therefore, the use of nucleosides that have the fluorophore already attached was investigated. These new metabolic precursors allowed a signal to be detected immediately after their incorporation into the nascent DNA chain. To enable better incorporation yields, the fluorescent nucleosides were further evolved into pro-nucleotides. Nuclear staining in proliferating living cells could be observed, even though it required incubation times of several hours. In summary, all adenosine derivatives label proliferating cells. Current challenges for more demanding applications are poor spatial and temporal resolution for all metabolic approaches. In addition to low incorporation yields, the labeling is often also accompanied by undesirable cytotoxicity effects. If these drawbacks could be overcome, such could pave the way for observing processes that involve DNA synthesis in living cells. Examples of applications include the study of DNA replication, repair, or retroviral DNA synthesis.