<> "The repository administrator has not yet configured an RDF license."^^ . <> . . "Molecular probes for DNA labeling"^^ . "In my thesis, I worked on two labeling strategies for cellular DNA in fluorescence microscopy.\r\nDNA can be studied by fluorescence microscopy with the aid of stains, that can label the\r\ntotal cellular DNA content in live cells. But for some applications, it is desirable that DNA\r\nlabeling should only occur in specific regions. To this end, Hoechst33342 was photocaged and\r\nits in cell activation with light was investigated in a first project. The activatable DNA stain was\r\nsuccessfully applied to label specific regions of DNA in the nucleus of living cells and in clusters\r\nof cells in zebrafish larva.\r\nMetabolic DNA labeling is frequently applied to label newly synthesized DNA. In metabolic\r\nDNA labeling, the cell activates nucleotide precursors and incorporates them into the nascent\r\nDNA chain. In a subsequent labeling reaction, the precursors can be marked with a fluorophore.\r\nUntil now, the visualization of incorporated reporter nucleosides was only amenable in fixed\r\ncells. In a second project, different approaches for metabolic DNA labeling were employed and\r\nfurther improved for applications in living cells.\r\nInitially, a reported nucleoside building block, 5-vinyl-2’-deoxyuridine (VdU), which is marked\r\nin an inverse-electron demand Diels–Alder (iEDDA) reaction was explored. The building block\r\nhad previously been shown to be effective for labeling of DNA in fixed cells, but not in living cells.\r\nI describe the development of a probe for a proximity-enhanced iEDDA labeling of VdU-tagged\r\nDNA in living cells.\r\nTo overcome the slow labeling observed for vinyl-modified DNA, the screening of new metabolic\r\nprecursors was undertaken, in hope of finding a building block with higher reactivity. Therefore,\r\n7-deaza-2’-deoxyadenosine derivatives were modified with more reactive dienophiles and were\r\ninvestigated. Their incorporation into the DNA was verified by mass spectrometry. In fluorescence\r\nimaging, they provided the first signals in the nucleus of proliferating cells. Despite faster\r\nlabeling kinetics, the overall sample preparation takes long since the reporter is introduced first\r\nand visualization with a fluorophore is only possible in the second step.\r\nSuccessful live cell imaging demands labeling methods with high labeling efficiency and\r\nspeed. Therefore, the use of nucleosides that have the fluorophore already attached was investigated.\r\nThese new metabolic precursors allowed a signal to be detected immediately after\r\ntheir incorporation into the nascent DNA chain. To enable better incorporation yields, the fluorescent\r\nnucleosides were further evolved into pro-nucleotides. Nuclear staining in proliferating\r\nliving cells could be observed, even though it required incubation times of several hours.\r\nIn summary, all adenosine derivatives label proliferating cells. Current challenges for more\r\ndemanding applications are poor spatial and temporal resolution for all metabolic approaches.\r\nIn addition to low incorporation yields, the labeling is often also accompanied by undesirable\r\ncytotoxicity effects. If these drawbacks could be overcome, such could pave the way for observing\r\nprocesses that involve DNA synthesis in living cells. Examples of applications include\r\nthe study of DNA replication, repair, or retroviral DNA synthesis."^^ . "2022" . . . . . . . "Carina"^^ . "Lämmle"^^ . "Carina Lämmle"^^ . . . . . . "Molecular probes for DNA labeling (PDF)"^^ . . . "20211202_PhD_Thesis_CL_final.pdf"^^ . . . "Molecular probes for DNA labeling (Other)"^^ . . . . . . "lightbox.jpg"^^ . . . "Molecular probes for DNA labeling (Other)"^^ . . . . . . "preview.jpg"^^ . . . "Molecular probes for DNA labeling (Other)"^^ . . . . . . "medium.jpg"^^ . . . "Molecular probes for DNA labeling (Other)"^^ . . . . . . "small.jpg"^^ . . . "Molecular probes for DNA labeling (Other)"^^ . . . . . . "indexcodes.txt"^^ . . "HTML Summary of #31478 \n\nMolecular probes for DNA labeling\n\n" . "text/html" . . . "540 Chemie"@de . "540 Chemistry and allied sciences"@en . . . "570 Biowissenschaften, Biologie"@de . "570 Life sciences"@en . .