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%0 Thesis
%A Käver, Larissa
%T Entwicklung einer orthogonalen Markierungsmethode für die Fluoreszenzdetektion epigenetischer DNA-Modifikationen
%I RWTH Aachen University
%V Dissertation
%C Aachen
%M RWTH-2022-02863
%P 1 Online-Ressource : Illustrationen, Diagramme
%D 2022
%Z Veröffentlicht auf dem Publikationsserver der RWTH Aachen University
%Z Dissertation, RWTH Aachen University, 2022
%X 5-Methylcytosin (5mC) and its oxidation product 5-Hydroxymethylcytosin (5hmC) are epigenetic DNA modifications responsible for gene regulation processes and also known for different types of cancer because of alterations in DNA methylation and DNA hydroxymethylation. [1–4] Parallel analysis of the levels of 5mC and 5hmC can potentially serve as biomarkers for early cancer detection, monitoring of disease progression and response to treatment. Current techniques for global quantification of 5mC and 5hmC are not yet suitable for clinical applications because this would require very rapid quantification of both modifications in the specific tissue using two different fluorophores. Therefore, the aim of this work was to develop an orthogonal method for parallel global fluorescent labeling of the epigenetic modifications 5mC and 5hmC. First focus of this work was the synthesis of analogs of the natural cofactor AdoMet (1) that exhibited different vinyl modifications and could be applied in two-step labeling reactions. Furthermore, the enzymatic activity of the new cofactor analogues was investigated in activity assays with different adenine- and cytosine-specific DNA-MTases. Based on this, the two-step DNA labeling reaction for methylation detection was studied using the different vinylic cofactor analogues and several tetrazine conjugates by analysis of fluorescent labeling with high and low label density. The analysis of the fluorescently labeled DNA was performed on immobilized and stretched single DNA-molecules (DNA combing) using the institute's own fluorescence microscope. Furthermore, methylation detection was additionally examined with one-step labeling reaction by sequence-specific modification of different DNA substrates using fluorescent cofactor analogues. Due to the important role of the epigenetic modifications 5mC and 5hmC in the early detection of cancer, a genomic human DNA sample (breast cancer cell line, UK Aachen) was also labeled and studied by our cooperation partners at Tel Aviv University in Israel with regard to epigenetic information. Besides, in addition to methylation detection, hydroxymethylation was analyzed in a two-step labeling reaction, as well as the orthogonal determination of 5mC and 5hmC by fluorescence microscopy. Both single detections as well as the orthogonal determination were additionally quantified with respect to reaction yields on short duplex ODN by rp-HPLC.[1]P. A. Jones, S. B. Baylin, Cell 2007, 128, 683.[2]M. C. Haffner, A. Chaux, A. K. Meeker, D. M. Esopi, J. Gerber, L. G. Pellakuru, A. Toubaji, P. Argani, C. Iacobuzio-Donahue, W. G. Nelson, G. J. Netto, A. M. de Marzo, S. Yegnasubramanian, Oncotarget 2011, 2, 627.[3]M. Ko, Y. Huang, A. M. Jankowska, U. J. Pape, M. Tahiliani, H. S. Bandukwala, J. An, E. D. Lamperti, K. P. Koh, R. Ganetzky, X. S. Liu, L. Aravind, S. Agarwal, J. P. Maciejewski, A. Rao, Nature 2010, 468, 839.[4]A. P. Feinberg, R. Ohlsson, S. Henikoff, Nat. Rev. Genet. 2006, 7, 21.
%F PUB:(DE-HGF)11
%9 Dissertation / PhD Thesis
%R 10.18154/RWTH-2022-02863
%U https://publications.rwth-aachen.de/record/843084