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TY  - THES
AU  - Eiden, Sophia Charlotte
TI  - Charakterisierung der Bindung humaner Proteine an G-Quadruplex-DNA und -RNA
PB  - RWTH Aachen University
VL  - Dissertation
CY  - Aachen
M1  - RWTH-2022-10395
SP  - 1 Online-Ressource : Illustrationen, Diagramme
PY  - 2022
N1  - Veröffentlicht auf dem Publikationsserver der RWTH Aachen University
N1  - Dissertation, RWTH Aachen University, 2022
AB  - The doctoral project presented in this work was dedicated to the characterization of the binding properties of nuclear proteins in relation to so-called G-quadruplex nucleic acid structures. The G-quadruplex is a special secondary structure of nucleic acids in which four guanine molecules within a sequence come together to form a planar tetrad. Different numbers of such G-tetrads combine to form different types of this unusual secondary structure. The binding of the proteins was analyzed with regard to G-quadruplex structures (Myc22-DNA, Myc22-RNA, CEB25-L111, CEB25-L191) and non-G-quadruplex structures (guanine-rich ssDNA and dsDNA). Both the deoxyribooligonucleotide and the ribooligonucleotide of the Myc22 sequence already used by Rauser (Valerie Rauser, PhD thesis RWTH, 2019) were used here. Analogously to the method described by Rauser for generating monomeric structures of the G-quadruplex (G4) Pu27 DNA or Myc22 DNA under basic conditions, a method for generating monomeric Myc22 RNA structures was first established at the beginning of the project. The success of each treatment approach was verified by analytical methods such as size exclusion chromatography (SEC) and polyacrylamide gel electrophoresis (PAGE). The pretreated oligonucleotides, folded into monomeric G4 structures, were then used for binding studies with certain predominantly nuclear proteins - starting from proteins that were identified by Rauser in 2019 as promising G4 binding candidates through proteomic analyzes of ovarian cancer cell lysates. As preliminary work for these binding studies, some of the potential G4-binding proteins (EED, hnRNPU and YBX1), each equipped with a His tag, were overexpressed in E. coli cells and purified using affinity chromatography (Ni-NTA column) and gel filtration. The characterization of the protein binding properties to the various DNA and RNA structures was carried out using biochemical and biophysical methods in order to enable the most accurate possible verification of the results. The pulldown assay and the EMSA (electrophoretic mobility shift assay) were used as biochemical methods. The pulldown assay is a type of affinity chromatographic isolation method in which proteins can be immobilized by their binding to oligonucleotide-coated magnetic particles. The protein-DNA complexes formed during the binding reaction can be dissociated after repeated washing of the samples with a sodium dodecyl sulfate solution (sodium dodecyl sulfate, SDS) and the fixed proteins can be identified and quantified by denaturing polyacrylamide gel electrophoresis (SDS-PAGE). In EMSA, a binding reaction with protein and fluorophore-labeled (Cy5) oligonucleotide is set up and incubated. The entire binding mixture is then applied to a native polyacrylamide gel, which typically reveals the eponymous shift of the oligonucleotide bands compared to the G4-protein complex bands. The FID assay (fluorescent intercalator displacement assay, FIDA) and MST (microscale thermophoresis) were used as biophysical methods. For the FIDA, the unmodified oligonucleotide is first incubated with a fluorophore (here acridine orange or thiazole orange) and this mixture is then gradually titrated with increasing concentrations of protein. Here, the successive decrease in fluorescence due to the displacement of the fluorophore by the added protein is recorded. In principle, microscale thermophoresis is also based on the titration of a Cy5-modified oligonucleotide of constant concentration with different protein concentrations. The evaluation is carried out by detecting the change in fluorescence or by quantifying the G4-protein complex formed as a function of the protein concentration used. After carrying out the various binding assays, the respective results for each protein to be characterized were analyzed, compared and classified according to G4 selectivity. It then finally became apparent whether the potential G4-binding proteins from previous proteomic analyzes are in fact G4 binders or whether there are deviations. In the end, a clearly recognizable G4 selectivity could only be seen for some of the proteins examined. Some of the other candidates showed an increased guanine selectivity, some showed a complete lack of selectivity in relation to the nucleic acid structures used.
LB  - PUB:(DE-HGF)11
DO  - DOI:10.18154/RWTH-2022-10395
UR  - https://publications.rwth-aachen.de/record/855732
ER  -