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@PHDTHESIS{Eiden:855732,
author = {Eiden, Sophia Charlotte},
othercontributors = {Weinhold, Elmar and Albrecht, Markus},
title = {{C}harakterisierung der {B}indung humaner {P}roteine an
{G}-{Q}uadruplex-{DNA} und -{RNA}},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
publisher = {RWTH Aachen University},
reportid = {RWTH-2022-10395},
pages = {1 Online-Ressource : Illustrationen, Diagramme},
year = {2022},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University; Dissertation, RWTH Aachen University, 2022},
abstract = {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.},
cin = {152620 / 150000},
ddc = {540},
cid = {$I:(DE-82)152620_20140620$ / $I:(DE-82)150000_20140620$},
typ = {PUB:(DE-HGF)11},
doi = {10.18154/RWTH-2022-10395},
url = {https://publications.rwth-aachen.de/record/855732},
}
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