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@PHDTHESIS{Rauser:789863,
author = {Rauser, Miriam},
othercontributors = {Weinhold, Elmar and Albrecht, Markus},
title = {{S}equenzspezifische {S}ynthese und {A}nwendung von
kovalenten {DNA}-{P}rotein- und
{DNA}-{O}ligonukleotid-{K}onjugaten},
school = {RWTH Aachen University},
type = {Dissertation},
address = {Aachen},
reportid = {RWTH-2020-05500},
pages = {1 Online-Ressource (V, 132 Seiten) : Illustrationen,
Diagramme},
year = {2020},
note = {Veröffentlicht auf dem Publikationsserver der RWTH Aachen
University; Dissertation, RWTH Aachen University, 2020},
abstract = {DNA-protein conjugates can be used in several fields of
molecular diagnostics and biotechnology by combination of
structural or encoded abilities of DNA with versatile
functionality of proteins. Methyltransferase-directed
transfer of activated groups (mTAG) method was combined with
the SNAP-tag technology for the synthesis of such
sequence-specific covalent DNA-protein conjugates.
Therefore, long DNA was sequence-specifically modified with
the O6-benzylguanine (BG) side chain of a cofactor AdoYnBG35
by DNA methyltransferase (DNA MTase) in the first step.
Through SNAP-tag technology BG modified DNA could be labeled
with different SNAP-tag fusion proteins in the second step.
The SNAP-tag is a mutant of the human O6-alkylguanine DNA
alkyl transferase and can be genetically fused to any DNA
MTases or fluorescent proteins. It reacts specifically with
different BG substrates.An application of covalent
DNA-protein conjugates deals with targeted DNA methylation
and targeted DNA labeling. As a result of a covalent bond of
DNA cytosine-C5 MTase SNAP-M.MpeI to one position on plasmid
DNA Litcon30, SNAP-M.MpeI was only able to methylate
recognition sequences near the covalent binding site. The
comparison of the three plasmid DNA conformations (linear,
nicked and supercoiled) revealed that targeted DNA
methylation increased, and unspecific DNA methylation
decreased from supercoiled to nicked to linear. That can be
explained by a more compact structure of the supercoiled
form allowing the covalently bound SNAP-M.MpeI not only to
methylate nearby sites, but also methylate sites far away in
sequence but close in space. On this basis, targeted DNA
labeling included the transfer of a fluorophore in the side
chain of cofactor AdoYnTAMRA to DNA instead of a methyl
group. Linear T7 DNA was three times covalently labeled with
DNA adenine-N6 MTase SNAP-M.TaqI. Only one of the covalently
bound SNAP-M.TaqI was able to reach and to label three
recognition sequences with the fluorophore. The use of
fluorophores and T7 DNA, which is many times longer than
plasmid DNA, enabled the analysis with a fluorescence
microscope. Targeted DNA labeling of the TaqI sequences,
which were close to the SNAP-M.TaqI binding site, could be
statistically verified through fluorescence images. The T7
DNA was visualized by a DNA intercalator.Plasmid DNA pUC19
and T7 DNA could be also sequence-specific labeled by
fluorescent protein due to covalent conjugate synthesis
using a fusion protein made of SNAP-tag and the yellow
fluorescent protein (YFP). DNA MTases cytosine-C5 M.HhaI and
adenine-N6 M.TaqI were used for BG modification. The
SNAP-YFP labeled HhaI sequences on pUC19 was verified by
electro mobility shift assays (EMSA). T7 DNA has many TaqI
sequences spread over the whole strand, resulting in
SNAP-YFP fluorescence over the whole strand. Thus,
fluorescence images of T7 DNA with SNAP-YFP labeled TaqI
sequences showed T7 DNA without any DNA intercalator. The
sequence pattern of TaqI was mostly verified by fluorescent
SNAP-YFP. The development of a method for CpG methylation
detection is promising because CpG methylation in mammalian
plays an important part in gene expression. Moreover, there
is no restriction enzyme, which recognizes the dinucleotide
CpG. DNA oligonucleotide (ODN) conjugates were used for this
method. These conjugates were synthesized by
sequence-specific transfer of a ODN side chain to plasmid
DNA pBR322 by CpG sensitive DNA adenine-N6 MTase M.TaqI.
M.TaqI recognize the sequence TCGA. ODN modified pBR322 were
then sequenced by Sanger sequencing. DNA polymerase
synthesized the complement strand during the Sanger
sequencing until it reached the ODN labeled sequence of
M.TaqI. The ODN blocked further synthesis, which led to a
decreased intensity in the sequencing data after the TaqI
sequence. Using three ODN side chains with two, eight or
eleven nucleotides, the longer side chains ODN8 and ODN11
resulted in a stronger decline in intensity than ODN2. If
the cytosine within the TaqI sequence is methylated, M.TaqI
is not able to transfer a ODN side chain to adenine.
Therefore, no decreased intensity could be detected in the
sequencing data and the CpG methylation is confirmed.},
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-2020-05500},
url = {https://publications.rwth-aachen.de/record/789863},
}
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