Sequenzspezifische Synthese und Anwendung von kovalenten DNA-Protein- und DNA-Oligonukleotid-Konjugaten

  • Sequence-specific synthesis and application of covalent DNA-protein and DNA-oligonucleotide conjugates

Rauser, Miriam; Weinhold, Elmar (Thesis advisor); Markus, Albrecht (Thesis advisor)

Aachen (2020)
Dissertation / PhD Thesis

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.

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