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

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 ...