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TY  - THES
AU  - Martin, Michael
TI  - Analoga des S-Adenosyl-l-methionins zur gezielten Markierung von Nukleinsäuren und Proteinen durch Methyltransferasen
CY  - Aachen
PB  - Publikationsserver der RWTH Aachen University
M1  - RWTH-CONV-144731
SP  - XII, 128 S. : Ill.
PY  - 2013
N1  - Aachen, Techn. Hochsch., Diss., 2013
AB  - In the present dissertation new analogues of the natural cofactor S-adenosyl-L-methionine (AdoMet) were synthesized and the activity of DNA- or protein methyl transferases (MTases) were testet in biological experiments. The new cofactor-analogues carry other side chains with functional groups instead of the methyl group. In three cofactor-analogues the sulfonium center was replaced by a selenonium center as well. In total 15 new cofactors with 12 new side chains were manufactured. A stable double-activated cofactor was developed which carries a terminal alkyne moiety that can be transferred sequence-specifically by MTases onto DNA, RNA and proteins. Because of the shortness of the side chain the cofactor is being accepted by a multitude of MTases. The replacement of sulfur against selenium results in a considerable increase of activity since the selenium analogue is inactivated by another and slower mechanism than its sulfur analogue. This is highly advantageous for the two-stage sequence-specific labeling of biomolecules. Aside other analogues of AdoMet with further functional groups (azides, alkenes, amines) which are suitable as well for the two-stage labeling procedure were manufactured. Furthermore a cofactor was synthesized whose transferable group (an aromatic azide) generates a covalent bond with the active site of the transfering DNA-MTase through irradiation of UV light. The goal to produce a covalent MTase-substrate complex with bound cofactor side chain and to provide evidence for it by electro mobility tests was achieved with two DNA-MTases. The yield of the photo crosslinking with this cofactor as well as the acitivity of DNA-MTases is higher-than-average. Cofactors with aromatic side chains are also suitable to elucidate more closely the transition state during the enzymatic modification reaction. In the last section two AdoMet analogues with different linker lengths were developed which transfer a reporter molecule, biotin, in a one-stage process onto DNA. This was possible only in two consecutive steps until this point of time. The cofactors which carry biotin in the side chain werde compared to each other regarding synthesis yield and acitivity with DNA-MTases. The successful transfer of biotin onto DNA was proved in electromobility-shift-assays through the interaction with streptavidin. In principle arbitrary reporter molecules can be attached using click chemistry or the formation of an amide bond respectively so that the library of cofactors for one-stage labeling processes can be extended rapidly.
KW  - DNA-Methyltransferase (SWD)
KW  - Adenosylmethionin (SWD)
KW  - AdoMet (SWD)
KW  - Alkinylierung (SWD)
KW  - Photovernetzung (SWD)
LB  - PUB:(DE-HGF)11
UR  - https://publications.rwth-aachen.de/record/229839
ER  -