Kupplung der DNA-Methyltransferase M.SssI mit Triplehelix-bildenden Oligodesoxynucleotiden
DNA modifying enzymes, like DNA methyltransferases (DNA MTases) and restriction endonucleases (REases), could in principle be used to map or manipulate genomes. However, these enzymes are inappropriate for that purpose because of their generally short recognition sequences. An increase of their sequence specificity would therefore be desirable. One strategy to produce megaspecific DNA modifying enzymes is to couple them with triple helix forming oligodeoxynucleotide (TFO). These additional DNA recognition units should address the enzymes only to predetermined DNA recognition sequences and result in an increase in their sequence specificity. In this research, the DNA cytosin-C5-MTase M.SssI from Spiroplasma sp. strain MQ1 was coupled with TFO. M.SssI is the only known prokaryotic DNA cytosin-C5-MTase, which methylates cytosin within the recognition sequence 5' CG-3' like mammalian DNA-MTases. Such a megaspecific M.SssI could then be used to silence specific genes. The fact that the EpCAM gene is overexpressed in a variety of carcinome and is involved in carcinogenesis makes it an interesting target. In the first phase of this research, M.SssI was coupled with TFO by native chemical peptide ligation (NCPL), thiazolidine ligation, expressed protein ligation (EPL) and heterobifunctional crosslinking. The different methods were optimised and compared with one another. Furthermore, the variant M.SssI-C141S, which doesn’t possess a catalytic cysteine residue but still shows a residual DNA MTase activity of approximately 0,3%, was coupled with a TFO, which was specifically designed for the EpCAM promotor. The produced M.SssI-TFO conjugate was purified to more than 95% by anion exchange chromatography. In a second phase, the group of Dr. P. McLaughlin demonstrated the profection of M.SssI in the cell nucleus with a cationic complex formed of N-Methyl-4-(dioleoyl)methyl-pyridinium-chlorid (SAINT-2) and 1,2-Dioleoyl-sn-glycero-3-phosphoethanolamin (DOPE) .
info:eu-repo/classification/ddc/540, Chemie, DNS-Methyltransferase, M.SssI, Triplehelix, Oligodesoxynucleotid, triple helix, oligodeoxynucleotide
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