Dysregulation of DNA methylation in eukaryotes, which requires the universal cofactor donor S-adenosyl-L-methionine (SAM), can result in severe alterations in cellular function and often lead to severe diseases, including carcinogenesis. Although there are many techniques to detect global DNA methylation, the development of site-specific DNA methylation identification techniques is still shortcoming. Herein, we report the examination of novel N-mustard analogs of S-adenosyl-L-methionine (SAM) as DNA methylation probe. Critical to the advancement of these analogs has been the functionalization with azides and alkynes capable of undergoing chemoselective ligations. While the successful utility of these N-mustard analogs has been demonstrated with a small panel of prokaryotic DNA methyltransferases (MTases) using a model plasmid DNA, the transition to eukaryotic DNA MTases have proven challenging owing to the simple recognition sequence (CpG) compared to most prokaryotic enzymes. In this work, we seek to validate the utility of our SAM analogs with eukaryotic DNA MTases to demonstrate that N-mustard analogs are transferred to a synthetic DNA substrate. Confirmation of transfer is measured using a fluorescence-based magnetic bead assay where analog-modified oligonucleotides are enriched and fluorescence quantitated following restriction digestion. Following method optimization with M.SssI, the only known prokaryotic DNA MTases with the same specificity as those found in eukaryotes, the fluorescence-based magnetic bead assay was used to evaluate the utility of SAM analogs with eukaryotic DNA methyltransferase1 (DNMT1). Results indicate that the SAM analog modification by DNMT 1 is limited by the location and size of the functional groups within the enzyme active site.

Modification of cytosine-targeted DNA using novel N-mustard analogs of S-Adenosyl-L-Methionine as DNA methylation probes

256th National Meeting (2018)

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