Methyltransferases (MTases) catalyze targeted methyl group transfers from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) to a multitude of biological targets in the cell. Methylation of cytosine and adenine residues in DNA is part of epigenetic regulation of gene function in vertebrates and can often be associated with the occurrence and progression of human disease. We went on to redesign the methyltransferase reactions for targeted transfer of larger chemical moieties by employing synthetic AdoMet analogues in which the sulfonium-bound methyl group is replaced by an extended side chain that typically carries a transferable propargylic moiety, a linear linker and a terminal reporter/functional group. Using a series of such AdoMet analogues and natural or engineered variants of appropriate MTases as targeting vehicles we achieved precise covalent functionalization and labeling of DNA. Practical utility of this labeling platform was demonstrated by single molecule sub-megabase scale genotyping and economical DNA epigenome profiling in vitro and ex vivo. Furthermore, we CRISPR-engineered the Dnmt1 locus in mouse embryonic stem cells and embryonic fibroblasts to install the engineered codons, which, following pulse-internalization of the synthetic cofactor analog AdoX by electroporation, permitted selective azide-tagging of Dnmt1-specific genomic targets in vivo. The deposited chemical groups can be exploited as click handles for precise mapping of the tagged methylation sites in the genomic sequence or spatial visualization in fixed nuclei, enabling selective high-resolution temporal genome-wide tracking of the Dnmt1 catalysis in live mammalian cells (Fig.1). Altogether, these engineered chemically-controllable systems offer unprecedented inroads into deciphering and altering the mammalian epigenetic mechanisms and facilitate the development of next generation diagnostics.

Chemically-controlled enzymatic reactions for targeted derivatization and analysis of DNA in vitro and in live mammalian cells

ACS Spring 2022

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