Carbamoyltransferase Enzyme Assay: In vitro Modification of 5-hydroxymethylcytosine (5hmC) to 5-carbamoyloxymethylcytosine (5cmC)

Abstract

Nucleic acids in living organisms are more complex than the simple combinations of the four canonical nucleotides. Recent advances in biomedical research have led to the discovery of numerous naturally occurring nucleotide modifications and enzymes responsible for the synthesis of such modifications. In turn, these enzymes can be leveraged towards toolkits for DNA and RNA manipulation for epigenetic sequencing or other biotechnological applications. Here, we present the protocol to obtain purified 5-hydroxymethylcytosine carbamoyltransferase enzymes and the associated assays to convert 5-hydroxymethylcytosine to 5-carbamoyloxymethylcytosine in vitro. We include detailed assays using DNA, RNA, and single nucleotide/deoxynucleotide as substrates. These assays can be combined with downstream applications for genetic/epigenetic regulatory mechanism studies and next-generation sequencing purposes.