Figure 1. Direct Methylation Sequencing (DM-Seq) is enabled by 5cxmC generation.

a) Top: Sequencing methods for localizing C, 5mC, and 5hmC differ in their use of chemical (e.g. BS-Seq) or enzymatic (e.g. ACE-Seq) deamination, with 5mC signal confounded by either 5hmC or C (boxes with dashed lines). Bottom: Proposed workflow for DM-Seq. DM-Seq was envisioned as an all-enzymatic workflow for the direct detection of only 5mC. This goal could be realized by coupling an engineered DNA MTase (MTase*) with a SAM analog to create a sterically bulky cytosine base that resists deamination by APOBEC3A (A3A). C*, modified C generated from MTase and SAM analog; 5ghmC, Glucosylated 5hmC. In the proposed workflow, only 5mC alone is converted T at CpG sites. b) MTase variants and SAM analogs including two candidates for DM-Seq. c) Restriction enzyme coupled assay for assessing A3A deamination of unnatural cytosine analogs. An oligonucleotide with a single Taq^αI restriction site (TCGA) is modified by the appropriate MTase variant to create 5mC, 5bC, or 5cxmC (dashed lines). Modified DNA is then deaminated by A3A. Taq^αI only cleaves DNA if C is protected from A3A deamination. Experiment was performed twice with similar results. See Extended Data Fig. 2 for assay schematic and ESI-MS validation of 5bC and 5cxmC substrates. d) Summary of reactivity of various cytosine derivatives towards A3A. Left: Cytosine (C) is modified to 5-methylcytosine (5mC) by WT MTases and SAM. Both C and 5mC are favorable substrates for enzymatic deamination by A3A. Right: structures of 5-(but-2-ynyl)cytosine (5bC) and 5-carboxymethylcytosine (5cxmC), with previously uncharacterized reactivity towards A3A. Box: 5cxmC satisfies the criteria required for the DM-Seq strategy: efficient MTase* transfer and complete protection from A3A deamination.