Figure 2. The challenge of asymmetric DNA carboxymethylation is overcome with copy-strand synthesis enabled by 5pyC adapters.

a) Lambda gDNA sequencing experiment. Lambda gDNA was incubated with WT M.MpeI or M.MpeI N374K in the presence of no SAM, SAM, or CxSAM. DNA was treated with bisulfite (BS) before PCR and Illumina sequencing (n=2 independent experiments). Data are presented as mean values +/− SD. b) Scatter plot showing relationship between CpGs on opposite strands within the same dyad. Data is filtered for CpGs with at least 5 sequencing reads.$$c) Top: Oligonucleotide modification assay (see Extended Data Fig. 3). An oligonucleotide with a labelled top-strand containing an unmodified CpG across from either a 5mCpG or unmodified CpG was reacted with M.MpeI N374K and no SAM, SAM, or CxSAM. Shown is the denaturing gel after digestion with a modification-sensitive restriction enzyme that reports on the modification status of the top strand. Experiment was performed twice with similar results. Bottom: Model for incomplete transfer. Symmetrical modification proceeds readily with SAM but is slow with CxSAM due to inefficient transfer across from a 5cxmC. Carboxymethylation is efficient when the opposite strand contains a 5mC. d) Left: Envisioned scheme where A3A-resistant adapters (purple) initiate universal copy strand synthesis. A copy strand (dotted line) containing 5mCs serves as a favorable substrate for DNA carboxymethylation. Right: Structures of unnatural cytosine analogs explored. The 5-position modifications are anticipated to interact with the steric gate Tyr-130 residue in A3A (orange) (see Extended Data Fig. 4). e) Left: PCR product is generated using modified-dCTPs in place of dCTP. dsDNA is deaminated, PCR amplified, and restriction digested to analyze deamination status at a single TCGA Taq^αI site. Right: Deep sequencing of same PCR products as in gel (n=2). f) Scatter plot as in b) showing improvement of carboxymethylation with copy strand synthesis. Data corresponds to Extended Data Fig. 6. g) Full DM-Seq workflow. Sheared gDNA is end-prepped and adapted to A3A resistant 5pyC adapters. A copy strand made with 5mCTPs is synthesized before glucosylation and carboxymethylation. A3A deaminates 5mCpGs to Ts which can be detected upon PCR amplification. Box: 5pyC adapters are synthetically accessible and permissive for A3A-dependent sequencing.