Extended Data Fig. 2: TaqαI assay for assessment of modified cytosine deamination by A3A.
From: Direct enzymatic sequencing of 5-methylcytosine at single-base resolution
a) A fluorophore-labelled top-strand is duplexed to a complementary bottom−strand containing a methylated cytosine. The methylated cytosine is represented with a black oval. The substrate is reacted with either WT M.MpeI + SAM, eM.MpeI + bSAM, or M.MpeI N374K + CxSAM. The half-purple/half-orange oval represents a modified cytosine that can either be 5mC, 5bC, or 5cxmC after the action of the MTase variant and SAM analog. The substrate is then deaminated with A3A before duplexing a complement strand. The restriction enzyme TaqαI only cleaves DNA if C is protected from A3A deamination. b) ESI-MS validating generation of 5bC and 5cxmC substrates before A3A reaction. No unmodified C substrate was detected.
