A Mutation‐Based Method for Pinpointing a DNA N6‐Methyladenine Methyltransferase Modification Site at Single Base Resolution

DNA N6‐methyladenine (6mA) has recently received notable attention due to an increased finding of its functional roles in higher eukaryotes. Here we report an enzyme‐assisted chemical labeling method to pinpoint the DNA 6mA methyltransferase (MTase) substrate modification site at single base resolution. A designed allyl‐substituted MTase cofactor was applied in the catalytic transfer reaction, and the allyl group was installed to the N6‐position of adenine within a specific DNA sequence to form N6‐allyladenine (6aA). The iodination of 6aA allyl group induced the formation of 1, N6‐cyclized adenine which caused mutations during DNA replication by a polymerase. Thus the modification site could be precisely detected by a mutation signal. We synthesized 6aA deoxynucleoside and deoxynucleotide model compounds and a 6aA‐containing DNA probe, and screened nine DNA polymerases to define an optimal system capable of detecting the substrate modification site of a DNA 6mA MTase at single‐base resolution. An enzyme‐assisted chemical labeling method to pinpoint the DNA 6mA methyltransferase (MTase) substrate modification site at single‐base resolution is described.