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.