Evaluation of N6-methyldeoxyadenosine antibody-based genomic profiling in eukaryotes

Low-level DNA N-6-methyldeoxyadenosine (DNA-m6A) has recently been reported across various eukaryotes. Although anti-m6A antibody-based approaches are commonly used to measure DNA-m6A levels, this approach is known to be confounded by DNA secondary structures, RNA contamination, and bacterial contamination. To evaluate for these confounding features, we introduce an approach for systematically validating the selectivity of antibody-based DNA-m6A methods and use a highly selective anti-DNA-m6A antibody to reexamine patterns of DNA-m6A in C. reinhardtii, A. thaliana, and D. melanogaster. Our findings raise caution about the use of antibody-based methods for endogenous m6A quantification and mapping in eukaryotes.

Automated summary

Low-level DNA N-6-methyldeoxyadenosine (DNA-m6A) has been recently discovered in various eukaryotes. However, the commonly used anti-m6A antibody-based methods for measuring DNA-m6A levels are known to be affected by DNA secondary structures, RNA contamination, and bacterial contamination. This study introduces an approach to validate the selectivity of antibody-based DNA-m6A methods and raises caution about their use for endogenous m6A quantification and mapping in eukaryotes.