Detecting and Mapping N6-Methyladenosine on RNA/DNA Hybrids

N6-methyladenosine (m⁶A) is an RNA modification essential for posttranscriptional regulation of gene expression in eukaryotes. We recently demonstrated that m⁶A decorates the RNA components of R-loops, specific nucleic acid structures consisting of an RNA/DNA hybrid and a single strand of non-template DNA, that represent a major source of genetic instability and, at the same time, contribute to regulation of gene expression in mammalian cells. According to growing body of experimental evidence, adenosine methylation affects stability of these structures and potentially influences various aspects of their metabolism. Here, we present two methods for detection and analysis of m⁶A-containing RNA/DNA hybrids: an immunostaining protocol allowing investigation of their spatial distribution in eukaryotic cells and m⁶A-DNA immunoprecipitation (DIP), an antibody-based technique that permits their genome mapping and locus-specific analysis. In addition to the m⁶A-focused studies, these methodologies can also contribute to elucidating the functional roles of other RNA modifications in R-loop biology.