DART-seq: an antibody-free method for global m⁶A detection.

Abstract

N⁶-methyladenosine (m⁶A) is a widespread RNA modification that influences nearly every aspect of the messenger RNA lifecycle. Our understanding of m⁶A has been facilitated by the development of global m⁶A mapping methods, which use antibodies to immunoprecipitate methylated RNA. However, these methods have several limitations, including high input RNA requirements and cross-reactivity to other RNA modifications. Here, we present DART-seq (deamination adjacent to RNA modification targets), an antibody-free method for detecting m⁶A sites. In DART-seq, the cytidine deaminase APOBEC1 is fused to the m⁶A-binding YTH domain. APOBEC1-YTH expression in cells induces C-to-U deamination at sites adjacent to m⁶A residues, which are detected using standard RNA-seq. DART-seq identifies thousands of m⁶A sites in cells from as little as 10 ng of total RNA and can detect m⁶A accumulation in cells over time. Additionally, we use long-read DART-seq to gain insights into m⁶A distribution along the length of individual transcripts.