Improved Methods for Deamination-Based m⁶A Detection

N ⁶-methyladenosine (m⁶A) is a critical regulator of gene expression and cellular function. Much of our knowledge of m⁶A has been enabled by the identification of m⁶A sites transcriptome-wide. However, global m⁶A profiling methods require high amounts of input RNA to accurately identify methylated RNAs, making m⁶A profiling from rare cell types or scarce tissue samples infeasible. To overcome this issue, we previously developed DART-seq, which relies on the expression of a fusion protein consisting of the APOBEC1 cytidine deaminase tethered to the m⁶A-binding YTH domain. APOBEC1-YTH directs C-to-U mutations adjacent to m⁶A sites, therefore enabling single nucleotide-resolution m⁶A mapping. Here, we present an improved version of DART-seq which utilizes a variant of the YTH domain engineered to achieve enhanced m⁶A recognition. In addition, we develop in vitro DART-seq and show that it performs similarly to cellular DART-seq and can map m⁶A in any sample of interest using nanogram amounts of total RNA. Altogether, these improvements to the DART-seq approach will enable better m⁶A detection and will facilitate the mapping of m⁶A in samples not previously amenable to global m⁶A profiling.