Improved Methods for Deamination-Based mA Detection.

-methyladenosine (mA) is a critical regulator of gene expression and cellular function. Much of our knowledge of mA has been enabled by the identification of mA sites transcriptome-wide. However, global mA profiling methods require high amounts of input RNA to accurately identify methylated RNAs, making mA 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 mA-binding YTH domain. APOBEC1-YTH directs C-to-U mutations adjacent to mA sites, therefore enabling single nucleotide-resolution mA mapping. Here, we present an improved version of DART-seq which utilizes a variant of the YTH domain engineered to achieve enhanced mA recognition. In addition, we develop DART-seq and show that it performs similarly to cellular DART-seq and can map mA in any sample of interest using nanogram amounts of total RNA. Altogether, these improvements to the DART-seq approach will enable better mA detection and will facilitate the mapping of mA in samples not previously amenable to global mA profiling.