Improved Methods for Deamination-Based m6A Detection
Huanyu Zhu, Xinhe Yin, Christopher L. Holley, Kate D. Meyer
Abstract
N 6 -methyladenosine (m 6 A) is a critical regulator of gene expression and cellular function. Much of our knowledge of m 6 A has been enabled by the identification of m 6 A sites transcriptome-wide. However, global m 6 A profiling methods require high amounts of input RNA to accurately identify methylated RNAs, making m 6 A 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 m 6 A-binding YTH domain. APOBEC1-YTH directs C-to-U mutations adjacent to m 6 A sites, therefore enabling single nucleotide-resolution m 6 A mapping. Here, we present an improved version of DART-seq which utilizes a variant of the YTH domain engineered to achieve enhanced m 6 A recognition. In addition, we develop in vitro DART-seq and show that it performs similarly to cellular DART-seq and can map m 6 A in any sample of interest using nanogram amounts of total RNA. Altogether, these improvements to the DART-seq approach will enable better m 6 A detection and will facilitate the mapping of m 6 A in samples not previously amenable to global m 6 A profiling.