Development of Site-Specific and Quantitative N6-Methyl Adenosine (m6A) Profiling Methods

N6-methyladenosine (m6A) is the most abundant mRNA modification in mammalian cells, consisting of 0.1%-0.4% of total adenosine residues. m6A regulates mRNA stability and translation, pre-mRNA splicing, miRNA biogenesis, lncRNA binding, and many other physiological and pathological processes. While the majority of m6As occur at a consensus motif of DRm6ACH (D = A/G/U, R = A/G, H = U/A/C), the presence of such motif does not guarantee methylation. Different copies of the same transcript are not uniformly methylated either. Within a single transcript m6As are not evenly distributed, showing an enrichment in long internal exons and terminal exons. These characteristics of m6A deposition calls for sequencing methods that not only pinpoint m6A sites at base resolution, but also quantitates the abundance of methylation across different RNA copies. We developed m6A-SAC-seq (m6A-selective allyl chemical labeling and sequencing) and eTAM-seq (evolved TadA-assisted N6-methyladenosine sequencing), both being site-specific and quantitative m6A profiling methods based on next-generation sequencing. Optimizations were made to the shared library construction strategy, which significantly improved the result and reduced the amount of sample requirement. Both methods were able to identify more than 10-thousands of m6A sites across the transcriptome of HeLa cells, being applicable to various biological systems including cell lines and tissue samples. Efforts had been made to extend both methods to high-throughput single-cell m6A profiling. Finally, a modified version of eTAM-seq was also applied to the study of transcriptome-wide in-vivo accessibility of RNA.