Atomic Mutagenesis of N⁶-Methyladenosine Reveals Distinct Recognition Modes by Human m⁶A Reader and Eraser Proteins

N⁶-methyladenosine (m⁶A) is an important modified nucleoside in cellular RNA associated with multiple cellular processes and is implicated in diseases. The enzymes associated with the dynamic installation and removal of m⁶A are heavily investigated targets for drug research, which requires detailed knowledge of the recognition modes of m⁶A by proteins. Here, we use atomic mutagenesis of m⁶A to systematically investigate the mechanisms of the two human m⁶A demethylase enzymes FTO and ALKBH5 and the binding modes of YTH reader proteins YTHDF2/DC1/DC2. Atomic mutagenesis refers to atom-specific changes that are introduced by chemical synthesis, such as the replacement of nitrogen by carbon atoms. Synthetic RNA oligonucleotides containing site-specifically incorporated 1-deaza-, 3-deaza-, and 7-deaza-m⁶A nucleosides were prepared by solid-phase synthesis and their RNA binding and demethylation by recombinant proteins were evaluated. We found distinct differences in substrate recognition and transformation and revealed structural preferences for the enzymatic activity. The deaza m⁶A analogues introduced in this work will be useful probes for other proteins in m⁶A research.