Flexible Binding of m⁶A Reader Protein YTHDC1 to Its Preferred RNA Motif

N⁶-Methyladenosine (m⁶A) is the most prevalent chemical modification in human mRNAs. Its recognition by reader proteins enables many cellular functions, including splicing and translation of mRNAs. However, the binding mechanisms of m⁶A-containing RNAs to their readers are still elusive due to the unclear roles of m⁶A-flanking ribonucleotides. Here, we use a model system, YTHDC1 with its RNA motif 5'-G_-2G_-1(m⁶A)C₊₁U₊₂-3', to investigate the binding mechanisms by atomistic simulations, X-ray crystallography, and isothermal titration calorimetry. The experimental data and simulation results show that m⁶A is captured by an aromatic cage of YTHDC1 and the 3' terminus nucleotides are stabilized by cation-π-π interactions, while the 5' terminus remains flexible. Notably, simulations of unbound RNA motifs reveal that the methyl group of m⁶A and the 5' terminus shift the conformational preferences of the oligoribonucleotide to the bound-like conformation, thereby facilitating the association process. The binding mechanisms may help in the discovery of chemical probes against m⁶A reader proteins.