A DNA‐Based Binding Assay for the m ⁶ A‐RNA Reader Proteins

N6-methyladenosine (m⁶A) is the most prevalent internal modification in eukaryotic messenger RNA. Dysregulation of m⁶A-RNA signaling has been implicated in a wide range of human diseases. The N6-methyladenosine modifications in DNA (6mA) is much rarer, and its role is still debated. Here, we report the first holo crystal structure of the m⁶A-RNA reader YTHDC2. The 1.6 Å resolution structure of YTHDC2 bound to the single-strand (ss) hexanucleotide GG(6mA)CTA-DNA shows an essentially identical binding mode of (6mA)CT-DNA as (m⁶A)CU-RNA in the other four human reader proteins YTHDC1 and YTHDF1-3. Comparative analysis of the binding of fluorescent-labeled 6mA-ssDNA and m⁶A-RNA revealed that the five human m⁶A-RNA readers exhibit slightly stronger binding affinity for 6mA-modified DNA with a factor ranging from about 1.3 for YTHDC1 to 30 for YTHDC2. Given the similar affinity and the similar binding mode of 6mA-ssDNA and m⁶A-RNA, we set up to develop a fluorescence polarization (FP) binding assay that uses a fluorescent-labeled 6mA-containing ssDNA as probe. The DNA-based FP assay shows high stability and robustness, making it suitable for high-throughput screening applications. The assay provides a powerful and cost-efficient platform to accelerate the discovery of small-molecule modulators targeting m⁶A-RNA reader proteins.