N6‐methyladenosine (m 6 A) is the most prevalent internal modification in eukaryotic messenger RNA. Dysregulation of m 6 A‐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 m 6 A‐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 (m 6 A)CU‐RNA in the other four human reader proteins YTHDC1 and YTHDF1‐3. Comparative analysis of the binding of fluorescent‐labeled 6mA‐ssDNA and m 6 A‐RNA revealed that the five human m 6 A‐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 m 6 A‐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 m 6 A‐RNA reader proteins.
Bedi et al. (Sat,) studied this question.