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.
BT - Chembiochem C1 - 0318 DA - 2026 Mar 27 DO - 10.1002/cbic.202500897 IS - 6 J2 - ChemBioChem LA - eng M3 - Research Article N2 -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.
PY - 2026 EP - e202500897 T2 - Chembiochem TI - A DNA-Based Binding Assay for the m6A-RNA Reader Proteins. UR - https://chemistry-europe.onlinelibrary.wiley.com/doi/10.1002/cbic.202500897 VL - 27 ER -