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Published on: July 2, 2010
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A DNA-Based Binding Assay for the m6A-RNA Reader Proteins.
Rajiv Kumar Bedi1, Amedeo Caflisch1
1Department of Biochemistry, University of Zurich, Zurich, Switzerland.
Chembiochem : a European Journal of Chemical Biology
|March 21, 2026
Summary
Researchers determined the crystal structure of the m6A-RNA reader YTHDC2, revealing a similar binding mode for both m6A-RNA and 6mA-DNA. They developed a robust DNA-based assay for high-throughput screening of modulators targeting these reader proteins.
Area of Science:
- Epigenetics and Molecular Biology
- Structural Biology
- Biochemistry
Background:
- N6-methyladenosine (m6A) is a prevalent RNA modification crucial for gene regulation, with its dysregulation linked to various human diseases.
- The role of N6-methyladenosine modifications in DNA (6mA) remains less understood and is an area of active research.
- Understanding the interaction of m6A-RNA reader proteins with modified nucleic acids is vital for deciphering their biological functions.
Purpose of the Study:
- To determine the first holo crystal structure of the m6A-RNA reader YTHDC2 bound to a modified DNA.
- To compare the binding affinities and modes of m6A-RNA and 6mA-DNA with human reader proteins.
- To develop a novel, high-throughput screening assay for identifying modulators of m6A-RNA reader proteins.
Main Methods:
- Holo crystal structure determination of YTHDC2 complexed with a single-strand hexanucleotide GG(6mA)CTA-DNA at 1.6 Å resolution.
- Comparative analysis using fluorescently labeled 6mA-ssDNA and m6A-RNA to assess binding affinities with five human m6A-RNA readers.
- Development and validation of a fluorescence polarization (FP) binding assay utilizing a 6mA-containing ssDNA probe.
Main Results:
- The crystal structure revealed an identical binding mode of 6mA-DNA to YTHDC2 as observed for m6A-RNA in other reader proteins.
- Human m6A-RNA readers exhibited slightly stronger binding affinity for 6mA-modified DNA compared to m6A-RNA, with YTHDC2 showing a 30-fold increase.
- A stable and robust DNA-based FP assay was successfully developed, demonstrating suitability for high-throughput screening.
Conclusions:
- The structural and binding data suggest a conserved interaction mechanism between m6A-RNA readers and both m6A-RNA and 6mA-DNA.
- The developed DNA-based FP assay offers a powerful and cost-efficient platform for discovering small-molecule modulators targeting m6A-RNA reader proteins.
- This work facilitates further investigation into the roles of 6mA-DNA and m6A-RNA signaling in human health and disease.
