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Small-molecule-catalysed deamination enables transcriptome-wide profiling of N6-methyladenosine in RNA

Nature Chemistry volume 17, pages 1042–1052 (2025)Cite this article

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Abstract

The deamination reaction is important to both fundamental organic chemistry and biochemistry. Traditional chemical methods of deamination rely on the use of aryldiazonium salts under harsh acidic conditions, which limits the application scope for most biological substrates. Here we present an N-nitrosation strategy for deamination under mild conditions that DNA and RNA biological macromolecules can tolerate. Cooperative catalysis combining a carbonyl organocatalyst with a Lewis acid catalyst facilitates the formation of a carbon–nitro intermediate from a primary amine, which, on rearrangement into N-nitrosamine, leads to the selective deamination of unsubstituted canonical DNA/RNA bases under mild conditions. We used this approach to deaminate adenine into hypoxanthine, read as guanine by reverse transcriptases or DNA polymerases, while N6-methyladenosine sites resist deamination and remain identified as adenine. This reactivity enables a chemically mild, low-input detection method for sequencing of adenosine methylation at base resolution, named chemical cooperative catalysis-assisted N6-methyladenosine sequencing.

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Fig. 1: Cooperative deamination of primary amines catalysed by a carbonyl organocatalyst and Lewis acid.
Fig. 2: Deamination of nucleobases catalysed by a organocatalyst combined with a Lewis acid.
Fig. 3: Mechanistic investigations and catalytic cycle.
Fig. 4: Optimization of deamination in oligonucleotides.
Fig. 5: Optimization of the CAM-seq protocol.
Fig. 6: Accurate quantification of m6A modification reveals m6A deposition at the motif level.
Fig. 7: Factors affecting m6A variation.

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Data availability

Sequencing data have been deposited in the Gene Expression Omnibus (GEO) with the following accession numbers: GSE268871 for the human samples, GSE268872 for the maize samples and GSE268873 for the Arabidopsis samples. All other data are available in the paper or the Supplementary Information. Source data are provided with this paper.

Code availability

Reads mapping and m6A sites detection scripts are available at GitHub via https://github.com/y9c/m6A-CAMseq (ref. 33).

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Acknowledgements

We thank Y. Ge, Y. Gao and Y. Xiao for discussions. We thank the Genomics Facility of the University of Chicago and the University of Chicago Comprehensive Cancer Center DNA Sequencing and Genotyping Facility for assistance with sequencing. This study was supported by the National Institutes of Health (grant HG008935, C.H.). C.H. is an investigator of the Howard Hughes Medical Institute.

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Author notes

  1. These authors contributed equally: Pingluan Wang, Chang Ye, Michelle Zhao.

Authors and Affiliations

  1. Department of Chemistry, The University of Chicago, Chicago, IL, USA

    Pingluan Wang, Chang Ye, Michelle Zhao, Bochen Jiang & Chuan He

  2. Howard Hughes Medical Institute, The University of Chicago, Chicago, IL, USA

    Pingluan Wang, Chang Ye, Bochen Jiang & Chuan He

  3. USA Institute for Biophysical Dynamics, The University of Chicago, Chicago, IL, USA

    Chuan He

  4. Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, IL, USA

    Chuan He

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Contributions

C.H., P.W. and C.Y. conceived the original idea and designed the original studies; P.W. performed most of the experiments with help from M.Z. and B.J; C.Y. performed most of the bioinformatics analyses with input from P.W; B.J. provided the plant materials. All authors approved the final paper.

Corresponding author

Correspondence to Chuan He.

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Competing interests

C.H. is a scientific founder, a member of the scientific advisory board and equity holder of Aferna Bio and Ellis Bio, a scientific cofounder and equity holder of Accent Therapeutics, and a member of the scientific advisory board of Rona Therapeutics and Element Biosciences. The other authors declare no competing interests.

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Nature Chemistry thanks Thomas Carell and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

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Supplementary Materials and Methods, Figs. 1–18, Tables 1–3, Notes I and II, and references.

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Source Data Fig. 4

Statistical source data.

Source Data Fig. 5

Unprocessed gels.

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Wang, P., Ye, C., Zhao, M. et al. Small-molecule-catalysed deamination enables transcriptome-wide profiling of N6-methyladenosine in RNA. Nat. Chem. 17, 1042–1052 (2025). https://doi.org/10.1038/s41557-025-01801-3

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