Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine

Abstract

The inert chemical property of RNA modification N6-methyladenosine (m6A) makes it very challenging to detect. Most m6A sequencing methods rely on m6A-antibody immunoprecipitation and cannot distinguish m6A and N6,2′-O-dimethyladenosine modification at the cap +1 position (cap m6Am). Although the two antibody-free methods (m6A-REF-seq/MAZTER-seq and DART-seq) have been developed recently, they are dependent on m6A sequence or cellular transfection. Here, we present an antibody-free, FTO-assisted chemical labeling method termed m6A-SEAL for specific m6A detection. We applied m6A-SEAL to profile m6A landscapes in humans and plants, which displayed the known m6A distribution features in transcriptome. By doing a comparison with all available m6A sequencing methods and specific m6A sites validation by SELECT, we demonstrated that m6A-SEAL has good sensitivity, specificity and reliability for transcriptome-wide detection of m6A. Given its tagging ability and FTO’s oxidation property, m6A-SEAL enables many applications such as enrichment, imaging and sequencing to drive future functional studies of m6A and other modifications.

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Fig. 1: FTO-assisted selective chemical labeling of m6A.
Fig. 2: m6A-SEAL on model RNA and human poly(A)+ RNA.
Fig. 3: m6A-SEAL-seq uncovers the transcriptome-wide m6A methylome in HEK293T cells.
Fig. 4: Comparison of m6A methylome in human HEK293T cells and rice detected by m6A-SEAL-seq and all available m6A-seq methods.

Data availability

Sequencing data have been deposited in GSE129979.

Code availability

Custom Bash, Perl and R codes used for data analysis are available at https://github.com/WYeast.

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Acknowledgements

We acknowledge X. Lei for discussing the thiol-addition reaction mechanism and S. Liu, J. Meng and H. Liu for helping with data analysis. This work was supported by the National Basic Research Program of China (grant nos. 2019YFA0802201 and 2017YFA0505201), the National Natural Science Foundation of China (nos. 21822702, 21820102008 and 21432002) and the State Key Laboratory of Drug Research.

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G.J. and Y.W. conceived the project and designed the experiments. Y.W. performed the experiments and data analysis with the help of Y.X., S.D. and Q.Y. G.J. and Y.W. wrote the manuscript.

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Correspondence to Guifang Jia.

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A patent application has been filed by Peking University for the technology disclosed in this publication.

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Supplementary Table 1–3, Figs. 1–25 and Notes 1–3.

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Supplementary Dataset 1

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Wang, Y., Xiao, Y., Dong, S. et al. Antibody-free enzyme-assisted chemical approach for detection of N6-methyladenosine. Nat Chem Biol (2020). https://doi.org/10.1038/s41589-020-0525-x

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