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Keth-seq for transcriptome-wide RNA structure mapping


RNA secondary structure is critical to RNA regulation and function. We report a new N3-kethoxal reagent that allows fast and reversible labeling of single-stranded guanine bases in live cells. This N3-kethoxal-based chemistry allows efficient RNA labeling under mild conditions and transcriptome-wide RNA secondary structure mapping.

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Fig. 1: N3-kethoxal and experimental evaluation of its selectivity, cell permeability and reversibility.
Fig. 2: Keth-seq method and the profile around rG4 regions.

Data availability

All genomic data sets have been deposited in the Gene Expression Omnibus under accession number GSE122096. Other data and materials are available from the authors upon reasonable request.

Code availability

All custom codes used in this study are available at


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This work was supported by the National Natural Science Foundation of China (grant nos. 21572172, 21778040 and 21822704 to X.W.; 21432008, 91753201 and 21721005 to X.Z.; 31671355, 91740204 and 31761163007 to Q.C.Z.) and National Institutes of Health grant no. HG008935 (C.H.). C.H. is an investigator of the Howard Hughes Medical Institute. X.W. was supported by China Scholarship Council (CSC) during his visit to the University of Chicago. We acknowledge S. Frank, who edited the manuscript.

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Authors and Affiliations



X.W., Q.C.Z., X.Z. and C.H. conceived the project, designed the experiments and wrote the manuscript. X.W., Y.C. and T.W. performed the experiments with the help of F.W., S.Y., Y.Y., G.L., K.C., L.H., H.M. and P.W. J.G. and Q.C.Z. designed and performed the bioinformatics analysis.

Corresponding authors

Correspondence to Qiangfeng Cliff Zhang, Xiang Zhou or Chuan He.

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Competing interests

C.H. is a scientific founder and a member of the scientific advisory board of Accent Therapeutics, Inc., and a shareholder of Epican Genetech.

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Supplementary Figs. 1–15 and Notes 1 and 2.

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Weng, X., Gong, J., Chen, Y. et al. Keth-seq for transcriptome-wide RNA structure mapping. Nat Chem Biol 16, 489–492 (2020).

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