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Nm-Mut-seq: a base-resolution quantitative method for mapping transcriptome-wide 2′-O-methylation

Cell Research volume 33, pages 727–730 (2023)Cite this article

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Dear Editor,

To develop an RT that is mutagenic at Nm sites, we employed the fluorescence-based RT selection platform we previously developed (RT-PCR-IVT assay) (Supplementary information, Fig. S1a and Data S1).8 Briefly, we utilized a synthetic RNA oligo,9 a 33-mer fluorogenic Broccoli aptamer with an Am15 modification, to select the RT variant capable of incorporating a mutation at Nm site during the reverse transcription reaction. We began with the previously developed RT-1306,8 an evolved variant of the p66 subunit of HIV RT with six mutations (V75F, D76A, F77A, R78K, W229Y and M230L) due to its improved basal Am detection compared to commercially available RTs (Supplementary information, Fig. S1b and Data S2). Iterative rounds of selection yielded our lead RT, the variant RT-41B4, which contains F61S and A62V substitutions in addition to the mutagenic background of RT-1306 (Fig. 1b; Supplementary information, Fig. S1c, d and Data S3). Further in vitro biochemical characterization of purified RT confirmed that RT-41B4 produces higher fluorescence intensity as well as improved mutagenic efficiency than RT-1306 (Fig. 1c; Supplementary information, Fig. S1e, f). Moreover, steady-state kinetics analyses and Sanger sequencing results of RT-41B4 revealed that dATP, rather than dCTP and dGTP, is the optimal nucleotide substrate for misincorporation at Nm sites during reverse transcription reaction (Supplementary information, Fig. S1g, h). After screening a suite of dNTP concentrations (Supplementary information, Fig. S1i), we found that a “restrictive RT condition” — consisting of a low concentration of dNTP (10 μM) and a high concentration of dATP (1 mM) — significantly increases the fluorescence response (Fig. 1d) and generates an over 50% Nm-to-T mutation rate at the modified sites, with no detectable mutation at any of the Nm sites under the “permissive RT condition” (1 mM dNTP) (Fig. 1e). Furthermore, the readthrough efficiency of RT-41B4 under the restrictive condition is considerably higher than that of RT-1306 (Supplementary information, Fig. S1j), indicating the potential application of the evolved RT in mapping Nm modifications.

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Fig. 1: Base-resolution quantitative mapping of transcriptome-wide 2′-O-methylation using Nm-Mut-seq.

Data availability

Sequencing data listed in Supplementary information, Table S10 are available in the Gene Expression Omnibus database under the accession number of GSE174518.

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Acknowledgements

This work was supported by the National Human Genome Research Institute (RM1 HG008935 to C.H. and B.C.D.) and the National Institute of Mental Health (R01 MH122142 to B.C.D.) of the National Institutes of Health (NIH), the University of Chicago Medicine Comprehensive Cancer Center (P30 CA14599) and the international postdoctoral exchange fellowship program (20190048) (L.C.). The evolved RT biochemistry work was partially supported by the National Key R&D Program of China (2018YFA0903200 to C.Z.). C.H. is an investigator at Howard Hughes Medical Institute. We thank Dr. Qing Dai for his help and suggestions on library construction. We thank Dr. Pieter W. Faber in Genomics Facility of the University of Chicago for help with high-throughput sequencing. We thank S. Ahmadiantehrani for editing the manuscript.

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Author notes

  1. Huiqing Zhou

    Present address: Chemistry Department, Boston College, Chestnut Hill, MA, USA

  2. These authors contributed equally: Li Chen, Li-Sheng Zhang, Chang Ye.

Authors and Affiliations

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

    Li Chen, Li-Sheng Zhang, Chang Ye, Huiqing Zhou, Bei Liu, Chuan He & Bryan C. Dickinson

  2. Department of Geriatrics, Zhongnan Hospital of Wuhan University, School of Pharmaceutical Sciences, Wuhan University, Wuhan, Hubei, China

    Li Chen, Zixin Deng & Changming Zhao

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

    Li-Sheng Zhang, Chang Ye, Bei Liu, Boyang Gao & Chuan He

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

    Boyang Gao & Chuan He

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Contributions

B.C.D., C.H. and L.C. conceived and planned the experiments. L.C. performed the RT screening experiments with suggestions from H.Z. L.C. evolved the RT enzyme, optimized RT conditions, carried out NGS library preparations, and characterized Nm methyltransferase. L.S.Z. performed bioinformatic analyses of Nm-Mut-seq data. C.Y. performed computational analyses on calibration curve and snoRNA prediction, with the suggestions from B.L. B.G. performed mass spectrometry measurement. L.C. performed RT enzyme kinetics and Nm site validation analyses under guidance from C.Z. and Z.D. L.C., C.H. and B.C.D. wrote the manuscript with input from all authors.

Corresponding authors

Correspondence to Changming Zhao, Chuan He or Bryan C. Dickinson.

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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, Inc. and AccuraDX Inc., a scientific cofounder and equity holder of Accent Therapeutics, Inc., and a member of the scientific advisory board of Rona Therapeutics. B.C.D. is a founder and holds equity in Tornado Bio, Inc.

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Chen, L., Zhang, LS., Ye, C. et al. Nm-Mut-seq: a base-resolution quantitative method for mapping transcriptome-wide 2′-O-methylation. Cell Res 33, 727–730 (2023). https://doi.org/10.1038/s41422-023-00836-w

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