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Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling

Nature Chemical Biology volume 10, pages 1049–1054 (2014)Cite this article

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Abstract

Probes that form covalent bonds with RNA molecules on the basis of their chemical reactivity would advance our ability to study the transcriptome. We developed a set of electrophilic activity-based RNA probes designed to react with unusually nucleophilic RNAs. We used these probes to identify reactive genome-encoded RNAs, resulting in the discovery of a 42-nt catalytic RNA from an archaebacterium that reacts with a 2,3-disubstituted epoxide at N7 of a specific guanosine. Detailed characterization of the catalytic RNA revealed the structural requirements for reactivity. We developed this catalytic RNA into a general tool to selectively conjugate a small molecule to an RNA of interest. This strategy enabled up to 500-fold enrichment of target RNA from total mammalian RNA or from cell lysate. We demonstrated the utility of this approach by selectively capturing proteins in yeast cell lysate that bind the ASH1 mRNA.

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Figure 1: Electrophilic probes for the discovery of unusually nucleophilic RNA.
Figure 2: A catalytic RNA from the A. pernix genome that reacts with a disubstituted epoxide.
Figure 3: Epoxide substrate selectivity of the catalytic RNA.
Figure 4: Application of the epoxide-opening catalytic RNA to enrich RNAs of interest from total cellular RNA and to capture RNA-binding proteins.

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Acknowledgements

This work was supported by US National Institutes of Health (NIH)/ National Institute of General Medical Sciences R01 GM065865 and the Howard Hughes Medical Institute. R.I.M. and S.M. were supported by a NIH National Research Service Award Postdoctoral Fellowship (F32GM099359 and F32GM101751). We thank L. Goff for assistance with bioinformatics analysis, D. Engelke (University of Michigan, Ann Arbor) for providing the 5S rRNA plasmid and E. Weerapana (Boston College) for providing the fluorophosphonate probe. We are also grateful to C. Dumelin, A. Leconte, L. McGregor, D. Thompson and D. Usanov for helpful discussions.

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Authors and Affiliations

  1. Department of Chemistry and Chemical Biology, Harvard University, Cambridge, Massachusetts, USA

    Richard I McDonald, John P Guilinger, Edward A Curtis, Won I Lee & David R Liu

  2. Howard Hughes Medical Institute, Harvard University, Cambridge, Massachusetts, USA

    Richard I McDonald, John P Guilinger, Shankar Mukherji, Edward A Curtis, Won I Lee & David R Liu

  3. Department of Molecular and Cellular Biology, Harvard University, Cambridge, Massachusetts, USA

    Shankar Mukherji

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  1. Richard I McDonald
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Contributions

R.I.M. designed the research, prepared materials and performed experiments. J.P.G. and S.M. prepared materials and performed research. E.A.C. and W.I.L. prepared materials. D.R.L. designed and supervised the research. R.I.M. and D.R.L. wrote the manuscript.

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Correspondence to David R Liu.

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Supplementary Results, Supplementary Figures 1–15, Supplementary Tables 1 and 2 and Supplementary Note. (PDF 5739 kb)

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McDonald, R., Guilinger, J., Mukherji, S. et al. Electrophilic activity-based RNA probes reveal a self-alkylating RNA for RNA labeling. Nat Chem Biol 10, 1049–1054 (2014). https://doi.org/10.1038/nchembio.1655

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