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Light-activated chemical probing of nucleobase solvent accessibility inside cells

Nature Chemical Biology volume 14, pages 276–283 (2018)Cite this article

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An Erratum to this article was published on 14 February 2018

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Abstract

The discovery of functional RNAs that are critical for normal and disease physiology continues to expand at a breakneck pace. Many RNA functions are controlled by the formation of specific structures, and an understanding of each structural component is necessary to elucidate its function. Measuring solvent accessibility intracellularly with experimental ease is an unmet need in the field. Here, we present a novel method for probing nucleobase solvent accessibility, Light Activated Structural Examination of RNA (LASER). LASER depends on light activation of a small molecule, nicotinoyl azide (NAz), to measure solvent accessibility of purine nucleobases. In vitro, this technique accurately monitors solvent accessibility and identifies rapid structural changes resulting from ligand binding in a metabolite-responsive RNA. LASER probing can further identify cellular RNA–protein interactions and unique intracellular RNA structures. Our photoactivation technique provides an adaptable framework to structurally characterize solvent accessibility of RNA in many environments.

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Figure 1: Characterizing the reactivity of NAz.
Figure 2: Reactivity of NAz with SAM-I RNA.
Figure 3: Differential structure probing mapped onto the SAM-I crystal structure.
Figure 4: LASER probing inside living cells.
Figure 5: LASER probing of the U1 snRNP inside living cells.

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  • 22 January 2018

    In the version of this article initially published online, the submission date was incorrectly stated as 21 June 2016. The correct date is 15 June 2017. The error has been corrected in the PDF and HTML versions of this article.

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Acknowledgements

We thank members of the Spitale lab for their careful reading and critique of the manuscript. The Spitale lab is supported by startup funds from the University of California, Irvine, and the NIH (1DP2GM119164 RCS) and 1RO1MH109588 (RCS). F.F. and the computational work are supported by the US Department of Energy under Award DE-SC0008694. C.M.H. acknowledges financial support from the National Science Foundation (DMR-1212842 and CHE-1609889), as well as generous allocations of computational resources at the Ohio Supercomputer Center. Femtosecond TRIR experiments were performed at The Ohio State University's Center for Chemical and Biophysical Dynamics.

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Author notes

  1. Chao Feng and Dalen Chan: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, University of California, Irvine, Irvine, California, USA

    Chao Feng, Dalen Chan & Robert C Spitale

  2. Department of Chemistry and Biochemistry, Ohio State University, Columbus, Ohio, USA

    Jojo Joseph, William H Coldren & Christopher M Hadad

  3. Department of Chemistry, University of California, Irvine, Irvine, California, USA

    Mikko Muuronen, Filipp Furche & Robert C Spitale

  4. New England BioLabs, Ipswich, Massachusetts, USA

    Nan Dai & Ivan R Corrêa Jr

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Contributions

C.F. performed synthesis of the NAz probe and all positive controls, and did all analysis to determine reactivity. D.C. performed all RNA structure probing experiments. J.J. performed TRIR experiments with help from W.H.C. and C.M.H. M.M. performed density functional theory calculations and analyzed the results with C.F. and F.F. N.D. and I.R.C. assisted with HPLC and LC–MS experiments. J.J., W.H.C., and C.M.H. performed the CASSCF and B3LYP/6-31+G** using Gaussian09. C.F. and D.C. wrote the manuscript with help from R.C.S. All authors read and helped finalize the manuscript before submission.

Corresponding author

Correspondence to Robert C Spitale.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–8, Supplementary Figures 1–16 (PDF 2533 kb)

Life Sciences Reporting Summary (PDF 128 kb)

Supplementary Note 1 (PDF 1303 kb)

Supplementary Note 2 (PDF 393 kb)

Supplementary Data Set 1

Cartesian Coordinates of Structures presented in Supplementary Figures 1 and 2. (XLSX 69 kb)

Supplementary Data Set 2

Cartesian Coordinates of Structures presented in Supplementary Figures 3–6. (XLSX 47 kb)

Supplementary Data Set 3

LASER (+SAM) and corresponding solvent accessibility measurements at C8. Values are in A2. Solvent was contoured to 2 Å. (XLSX 44 kb)

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Feng, C., Chan, D., Joseph, J. et al. Light-activated chemical probing of nucleobase solvent accessibility inside cells. Nat Chem Biol 14, 276–283 (2018). https://doi.org/10.1038/nchembio.2548

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