Light-activated chemical probing of nucleobase solvent accessibility inside cells

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.

References

  1. 1

    Mattick, J.S. The functional genomics of noncoding RNA. Science 309, 1527–1528 (2005).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  2. 2

    Chappell, J. et al. The centrality of RNA for engineering gene expression. Biotechnol. J. 8, 1379–1395 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  3. 3

    Sharp, P.A. The centrality of RNA. Cell 136, 577–580 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  4. 4

    Tijerina, P., Mohr, S. & Russell, R. DMS footprinting of structured RNAs and RNA-protein complexes. Nat. Protoc. 2, 2608–2623 (2007).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  5. 5

    Wilkinson, K.A., Merino, E.J. & Weeks, K.M. Selective 2′-hydroxyl acylation analyzed by primer extension (SHAPE): quantitative RNA structure analysis at single nucleotide resolution. Nat. Protoc. 1, 1610–1616 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  6. 6

    McGinnis, J.L., Duncan, C.D. & Weeks, K.M. High-throughput SHAPE and hydroxyl radical analysis of RNA structure and ribonucleoprotein assembly. Methods Enzymol. 468, 67–89 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  7. 7

    Tullius, T.D. & Greenbaum, J.A. Mapping nucleic acid structure by hydroxyl radical cleavage. Curr. Opin. Chem. Biol. 9, 127–134 (2005).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  8. 8

    Adilakshmi, T., Lease, R.A. & Woodson, S.A. Hydroxyl radical footprinting in vivo: mapping macromolecular structures with synchrotron radiation. Nucleic Acids Res. 34, e64 (2006).

    PubMed  PubMed Central  Article  CAS  Google Scholar 

  9. 9

    Ramaswamy, P. & Woodson, S.A. S16 throws a conformational switch during assembly of 30S 5′ domain. Nat. Struct. Mol. Biol. 16, 438–445 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  10. 10

    Batey, R.T., Rambo, R.P. & Doudna, J.A. Tertiary motifs in RNA structure and folding. Angew. Chem. Int. Ed. Engl. 38, 2326–2343 (1999).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  11. 11

    Moras, D. & Poterszman, A. Getting into the major groove. Protein-RNA interactions. Curr. Biol. 6, 530–532 (1996).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  12. 12

    Varani, L., Spillantini, M.G., Goedert, M. & Varani, G. Structural basis for recognition of the RNA major groove in the tau exon 10 splicing regulatory element by aminoglycoside antibiotics. Nucleic Acids Res. 28, 710–719 (2000).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  13. 13

    Chen, L. & Frankel, A.D. A peptide interaction in the major groove of RNA resembles protein interactions in the minor groove of DNA. Proc. Natl. Acad. Sci. USA 92, 5077–5081 (1995).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  14. 14

    Lawley, P.D. & Brookes, P. Further studies on the alkylation of nucleic acids and their constituent nucleotides. Biochem. J. 89, 127–138 (1963).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. 15

    Lee, B. et al. Comparison of SHAPE reagents for mapping RNA structures inside living cells. RNA 23, 169–174 (2017).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  16. 16

    Klán, P. et al. Photoremovable protecting groups in chemistry and biology: reaction mechanisms and efficacy. Chem. Rev. 113, 119–191 (2013).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  17. 17

    Baker, A.S. & Deiters, A. Optical control of protein function through unnatural amino acid mutagenesis and other optogenetic approaches. ACS Chem. Biol. 9, 1398–1407 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  18. 18

    Song, C.-X. & He, C. Bioorthogonal labeling of 5-hydroxymethylcytosine in genomic DNA and diazirine-based DNA photo-cross-linking probes. Acc. Chem. Res. 44, 709–717 (2011).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  19. 19

    Wild, D. A novel pathway to the ultimate mutagens of aromatic amino and nitro compounds. Environ. Health Perspect. 88, 27–31 (1990).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  20. 20

    Xue, J., Du, L., Zhu, R., Huang, J. & Phillips, D.L. Direct time-resolved spectroscopic observation of arylnitrenium ion reactions with guanine-containing DNA oligomers. J. Org. Chem. 79, 3610–3614 (2014).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  21. 21

    Kuska, M.S. et al. Structural influence of C8-phenoxy-guanine in the NarI recognition DNA sequence. Chem. Res. Toxicol. 26, 1397–1408 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  22. 22

    Voskresenska, V. et al. Photoaffinity labeling via nitrenium ion chemistry: protonation of the nitrene derived from 4-amino-3-nitrophenyl azide to afford reactive nitrenium ion pairs. J. Am. Chem. Soc. 131, 11535–11547 (2009).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  23. 23

    Loeb, L.A. & Harris, C.C. Advances in chemical carcinogenesis: a historical review and prospective. Cancer Res. 68, 6863–6872 (2008).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  24. 24

    Brachet, E., Ghosh, T., Ghosh, I. & Konig, B. Visible light C-H amidation of heteroarenes with benzoyl azides. Chem. Sci. 6, 987–992 (2015).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  25. 25

    Kubicki, J. et al. Direct observation of acyl azide excited states and their decay processes by ultrafast time resolved infrared spectroscopy. J. Am. Chem. Soc. 131, 4212–4213 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  26. 26

    Spitale, R.C. et al. RNA SHAPE analysis in living cells. Nat. Chem. Biol. 9, 18–20 (2013).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  27. 27

    Desikan, V., Liu, Y., Toscano, J.P. & Jenks, W.S. Photochemistry of sulfilimine-based nitrene precursors: generation of both singlet and triplet benzoylnitrene. J. Org. Chem. 72, 6848–6859 (2007).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  28. 28

    Humphreys, W.G., Kadlubar, F.F. & Guengerich, F.P. Mechanism of C8 alkylation of guanine residues by activated arylamines: evidence for initial adduct formation at the N7 position. Proc. Natl. Acad. Sci. USA 89, 8278–8282 (1992).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  29. 29

    Reha, D. et al. Intercalators. 1. Nature of stacking interactions between intercalators (ethidium, daunomycin, ellipticine, and 4′,6-diaminide-2-phenylindole) and DNA base pairs. Ab initio quantum chemical, density functional theory, and empirical potential study. J. Am. Chem. Soc. 124, 3366–3376 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  30. 30

    Wentrup, C., Reisinger, A. & Kvaskoff, D. 4-Pyridylnitrene and 2-pyrazinylcarbene. Beilstein J. Org. Chem. 9, 754–760 (2013).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  31. 31

    Montange, R.K. & Batey, R.T. Structure of the S-adenosylmethionine riboswitch regulatory mRNA element. Nature 441, 1172–1175 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  32. 32

    Winkler, W.C., Nahvi, A., Sudarsan, N., Barrick, J.E. & Breaker, R.R. An mRNA structure that controls gene expression by binding S-adenosylmethionine. Nat. Struct. Biol. 10, 701–707 (2003).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  33. 33

    Hennelly, S.P. & Sanbonmatsu, K.Y. Tertiary contacts control switching of the SAM-I riboswitch. Nucleic Acids Res. 39, 2416–2431 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  34. 34

    Heppell, B. et al. Molecular insights into the ligand-controlled organization of the SAM-I riboswitch. Nat. Chem. Biol. 7, 384–392 (2011).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  35. 35

    Stoddard, C.D. et al. Free state conformational sampling of the SAM-I riboswitch aptamer domain. Structure 18, 787–797 (2010).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  36. 36

    Mortimer, S.A., Johnson, J.S. & Weeks, K.M. Quantitative analysis of RNA solvent accessibility by N-silylation of guanosine. Biochemistry 48, 2109–2114 (2009).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  37. 37

    Adams, P.L. et al. Crystal structure of a group I intron splicing intermediate. RNA 10, 1867–1887 (2004).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  38. 38

    Kubota, M., Tran, C. & Spitale, R.C. Progress and challenges for chemical probing of RNA structure inside living cells. Nat. Chem. Biol. 11, 933–941 (2015).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  39. 39

    Khatter, H., Myasnikov, A.G., Natchiar, S.K. & Klaholz, B.P. Structure of the human 80S ribosome. Nature 520, 640–645 (2015).

    CAS  PubMed  Article  Google Scholar 

  40. 40

    So, B.R. et al. A U1 snRNP-specific assembly pathway reveals the SMN complex as a versatile hub for RNP exchange. Nat. Struct. Mol. Biol. 23, 225–230 (2016).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  41. 41

    Du, H. & Rosbash, M. The U1 snRNP protein U1C recognizes the 5′ splice site in the absence of base pairing. Nature 419, 86–90 (2002).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  42. 42

    Kondo, Y., Oubridge, C., van Roon, A.M. & Nagai, K. Crystal structure of human U1 snRNP, a small nuclear ribonucleoprotein particle, reveals the mechanism of 5′ splice site recognition. eLife 4, e04986 (2015).

    PubMed Central  Article  Google Scholar 

  43. 43

    McConnell, T.S., Lokken, R.P. & Steitz, J.A. Assembly of the U1 snRNP involves interactions with the backbone of the terminal stem of U1 snRNA. RNA 9, 193–201 (2003).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  44. 44

    Das, R., Laederach, A., Pearlman, S.M., Herschlag, D. & Altman, R.B. SAFA: semi-automated footprinting analysis software for high-throughput quantification of nucleic acid footprinting experiments. RNA 11, 344–354 (2005).

    CAS  PubMed  PubMed Central  Article  Google Scholar 

  45. 45

    Tao, J., Perdew, J.P., Staroverov, V.N. & Scuseria, G.E. Climbing the density functional ladder: nonempirical meta-generalized gradient approximation designed for molecules and solids. Phys. Rev. Lett. 91, 146401 (2003).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  46. 46

    Weigend, F. & Ahlrichs, R. Balanced basis sets of split valence, triple zeta valence and quadruple zeta valence quality for H to Rn: Design and assessment of accuracy. Phys. Chem. Chem. Phys. 7, 3297–3305 (2005).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  47. 47

    Schäfer, A., Horn, H. & Ahlrichs, R. Fully optimized contracted Gaussian basis sets for atoms Li to Kr. J. Chem. Phys. 97, 2571–2577 (1992).

    Article  Google Scholar 

  48. 48

    Schäfer, A., Huber, C. & Ahlrichs, R. Fully optimized contracted Gaussian basis sets of triple zeta valence quality for atoms Li to Kr. J. Chem. Phys. 100, 5829–5835 (1994).

    Article  Google Scholar 

  49. 49

    Eichkorn, K., Weigend, F., Treutler, O. & Ahlrichs, R. Auxiliary basis sets for main row atoms and transition metals and their use to approximate Coulomb potentials. Theor. Chem. Acc. 97, 119–124 (1997).

    CAS  Article  Google Scholar 

  50. 50

    Weigend, F., Furche, F. & Ahlrichs, R. Gaussian basis sets of quadruple zeta valence quality for atoms H–Kr. J. Chem. Phys. 119, 12753–12762 (2003).

    CAS  Article  Google Scholar 

  51. 51

    Grimme, S., Antony, J., Ehrlich, S. & Krieg, H. A consistent and accurate ab initio parametrization of density functional dispersion correction (DFT-D) for the 94 elements H-Pu. J. Chem. Phys. 132, 154104 (2010).

    PubMed  Article  CAS  PubMed Central  Google Scholar 

  52. 52

    Schäfer, A., Klamt, A., Sattel, D., Lohrenz, J.C. & Eckert, F. COSMO Implementation in TURBOMOLE: Extension of an efficient quantum chemical code towards liquid systems. Phys. Chem. Chem. Phys. 2, 2187–2193 (2000).

    Article  Google Scholar 

  53. 53

    Grimme, S. Supramolecular binding thermodynamics by dispersion-corrected density functional theory. Chem. Eur. J. 18, 9955–9964 (2012).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  54. 54

    Furche, F. et al. Turbomole. Wiley Interdiscip. Rev. Comput. Mol. Sci. 4, 91–100 (2014).

    CAS  Article  Google Scholar 

  55. 55

    Sierka, M., Hogekamp, A. & Ahlrichs, R. Fast evaluation of the Coulomb potential for electron densities using multipole accelerated resolution of identity approximation. J. Chem. Phys. 118, 9136–9148 (2003).

    CAS  Article  Google Scholar 

  56. 56

    Weigend, F. Accurate Coulomb-fitting basis sets for H to Rn. Phys. Chem. Chem. Phys. 8, 1057–1065 (2006).

    CAS  PubMed  Article  PubMed Central  Google Scholar 

  57. 57

    Gritsan, N.P. & Pritchina, E.A. Are aroylnitrenes species with a singlet ground state? Mendeleev. Commun. 11, 94–95 (2001).

    Article  Google Scholar 

  58. 58

    Pritchina, E.A. et al. Matrix isolation, time-resolved IR, and computational study of the photochemistry of benzoyl azide. Phys. Chem. Chem. Phys. 5, 1010–1018 (2003).

    CAS  Article  Google Scholar 

  59. 59

    Staroverov, V.N., Scuseria, G.E., Tao, J. & Perdew, J.P. Comparative assessment of a new nonempirical density functional: Molecules and hydrogen-bonded complexes. J. Chem. Phys. 119, 12129–12137 (2003).

    CAS  Article  Google Scholar 

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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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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.

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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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