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Mutate-and-chemical-shift-fingerprint (MCSF) to characterize excited states in RNA using NMR spectroscopy

Nature Protocols volume 16, pages 5146–5170 (2021)Cite this article

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Abstract

It is important to understand the dynamics and higher energy structures of RNA, called excited states, to achieve better understanding of RNA function. R1ρ relaxation dispersion NMR spectroscopy (RD) determines chemical shift differences between the most stable, ground state and the short-lived, low-populated excited states. We describe a procedure for deducing the excited state structure from these chemical shift differences using the mutate-and-chemical-shift-fingerprint (MCSF) method, which requires ~2–6 weeks and moderate understanding of NMR and RNA structure. We recently applied the MCSF methodology to elucidate the excited state of microRNA 34a targeting the SIRT1 mRNA and use this example to demonstrate the analysis. The protocol comprises the following steps: (i) determination of the secondary structure of the excited state from RD chemical shift data, (ii) design of trapped excited state RNA, (iii) validation of the excited state structure by NMR, and (iv) MCSF analysis comparing the chemical shifts of the trapped excited state with the RD-derived chemical shift differences. MCSF enables observation of the short-lived RNA structures, which can be functionally and structurally characterized by entrapment.

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Fig. 1: RNA conformational rearrangements occur on different timescales.
Fig. 2: Overview of the procedure depicted with mock spectra.
Fig. 3: CS and its correlation to structure.
Fig. 4: BMRB CSs and example of inferring an ES structure on the miR-34a–mSIRT1 construct.
Fig. 5: Construct design.
Fig. 6: Example of trapped ES.
Fig. 7: Example of two single-point mutants failing to trap the ES.

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Data availability

The NMR resonance assignments of miR-34a–mSIRT1 bulge (entry 27226) and miR-34a–mSIRT1 trapped ES (entry 27229) that were used in the ‘Anticipated results’, originally published in ref. 17 are available in the BMRB. Figure 6b–d contains raw data from ref. 17.

Code availability

The custom code used for the local RNA BMRB SQL database query in the Procedure is available at https://github.com/PetzoldLab/mcsf-git along with the SQL database.

References

  1. Mattick, J. S. & Makunin, I. V. Non-coding RNA. Hum. Mol. Genet. 15, R17–R29 (2006).

    Article  CAS  PubMed  Google Scholar 

  2. Cech, T. R. & Steitz, J. A. The noncoding rna revolution—trashing old rules to forge new ones. Cell 157, 77–94 (2014).

    Article  CAS  PubMed  Google Scholar 

  3. Marek, M. S., Johnson-Buck, A. & Walter, N. G. The shape-shifting quasispecies of RNA: one sequence, many functional folds. Phys. Chem. Chem. Phys. 13, 11524–11537 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Ganser, L. R., Kelly, M. L., Herschlag, D. & Al-Hashimi, H. M. The roles of structural dynamics in the cellular functions of RNAs. Nat. Rev. Mol. Cell Biol. 20, 474–489 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Mustoe, A. M., Brooks, C. L. & Al-Hashimi, H. M. Hierarchy of RNA functional dynamics. Annu. Rev. Biochem. 83, 441–466 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Frauenfelder, H., Sligar, S. & Wolynes, P. The energy landscapes and motions of proteins. Science 254, 1598–1603 (1991).

    Article  CAS  PubMed  Google Scholar 

  7. Marušič, M., Schlagnitweit, J. & Petzold, K. RNA dynamics by NMR spectroscopy. ChemBioChem 20, 2685–2710 (2019).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  8. Zhao, B. & Zhang, Q. Measuring residual dipolar couplings in excited conformational states of nucleic acids by CEST NMR spectroscopy. J. Am. Chem. Soc. 137, 13480–13483 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Al-Hashimi, H. M. & Walter, N. G. RNA dynamics: it is about time. Curr. Opin. Struct. Biol. 18, 321–329 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Tian, S., Cordero, P., Kladwang, W. & Das, R. High-throughput mutate-map-rescue evaluates SHAPE-directed RNA structure and uncovers excited states. RNA 20, 1815–1826 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Mulder, F. A. A., Mittermaier, A., Hon, B., Dahlquist, F. W. & Kay, L. E. Studying excited states of proteins by NMR spectroscopy. Nat. Struct. Biol. 8, 932–935 (2001).

    Article  CAS  PubMed  Google Scholar 

  12. Bakolitsa, C. et al. Low-populated folding intermediates of Fyn SH3 characterized by relaxation dispersion NMR. Nature 430, 586–590 (2004).

    Article  CAS  Google Scholar 

  13. Nikolova, E. N. et al. Transient Hoogsteen base pairs in canonical duplex DNA. Nature 470, 498–502 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Kimsey, I. J., Petzold, K., Sathyamoorthy, B., Stein, Z. W. & Al-Hashimi, H. M. Visualizing transient Watson–Crick-like mispairs in DNA and RNA duplexes. Nature 519, 315–320 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Dethoff, E. A., Petzold, K., Chugh, J., Casiano-Negroni, A. & Al-Hashimi, H. M. Visualizing transient low-populated structures of RNA. Nature 491, 724–728 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Baisden, J. T., Boyer, J. A., Zhao, B., Hammond, S. M. & Zhang, Q. Visualizing a protonated RNA state that modulates microRNA-21 maturation. Nat. Chem. Biol. 17, 80–88 (2021).

    Article  CAS  PubMed  Google Scholar 

  17. Baronti, L. et al. Base-pair conformational switch modulates miR-34a targeting of Sirt1 mRNA. Nature 583, 139–144 (2020).

    Article  CAS  PubMed  Google Scholar 

  18. Ulrich, E. L. et al. BioMagResBank. Nucleic Acids Res. 36, D402–D408 (2008).

    Article  CAS  PubMed  Google Scholar 

  19. Rangadurai, A., Szymaski, E. S., Kimsey, I. J., Shi, H. & Al-Hashimi, H. M. Characterizing micro-to-millisecond chemical exchange in nucleic acids using off-resonance R 1ρ relaxation dispersion. Prog. Nucl. Mag. Res. Sp. 112–113, 55–102 (2019).

    Article  CAS  Google Scholar 

  20. Parisien, M. & Major, F. The MC-Fold and MC-Sym pipeline infers RNA structure from sequence data. Nature 452, 51–55 (2008).

    Article  CAS  PubMed  Google Scholar 

  21. Lukavsky, P. J. & Puglisi, J. D. Structure determination of large biological RNAs. Methods Enzymol. 394, 399–416 (2005).

    Article  CAS  PubMed  Google Scholar 

  22. Karlsson, H., Baronti, L. & Petzold, K. A robust and versatile method for production and purification of large-scale RNA samples for structural biology. RNA 26, 1023–1037 (2020).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Baronti, L., Karlsson, H., Marušič, M. & Petzold, K. A guide to large-scale RNA sample preparation. Anal. Bioanal. Chem. 410, 3239–3252 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Xue, Y. et al. Chapter two characterizing RNA excited states using NMR relaxation dispersion. Methods Enzymol. 558, 39–73 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Feyrer, H., Schlagnitweit, J. & Petzold, K. Practical aspects of sample preparation and setup of 1H R1ρ relaxation dispersion experiments of RNA. J. Vis. Exp. https://doi.org/10.3791/62470 (2021).

  26. Beckert, B. & Masquida, B. Synthesis of RNA by in vitro transcription. Methods Mol. Biol. 703, 29–41 (2010).

    Article  CAS  Google Scholar 

  27. Milligan, J. F., Groebe, D. R., Witherell, G. W. & Uhlenbeck, O. C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res. 15, 8783–8798 (1987).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Melton, D. A. et al. Efficient in vitro synthesis of biologically active RNA and RNA hybridization probes from plasmids containing a bacteriophage SP6 promoter. Nucleic Acids Res. 12, 7035–7056 (1984).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Feyrer, H., Munteanu, R., Baronti, L. & Petzold, K. One-pot production of RNA in high yield and purity through cleaving tandem transcripts. Molecules 25, 1142 (2020).

    Article  CAS  PubMed Central  Google Scholar 

  30. Nikonowicz, E. P. et al. Preparation of 13 C and 15 N labelled RNAs for heteronuclear multi-dimensional NMR studies. Nucleic Acids Res. 20, 4507–4513 (1992).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Wyatt, J. R., Chastain, M. & Puglisi, J. D. Synthesis and purification of large amounts of RNA oligonucleotides. Biotechniques 11, 764–769 (1991).

    CAS  PubMed  Google Scholar 

  32. Anderson, A. C., Scaringe, S. A., Earp, B. E. & Frederick, C. A. HPLC purification of RNA for crystallography and NMR. RNA 2, 110–117 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Murray, J. B., Collier, A. K. & Arnold, J. R. P. A general purification procedure for chemically synthesized oligoribonucleotides. Anal. Biochem. 218, 177–184 (1994).

    Article  CAS  PubMed  Google Scholar 

  34. Strebitzer, E., Nußbaumer, F., Kremser, J., Tollinger, M. & Kreutz, C. Studying sparsely populated conformational states in RNA combining chemical synthesis and solution NMR spectroscopy. Methods 148, 39–47 (2018).

    Article  CAS  PubMed  Google Scholar 

  35. Becette, O., Olenginski, L. T. & Dayie, T. K. Solid-phase chemical synthesis of stable isotope-labeled RNA to aid structure and dynamics studies by NMR spectroscopy. Molecules 24, 3476 (2019).

    Article  CAS  PubMed Central  Google Scholar 

  36. Strebitzer, E. et al. 5-Oxyacetic acid modification destabilizes double helical stem structures and favors anionic Watson–Crick like cmo5U-G base pairs. Chem. Eur. J. 24, 18903–18906 (2018).

    Article  CAS  PubMed  Google Scholar 

  37. Le, M. T., Brown, R. E., Simon, A. E. & Dayie, T. K. Chapter nineteen in vivo, large-scale preparation of uniformly 15N- and site-specifically 13C-labeled homogeneous, recombinant RNA for NMR studies. Methods Enzymol. 565, 495–535 (2015).

    Article  CAS  PubMed  Google Scholar 

  38. Fürtig, B., Richter, C., Wöhnert, J. & Schwalbe, H. NMR spectroscopy of RNA. ChemBioChem 4, 936–962 (2003).

    Article  PubMed  CAS  Google Scholar 

  39. Lee, J., Dethoff, E. A. & Al-Hashimi, H. M. Invisible RNA state dynamically couples distant motifs. Proc. Natl Acad. Sci. USA 111, 9485–9490 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Ebrahimi, P., Kaur, S., Baronti, L., Petzold, K. & Chen, A. A. A two-dimensional replica-exchange molecular dynamics method for simulating RNA folding using sparse experimental restraints. Methods 162–163, 96–107 (2019).

    Article  PubMed  CAS  Google Scholar 

  41. Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res. (2000) https://doi.org/10.1093/nar/28.1.235

  42. Aeschbacher, T., Shubert, M. & Allain, F. H.-T. A procedure to validate and correct the C chemical shift calibration of RNA datasets. J. Biomol. NMR 52, 179–190 (2012).

    Article  CAS  PubMed  Google Scholar 

  43. Wishart, D. S. et al. 1H, 13C and 15N chemical shift referencing in biomolecular NMR. J. Biomol. NMR 6, 135–140 (1995).

    Article  CAS  PubMed  Google Scholar 

  44. Popenda, M. et al. RNA FRABASE 2.0: an advanced web-accessible database with the capacity to search the three-dimensional fragments within RNA structures. BMC Bioinformatics 11, 231 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  45. Gruber, A. R., Bernhart, S. H. & Lorenz, R. RNA bioinformatics. Methods Mol. Biol. 1269, 307–326 (2014).

    Article  CAS  Google Scholar 

  46. Jin, X., Zhu, T., Zhang, J. Z. H. & He, X. A systematic study on RNA NMR chemical shift calculation based on the automated fragmentation QM/MM approach. RSC Adv. 6, 108590–108602 (2016).

    Article  CAS  Google Scholar 

  47. Hopkins, N. & Riad, M. Mutate-and-Chemical-Shift-Fingerprint (MCSF) to characterize excited states in RNA using NMR spectroscopy. Petzoldlab/mcsf-git, https://doi.org/10.5281/zenodo.4823710 (2021).

  48. Dominguez, C., Schubert, M., Duss, O., Ravindranathan, S. & Allain, F. H.-T. Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy. Prog. Nucl. Mag. Res. Sp. 58, 1–61 (2011).

    Article  CAS  Google Scholar 

  49. Farès, C., Amata, I. & Carlomagno, T. 13 C-Detection in RNA bases: revealing structure−chemical shift relationships. J. Am. Chem. Soc. 129, 15814–15823 (2007).

    Article  PubMed  CAS  Google Scholar 

  50. Schlagnitweit, J., Steiner, E., Karlsson, H. & Petzold, K. Efficient detection of structure and dynamics in unlabeled RNAs: the SELOPE approach. Chem. Eur. J. 24, 6067–6070 (2018).

    Article  CAS  PubMed  Google Scholar 

  51. Dingley, A. & Grzesiek, S. Direct observation of hydrogen bonds in nucleic acid base pairs by internucleotide (2)J(NN) couplings. J. Am. Chem. Soc. 120, 8293–8297 (1998).

    Article  CAS  Google Scholar 

  52. Wang, Y., Han, G., Jiang, X., Yuwen, T. & Xue, Y. Chemical shift prediction of RNA imino groups: application toward characterizing RNA excited states. Nat. Commun. 12, 1595 (2021).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Nikolova, E. N., Gottardo, F. L. & Al-Hashimi, H. M. Probing transient Hoogsteen hydrogen bonds in canonical duplex DNA using NMR relaxation dispersion and single-atom substitution. J. Am. Chem. Soc. 134, 3667–3670 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  54. Hansen, A. L., Nikolova, E. N., Casiano-Negroni, A. & Al-Hashimi, H. M. Extending the range of microsecond-to-millisecond chemical exchange detected in labeled and unlabeled nucleic acids by selective carbon R 1ρ NMR spectroscopy. J. Am. Chem. Soc. 131, 3818–3819 (2009).

    Article  CAS  PubMed  Google Scholar 

  55. Steiner, E., Schlagnitweit, J., Lundström, P. & Petzold, K. Capturing excited states in the fast-intermediate exchange limit in biological systems using 1H NMR spectroscopy. Angew. Chem. Int. Ed. 55, 15869–15872 (2016).

    Article  CAS  Google Scholar 

  56. Palmer, A. G. & Massi, F. Characterization of the dynamics of biomacromolecules using rotating-frame spin relaxation NMR spectroscopy. Chem. Rev. 106, 1700–1719 (2006).

    Article  CAS  PubMed  Google Scholar 

  57. Yamakuchi, M. & Lowenstein, C. J. MiR-34, SIRT1, and p53: the feedback loop. Cell Cycle 8, 712–715 (2009).

    Article  CAS  PubMed  Google Scholar 

  58. Korzhnev, D. M., Orekhov, V. Y. & Kay, L. E. Off-resonance R 1ρ NMR studies of exchange dynamics in proteins with low spin-lock fields: an application to a Fyn SH3 domain. J. Am. Chem. Soc. 127, 713–721 (2005).

    Article  CAS  PubMed  Google Scholar 

  59. McConnell, H. M. Reaction rates by nuclear magnetic resonance. J. Chem. Phys. 28, 430–431 (1958).

    Article  CAS  Google Scholar 

  60. Trott, O. & Palmer, A. G. R1ρ relaxation outside of the fast-exchange limit. J. Magn. Reson. 154, 157–160 (2002).

    Article  CAS  PubMed  Google Scholar 

  61. Dejaegere, A. P. & Case, D. A. Density functional study of ribose and deoxyribose chemical shifts. J. Phys. Chem. 102, 5280–5289 (1998).

    Article  CAS  Google Scholar 

  62. Ebrahimi, M., Rossi, P., Rogers, C. & Harbison, G. S. Dependence of 13C NMR chemical shifts on conformations of RNA nucleosides and nucleotides. J. Magn. Reson. 150, 1–9 (2001).

    Article  CAS  PubMed  Google Scholar 

  63. Xu, X.-P. & Au−Yeung, S. C. F. Investigation of chemical shift and structure relationships in nucleic acids using NMR and density functional theory methods. J. Phys. Chem. B 104, 5641–5650 (2000).

    Article  CAS  Google Scholar 

  64. Clay, M. C., Ganser, L. R., Merriman, D. K. & Al-Hashimi, H. M. Resolving sugar puckers in RNA excited states exposes slow modes of repuckering dynamics. Nucleic Acids Res. 45, e134 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Leontis, N. B. & Westhof, E. Geometric nomenclature and classification of RNA base pairs. RNA 7, 499–512 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  66. Antao, V. P., Lai, S. Y. & Tinoco, I. A thermodynamic study of unusually stable RNA and DNA hairpins. Nucleic Acids Res. 19, 5901–5905 (1991).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  67. Nozinovic, S., Fürtig, B., Jonker, H. R. A., Richter, C. & Schwalbe, H. High-resolution NMR structure of an RNA model system: the 14-mer cUUCGg tetraloop hairpin RNA. Nucleic Acids Res. 38, 683–694 (2010).

    Article  CAS  PubMed  Google Scholar 

  68. Varani, G., Cheong, C. & Tinoco, I. Solution structure of an unusually stable RNA hairpin, 5GGAC(UUCG)GUCC. Nature 346, 680–682 (1990).

    Article  PubMed  Google Scholar 

  69. Ghose, R., Marino, J. P., Wiberg, K. B. & Prestegard, J. H. Dependence of 13C chemical shifts on glycosidic torsional angles in ribonucleic acids. J. Am. Chem. Soc. 116, 8827–8828 (1994).

    Article  CAS  Google Scholar 

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Acknowledgements

K.P. acknowledges funding from the Swedish Research Council (grant numbers 2014-04303 and 2018-00250), the Swedish Foundation for Strategic Research (project number ICA14-0023), Harald och Greta Jeansson Stiftelse (JS20140009), Carl Tryggers stiftelse (CTS14-383 and 15-383), Eva och, Oscar Ahréns Stiftelse, Åke Wiberg Stiftelse (467080968 and M14-0109), Cancerfonden (CAN 2015/388), the Karolinska Institute Department of Medical Biochemistry and Biophysics (grant number KID 2-3707/2013 and support for the purchase of a 600-MHz Bruker NMR spectrometer) and Ragnar Söderberg Stiftelse (M91/14). J.S. acknowledges funding through a Marie Sklodowska-Curie Individual Fellowship (EU H2020/project number 747446).

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  1. Lorenzo Baronti

    Present address: Institute of Structural Biology, Helmholtz Zentrum München, Neuherberg, Germany

Authors and Affiliations

  1. Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden

    Magdalena Riad, Noah Hopkins, Lorenzo Baronti, Hampus Karlsson, Judith Schlagnitweit & Katja Petzold

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Contributions

M.R. and K.P. wrote the manuscript. N.H. and M.R. wrote the scripts based on earlier work done by N.H., H.K. and L.B. L.B. carried out most of the experiments of the original manuscript and data analysis, with assistance from K.P. and J.S. All authors contributed to the final version.

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Correspondence to Katja Petzold.

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Competing interests

K.P. is a consultant to Arrakis Therapeutics, an RNA-targeting drug-discovery company.

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Peer review information Nature Protocols thanks Diego Carnevale, Christoph Kreutz and the other, anonymous reviewer(s) for their contribution to the peer review of this work.

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Key references using this protocol

Baronti, L. et al. Nature 583, 139–144 (2020): https://doi.org/10.1038/s41586-020-2336-3

Schlagnitweit, J. et al. Chem. European J. 24, 6067–6070 (2018): https://doi.org/10.1002/chem.201800992

Steiner, E. et al. Angew. Chem. Int. Ed. 55, 15869–15872 (2016): https://doi.org/10.1002/anie.201609102

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Riad, M., Hopkins, N., Baronti, L. et al. Mutate-and-chemical-shift-fingerprint (MCSF) to characterize excited states in RNA using NMR spectroscopy. Nat Protoc 16, 5146–5170 (2021). https://doi.org/10.1038/s41596-021-00606-1

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