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

Abstract

It is important to understand the dynamics and higher energy structures of RNA, called excited states, to achieve better understanding of RNA function. R 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.

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.

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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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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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