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Site-Specific Spin Labeling of RNA for NMR and EPR Structural Studies

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RNA Spectroscopy

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2113))

Abstract

Many RNA architectures were discovered to be involved in essential biological pathways acting as catalysts and/or regulators of gene expression, transcription, translation, splicing, or viral infection. The key to understand their diverse biological functions is to investigate their structure and dynamic. Nuclear Magnetic Resonance (NMR) is a powerful method to gain insight into these properties. However, the study of high-molecular-weight RNAs by NMR remains challenging. Advances in biochemical and NMR methods over the recent years allow to overcome the limitation of NMR. In particular, the incorporation of paramagnetic probes, coupled to the measurement of the induced effects on nuclear spins, has become an efficient tool providing long-range distance restraints and information on dynamic in solution. At the same time, the use of spin label enabled the application of Electron Paramagnetic Resonance (EPR) to study biological macromolecules. Combining NMR and EPR is emerging as a new approach to investigate the architecture of biological systems.

Here, we describe an efficient protocol to introduce a paramagnetic probe into a RNA at a specific position. This method enables various combinations of isotopic labeling for NMR and is also of interest for EPR studies.

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References

  1. Joyce GF (2002) The antiquity of RNA-based evolution. Nature 418:214–221

    CAS  PubMed  Google Scholar 

  2. Kruger K, Grabowski PJ, Zaug AJ, Sands J, Gottschling DE, Cech TR (1982) Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena. Cell 31:147–157

    CAS  PubMed  Google Scholar 

  3. Doudna JA, Cech TR (2002) The chemical repertoire of natural ribozymes. Nature 418:222–228

    CAS  PubMed  Google Scholar 

  4. Walter NG (2007) Ribozyme catalysis revisited: is water involved? Mol Cell 28:923–929

    CAS  PubMed  PubMed Central  Google Scholar 

  5. Serganov A, Patel DJ (2007) Ribozymes, riboswitches and beyond: regulation of gene expression without proteins. Nat Rev Genet 8:776–790

    CAS  PubMed  PubMed Central  Google Scholar 

  6. Mandal M, Breaker RR (2004) Gene regulation by riboswitches. Nat Rev Mol Cell Biol 5:451–463

    CAS  PubMed  Google Scholar 

  7. Grosshans H, Filipowicz W (2008) Molecular biology: the expanding world of small RNAs. Nature 451:414–416

    CAS  PubMed  Google Scholar 

  8. Hannon GJ (2002) RNA interference. Nature 418:244–251

    CAS  PubMed  Google Scholar 

  9. Bevilacqua PC, Blose JM (2008) Structures, kinetics, thermodynamics, and biological functions of RNA hairpins. Annu Rev Phys Chem 59:79–103

    CAS  PubMed  Google Scholar 

  10. Brierley I, Digard P, Inglis SC (1989) Characterization of an efficient coronavirus ribosomal frameshifting signal: requirement for an RNA pseudoknot. Cell 57:537–547

    CAS  PubMed  PubMed Central  Google Scholar 

  11. Brunel C, Marquet R, Romby P, Ehresmann C (2002) RNA loop-loop interactions as dynamic functional motifs. Biochimie 84:925–944

    CAS  PubMed  Google Scholar 

  12. Derrigo M, Cestelli A, Savettieri G, Di Liegro I (2000) RNA-protein interactions in the control of stability and localization of messenger RNA (review). Int J Mol Med 5:111–123

    CAS  PubMed  Google Scholar 

  13. Kolb FA, Engdahl HM, Slagter-Jäger JG, Ehresmann B, Ehresmann C, Westhof E, Wagner EG, Romby P (2000) Progression of a loop-loop complex to a four-way junction is crucial for the activity of a regulatory antisense RNA. EMBO J 19:5905–5915

    CAS  PubMed  PubMed Central  Google Scholar 

  14. Tomizawa J, Som T (1984) Control of ColE1 plasmid replication: enhancement of binding of RNA I to the primer transcript by the Rom protein. Cell 38:871–878

    CAS  PubMed  Google Scholar 

  15. Allen M, Varani L, Varani G (2001) Nuclear magnetic resonance methods to study structure and dynamics of RNA-protein complexes. Methods Enzymol 339:357–376

    CAS  PubMed  Google Scholar 

  16. Latham MP, Brown DJ, McCallum SA, Pardi A (2005) NMR methods for studying the structure and dynamics of RNA. Chembiochem 6:1492–1505

    CAS  PubMed  Google Scholar 

  17. Fürtig B, Buck J, Manoharan V, Bermel W, Jäschke A, Wenter P, Pitsch S, Schwalbe H (2007) Time-resolved NMR studies of RNA folding. Biopolymers 86:360–383

    PubMed  Google Scholar 

  18. Getz M, Sun X, Casiano-Negroni A, Zhang Q, Al-Hashimi HM (2007) NMR studies of RNA dynamics and structural plasticity using NMR residual dipolar couplings. Biopolymers 86:384–402

    CAS  PubMed  Google Scholar 

  19. Dominguez C, Schubert M, Duss O, Ravindranathan S, Allain FH-T (2011) Structure determination and dynamics of protein–RNA complexes by NMR spectroscopy. Prog Nucl Magn Reson Spectrosc 58:1–61

    CAS  PubMed  Google Scholar 

  20. Duss O, Lukavsky PJ, Allain FH-T (2012) Isotope labeling and segmental labeling of larger RNAs for NMR structural studies. In: Atreya HS (ed) Isotope labeling in biomolecular NMR. Springer, Dordrecht, pp 121–144

    Google Scholar 

  21. Yadav DK, Lukavsky PJ (2016) NMR solution structure determination of large RNA-protein complexes. Prog Nucl Magn Reson Spectrosc 97:57–81

    CAS  PubMed  Google Scholar 

  22. Schlundt A, Tants J-N, Sattler M (2017) Integrated structural biology to unravel molecular mechanisms of protein-RNA recognition. Methods 118–119:119–136

    PubMed  Google Scholar 

  23. Barnwal RP, Yang F, Varani G (2017) Applications of NMR to structure determination of RNAs large and small. Arch Biochem Biophys 628:42–56

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Cai S, Zhu L, Zhang Z, Chen Y (2007) Determination of the three-dimensional structure of the Mrf2−DNA complex using paramagnetic spin labeling. Biochemistry 46:4943–4950

    CAS  PubMed  Google Scholar 

  25. Hennig J, Warner LR, Simon B, Geerlof A, Mackereth CD, Sattler M (2015) Structural analysis of protein–RNA complexes in solution using NMR paramagnetic relaxation enhancements. Methods Enzymol 558:333–362

    CAS  PubMed  Google Scholar 

  26. Ramos A, Varani G (1998) A new method to detect long-range protein−RNA contacts: NMR detection of electron−proton relaxation induced by nitroxide spin-labeled RNA. J Am Chem Soc 120:10992–10993

    CAS  Google Scholar 

  27. Wunderlich CH, Huber RG, Spitzer R, Liedl KR, Kloiber K, Kreutz C (2013) A novel paramagnetic relaxation enhancement tag for nucleic acids: a tool to study structure and dynamics of RNA. ACS Chem Biol 8:2697–2706

    CAS  PubMed  PubMed Central  Google Scholar 

  28. Iwahara J, Clore GM (2006) Detecting transient intermediates in macromolecular binding by paramagnetic NMR. Nature 440:1227–1230

    CAS  PubMed  Google Scholar 

  29. Helmling C, Bessi I, Wacker A, Schnorr KA, Jonker HRA, Richter C, Wagner D, Kreibich M, Schwalbe H (2014) Noncovalent spin labeling of riboswitch RNAs to obtain long-range structural NMR restraints. ACS Chem Biol 9:1330–1339

    CAS  PubMed  Google Scholar 

  30. Shelke SA, Sandholt GB, Sigurdsson ST (2014) Nitroxide-labeled pyrimidines for non-covalent spin-labeling of abasic sites in DNA and RNA duplexes. Org Biomol Chem 12:7366–7374

    CAS  PubMed  Google Scholar 

  31. Saha S, Hetzke T, Prisner TF, Sigurdsson ST (2018) Noncovalent spin-labeling of RNA: the aptamer approach. Chem Commun (Camb) 54:11749–11752

    CAS  Google Scholar 

  32. Macosko JC, Pio MS, Tinoco I, Shin YK (1999) A novel 5 displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA. RNA 5:1158–1166

    CAS  PubMed  PubMed Central  Google Scholar 

  33. Grant GPG, Qin PZ (2007) A facile method for attaching nitroxide spin labels at the 5′ terminus of nucleic acids. Nucleic Acids Res 35:e77

    PubMed  PubMed Central  Google Scholar 

  34. Höbartner C, Sicoli G, Wachowius F, Gophane DB, Sigurdsson ST (2012) Synthesis and characterization of RNA containing a rigid and nonperturbing cytidine-derived spin label. J Org Chem 77:7749–7754

    PubMed  Google Scholar 

  35. Gophane DB, Endeward B, Prisner TF, Sigurdsson ST (2018) A semi-rigid isoindoline-derived nitroxide spin label for RNA. Org Biomol Chem 16:816–824

    CAS  PubMed  Google Scholar 

  36. Shelke SA, Sigurdsson ST (2012) Site-directed spin labelling of nucleic acids. Eur J Org Chem 2012:2291–2301

    CAS  Google Scholar 

  37. Büttner L, Seikowski J, Wawrzyniak K, Ochmann A, Höbartner C (2013) Synthesis of spin-labeled riboswitch RNAs using convertible nucleosides and DNA-catalyzed RNA ligation. Bioorg Med Chem 21:6171–6180

    PubMed  Google Scholar 

  38. Wawrzyniak-Turek K, Höbartner C (2014) Deoxyribozyme-mediated ligation for incorporating EPR spin labels and reporter groups into RNA. Methods Enzymol 549:85–104

    CAS  PubMed  Google Scholar 

  39. Lebars I, Vileno B, Bourbigot S, Turek P, Wolff P, Kieffer B (2014) A fully enzymatic method for site-directed spin labeling of long RNA. Nucleic Acids Res 42:e117

    PubMed  PubMed Central  Google Scholar 

  40. Duss O, Yulikov M, Jeschke G, Allain FH-T (2014) EPR-aided approach for solution structure determination of large RNAs or protein–RNA complexes. Nat Commun 5:3669

    Google Scholar 

  41. Domnick C, Hagelueken G, Eggert F, Schiemann O, Kath-Schorr S (2019) Posttranscriptional spin labeling of RNA by tetrazine-based cycloaddition. Org Biomol Chem 17:1805.

    CAS  Google Scholar 

  42. Qin PZ, Dieckmann T (2004) Application of NMR and EPR methods to the study of RNA. Curr Opin Struct Biol 14:350–359

    CAS  PubMed  Google Scholar 

  43. Duss O, Yulikov M, Allain FHT, Jeschke G (2015) Combining NMR and EPR to determine structures of large RNAs and protein-RNA complexes in solution. Methods Enzymol 558:279–331

    CAS  PubMed  Google Scholar 

  44. Nielsen H (2011) Working with RNA. In: Nielsen H (ed) RNA. Humana Press, Totowa, pp 15–28

    Google Scholar 

  45. Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15:8783–8798

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Wyatt JR, Chastain M, Puglisi JD (1991) Synthesis and purification of large amounts of RNA oligonucleotides. BioTechniques 11:764–769

    CAS  PubMed  Google Scholar 

  47. Nelissen FHT, van Gammeren AJ, Tessari M, Girard FC, Heus HA, Wijmenga SS (2008) Multiple segmental and selective isotope labeling of large RNA for NMR structural studies. Nucleic Acids Res 36:e89

    PubMed  PubMed Central  Google Scholar 

  48. Romaniuk PJ, Uhlenbeck OC (1983) Joining of RNA molecules with RNA ligase. Methods Enzymol 100:52–59

    CAS  PubMed  Google Scholar 

  49. Bullard DR, Bowater RP (2006) Direct comparison of nick-joining activity of the nucleic acid ligases from bacteriophage T4. Biochem J 398:135–144

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Milligan JF, Uhlenbeck OC (1989) Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol 180:51–62

    CAS  PubMed  Google Scholar 

  51. Scott LG, Hennig M (2008) RNA structure determination by NMR. In: Keith JM (ed) Bioinformatics. Humana Press, Totowa, pp 29–61

    Google Scholar 

  52. Fürtig B, Richter C, Wöhnert J, Schwalbe H (2003) NMR spectroscopy of RNA. Chembiochem 4:936–962

    PubMed  Google Scholar 

  53. Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual, vol vol. 2, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor

    Google Scholar 

  54. Fajer PG (2006) Electron spin resonance spectroscopy labeling in peptide and protein analysis. In: Encyclopedia of analytical chemistry. https://doi.org/10.1002/9780470027318.a1609

    Chapter  Google Scholar 

  55. Oppenheim SF, Buettner GR, Rodgers VGJ (1996) Relationship of rotational correlation time from EPR spectroscopy and protein-membrane interaction. J Membr Sci 118:133–139

    CAS  Google Scholar 

  56. Stoll S, Schweiger A (2007) EasySpin: simulating cw ESR spectra. Magn Reson 27:299–321

    Google Scholar 

  57. Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 178:42–55

    CAS  PubMed  Google Scholar 

  58. Etienne E, Le Breton N, Martinho M, Mileo E, Belle V (2017) SimLabel: a graphical user interface to simulate continuous wave EPR spectra from site-directed spin labeling experiments. Magn Reson Chem 55:714–719

    CAS  PubMed  Google Scholar 

  59. Qin PZ, Butcher SE, Feigon J, Hubbell WL (2001) Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: a site-directed spin labeling study. Biochemistry 40:6929–6936

    CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Laboratoire des Propriétés Optiques et Magnétiques des Architectures Moléculaires, Institut de Chimie (UMR7177) Université de Strasbourg / CNRS, Strasbourg, France

    Bertrand Vileno

  2. French EPR Federation of Research (REseau NAtional de Rpe interDisciplinaire (RENARD), Fédération IR-RPE CNRS #3443), Strasbourg, France

    Bertrand Vileno

  3. Architecture et Réactivité de l’ARN - CNRS UPR 9002, Institut de Biologie Moléculaire et Cellulaire, Université de Strasbourg, Strasbourg, France

    Isabelle Lebars

Authors
  1. Bertrand Vileno
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  2. Isabelle Lebars
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Corresponding authors

Correspondence to Bertrand Vileno or Isabelle Lebars .

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Editors and Affiliations

  1. Université de Paris, Paris, France, Laboratoire Léon Brillouin LLB, CEA, CNRS UMR12, Université Paris Saclay, Gif-sur-Yvette, France

    Véronique Arluison

  2. Synchrotron SOLEIL L’Orme des Merisiers Saint Aubin, Gif-sur-Yvette, France

    Frank Wien

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Vileno, B., Lebars, I. (2020). Site-Specific Spin Labeling of RNA for NMR and EPR Structural Studies. In: Arluison, V., Wien, F. (eds) RNA Spectroscopy. Methods in Molecular Biology, vol 2113. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0278-2_15

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  • DOI: https://doi.org/10.1007/978-1-0716-0278-2_15

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0277-5

  • Online ISBN: 978-1-0716-0278-2

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