Advertisement

Journal of Biomolecular NMR

, Volume 71, Issue 3, pp 151–164 | Cite as

Isotope labeling for studying RNA by solid-state NMR spectroscopy

  • Alexander Marchanka
  • Christoph Kreutz
  • Teresa Carlomagno
Article

Abstract

Nucleic acids play key roles in most biological processes, either in isolation or in complex with proteins. Often they are difficult targets for structural studies, due to their dynamic behavior and high molecular weight. Solid-state nuclear magnetic resonance spectroscopy (ssNMR) provides a unique opportunity to study large biomolecules in a non-crystalline state at atomic resolution. Application of ssNMR to RNA, however, is still at an early stage of development and presents considerable challenges due to broad resonances and poor dispersion. Isotope labeling, either as nucleotide-specific, atom-specific or segmental labeling, can resolve resonance overlaps and reduce the line width, thus allowing ssNMR studies of RNA domains as part of large biomolecules or complexes. In this review we discuss the methods for RNA production and purification as well as numerous approaches for isotope labeling of RNA. Furthermore, we give a few examples that emphasize the instrumental role of isotope labeling and ssNMR for studying RNA as part of large ribonucleoprotein complexes.

Keywords

RNA structure and dynamics Solid-state NMR Chemical synthesis of RNA In vitro RNA transcription Atom selective-labelling of nucleotides 

Notes

Acknowledgements

T.C. thanks the DFG for support through Grant CA 294/10 − 1.

References

  1. Alvarado LJ et al (2014) Chemo-enzymatic synthesis of selectively 13C/15N-labeled RNA for NMR structural and dynamics studies. Methods Enzymol 549:133–162Google Scholar
  2. Anderson AC, Scaringe SA, Earp BE, Frederick CA (1996) HPLC purification of RNA for crystallography and NMR. RNA 2:110–117Google Scholar
  3. Asami S, Rakwalska-Bange M, Carlomagno T, Reif B (2013) Protein–RNA interfaces probed by 1H-detected MAS solid-state NMR spectroscopy. Angew Chem Int Ed 52:2345–2349Google Scholar
  4. Azarani A, Hecker KH (2001) RNA analysis by ion-pair reversed-phase high performance liquid chromatography. Nucleic Acids Res 29:e7Google Scholar
  5. Batey RT, Kieft JS (2007) Improved native affinity purification of RNA. RNA 13:1384–1389Google Scholar
  6. Batey RT, Inada M, Kujawinski E, Puglisi JD, Williamson JR (1992) Preparation of isotopically labeled ribonucleotides for multidimensional NMR-spectroscopy of RNA. Nucleic Acids Res 20:4515–4523Google Scholar
  7. Batey RT, Battiste JL, Williamson JR (1995) Preparation of isotopically enriched RNAs for heteronuclear NMR. Methods Enzymol 261:300–322Google Scholar
  8. Bayro MJ et al. (2009) Dipolar truncation in magic-angle spinning NMR recoupling experiments. J Chem Phys 130:114506ADSGoogle Scholar
  9. Beaucage SL, Iyer RP (1992) Advances in the synthesis of oligonucleotides by the phosphoramidite approach. Tetrahedron 48:2223–2311Google Scholar
  10. Beaucage SL, Reese CB (2009) Recent advances in the chemical synthesis of RNA. Curr Protoc Nucleic Acid Chem.  https://doi.org/10.1002/0471142700.nc0216s38 Google Scholar
  11. Cheong HK, Hwang E, Lee C, Choi BS, Cheong C (2004) Rapid preparation of RNA samples for NMR spectroscopy and X-ray crystallography. Nucleic Acids Res 32:e84Google Scholar
  12. Coleman TM, Wang GC, Huang FQ (2004) Superior 5′ homogeneity of RNA from ATP-initiated transcription under the T7 phi 2.5 promoter. Nucleic Acids Res 32:e14Google Scholar
  13. Cvetkovic MA, Wurm JP, Audin MJ, Schütz S, Sprangers R (2017) The Rrp4-exosome complex recruits and channels substrate RNA by a unique mechanism. Nat Chem Biol 13:522–528Google Scholar
  14. D’Souza V, Dey A, Habib D, Summers MF (2004) NMR structure of the 101-nucleotide core encapsidation signal of the Moloney murine leukemia virus. J Mol Biol 337:427–442Google Scholar
  15. Davis JH et al (2005) RNA helical packing in solution: NMR structure of a 30 kDa GAAA tetraloop-receptor complex. J Mol Biol 351:371–382Google Scholar
  16. Dayie KT (2008) Key labeling technologies to tackle sizeable problems in RNA structural biology. Int J Mol Sci 9:1214–1240Google Scholar
  17. De Paëpe G, Lewandowski JR, Loquet A, Boeckmann A, Griffin RG (2008) Proton assisted recoupling and protein structure determination. J Chem Phys 129:245101ADSGoogle Scholar
  18. Dominissini D et al (2016) The dynamic N1-methyladenosine methylome in eukaryotic messenger RNA. Nature 530:441–446ADSGoogle Scholar
  19. Duss O, Maris C, von Schroetter C, Allain FHT (2010) A fast, efficient and sequence-independent method for flexible multiple segmental isotope labeling of RNA using ribozyme and RNase H cleavage. Nucleic Acids Res 38:e188Google Scholar
  20. Duss O, Lukavsky PJ, Allain FH (2012) Isotope labeling and segmental labeling of larger RNAs for NMR structural studies. In: Attrea HS (ed) Isotope labeling in biomolecular NMR. Springer, Dordrecht pp 121–144Google Scholar
  21. Duss O, Michel E, Konte NDD, Schubert M, Allain FH (2014a) Molecular basis for the wide range of affinity found in CSR/RSM protein-RNA recognition. Nucleic Acids Res 42:5332–5346Google Scholar
  22. Duss O et al (2014b) Structural basis of the non-coding RNA RsmZ acting as a protein sponge. Nature 509:588–592ADSGoogle Scholar
  23. Duss O, Diarra dit Konti N, Allain FHT (2015) Cut and paste RNA for nuclear magnetic resonance, paramagnetic resonance enhancement, and electron paramagnetic resonance structural studies. Methods Enzymol 565:537–562Google Scholar
  24. Ferre-D’Amare AR, Doudna JA (1996) Use of cis- and trans-ribozymes to remove 5′ and 3′ heterogeneities from milligrams of in vitro transcribed RNA. Nucleic Acids Res 24:977–978Google Scholar
  25. Ferre-D’Amare AR, Zhou KH, Doudna JA (1998) Crystal structure of a hepatitis delta virus ribozyme. Nature 395:567–574ADSGoogle Scholar
  26. Fürtig B, Richter C, Woehnert J, Schwalbe H (2003) NMR spectroscopy of RNA. ChemBioChem 4:936–962Google Scholar
  27. Graziadei A, Masiewicz P, Lapinaite A, Carlomagno T (2016) Archaea box C/D enzymes methylate two distinct substrate rRNA sequences with different efficiency. RNA 22:764–772Google Scholar
  28. Gullion T, Schaefer J (1989) Rotational-echo double-resonance NMR. J Magn Reson 81:196–200ADSGoogle Scholar
  29. Guo HCT, Collins RA (1995) Efficient trans-cleavage of A stem-loop RNA substrate by a ribozyme derived from neurospora vs RNA. EMBO J 14:368–376Google Scholar
  30. Helm M, Brule H, Giege R, Florentz C (1999) More mistakes by T7 RNA polymerase at the 5′ ends of in vitro-transcribed RNAs. RNA 5:618–621Google Scholar
  31. Hennig M, Scott LG, Sperling E, Bermel W, Williamson JR (2007) Synthesis of 5-fluoropyrimidine nucleotides as sensitive NMR probes of RNA structure. J Am Chem Soc 129:14911–14921Google Scholar
  32. Hoffman DW, Holland JA (1995) Preparation of C-13 labeled ribonucleotides using acetate as an isotope source. Nucleic Acids Res 23:3361–3362Google Scholar
  33. Houck-Loomis B et al. (2011) An equilibrium-dependent retroviral mRNA switch regulates translational recoding. Nature 480: 561–564ADSGoogle Scholar
  34. Huang XN, Yu PL, LeProust E, Gao XL (1997) An efficient and economic site-specific deuteration strategy for NMR studies of homologous oligonucleotide repeat sequences. Nucleic Acids Res 25:4758–4763Google Scholar
  35. Huang W, Varani G, Drobny GP (2010) C-13/N-15-F-19 intermolecular REDOR NMR study of the interaction of TAR RNA with tat peptides. J Am Chem Soc 132:17643–17645Google Scholar
  36. Huang W, Bardaro MF, Varani G, Drobny GP (2012) Preparation of RNA samples with narrow line widths for solid state NMR investigations. J Magn Reson 223:51–54ADSGoogle Scholar
  37. Jehle S et al (2010) Intermolecular protein-RNA interactions revealed by 2D P-31-N-15 magic angle spinning solid-state NMR spectroscopy. J Am Chem Soc 132:3842–3846Google Scholar
  38. Johnson JE, Julien KR, Hoogstraten CG (2006) Alternate-site isotopic labeling of ribonucleotides for NMR studies of ribose conformational dynamics in RNA. J Biomol NMR 35:261–274Google Scholar
  39. Juen MA et al (2016) Excited states of nucleic acids probed by proton relaxation dispersion NMR spectroscopy. Angew Chem Int Ed 55:12008–12012Google Scholar
  40. Kao C, Zheng M, Rudisser S (1999) A simple and efficient method to reduce nontemplated nucleotide addition at the 3′ terminus of RNAs transcribed by T7 RNA polymerase. RNA 5:1268–1272Google Scholar
  41. Keane SC et al (2015) Structure of the HIV-1 RNA packaging signal. Science 348:917–921ADSGoogle Scholar
  42. Kieft JS, Batey RT (2004) A general method for rapid and nondenaturing purification of RNAs. RNA 10:988–995Google Scholar
  43. Kim I, Lukavsky PJ, Puglisi JD (2002) NMR study of 100 kDa HCV IRES RNA using segmental isotope labeling. J Am Chem Soc 124:9338–9339Google Scholar
  44. Kremser J et al (2017) Chemical synthesis and NMR spectroscopy of long stable isotope labelled RNA. Chem Commun 53:12938–12941Google Scholar
  45. Krieg PA, Melton DA (1984) Functional messenger-RNAs are produced by SP6 invitro transcription of cloned CDNAs. Nucleic Acids Res 12:7057–7070Google Scholar
  46. Krieg PA, Melton DA (1987) In vitro RNA-synthesis with SP6-RNA polymerase. Methods Enzymol 155:397–415Google Scholar
  47. Lapham J, Crothers DM (1996) RNase H cleavage for processing of in vitro transcribed RNA for NMR studies and RNA ligation. RNA 2:289–296Google Scholar
  48. Lapinaite A et al (2013) The structure of the box C/D enzyme reveals regulation of RNA methylation. Nature 502:519–523ADSGoogle Scholar
  49. Latham MP, Brown DJ, McCallum SA, Pardi A (2005) NMR methods for studying the structure and dynamics of RNA. ChemBioChem 6:1492–1505Google Scholar
  50. Le MT, Brown RE, Simon AE, Dayie TK (2015) In vivo, large-scale preparation of uniformly N-15- and site-specifically C-13-labeled homogeneous, recombinant RNA for NMR studies. Methods Enzymol 565:495–535Google Scholar
  51. Leppert J et al (2004) Identification of NH … N hydrogen bonds by magic angle spinning solid state NMR in a double-stranded RNA associated with myotonic dystrophy. Nucleic Acids Res 32:1177–1183Google Scholar
  52. Lewandowski JR, De Paëpe G, Griffin RG (2007) Proton assisted insensitive nuclei cross polarization. J Am Chem Soc 129:728–729Google Scholar
  53. Liu Y et al (2015) Synthesis and applications of RNAs with position-selective labelling and mosaic composition. Nature 522:368–372ADSGoogle Scholar
  54. Liu Y, Sousa R, Wang YX (2016a) Specific labeling: an effective tool to explore the RNA world. Bioessays 38:192–200Google Scholar
  55. Liu Y et al (2016b) Applications of PLOR in labeling large RNAs at specific sites. Methods 103:4–10ADSGoogle Scholar
  56. Lu K, Miyazaki Y, Summers MF (2010) Isotope labeling strategies for NMR studies of RNA. J Biomol NMR 46:113–125Google Scholar
  57. Lu K et al (2011) NMR detection of structures in the HIV-1 5′-leader RNA that regulate genome packaging. Science 334:242–245ADSGoogle Scholar
  58. Lukavsky PJ, Puglisi JD (2004) Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA 10:889–893Google Scholar
  59. Marchanka A, Carlomagno T (2014) Solid-state NMR and RNA structure: a new partnership? eMagRes 3:119–128Google Scholar
  60. Marchanka A, Simon B, Carlomagno T (2013) A suite of solid-state NMR experiments for RNA resonance assignment in a 21 kDa protein-RNA complex. Angew Chem Int Ed 52:9996–10001Google Scholar
  61. Marchanka A, Simon B, Althoff-Ospelt G, Carlomagno T (2015) RNA structure determination by solid-state NMR spectroscopy. Nat Commun 6:7024ADSGoogle Scholar
  62. McKenna SA et al (2007) Purification and characterization of transcribed RNAs using gel filtration chromatography. Nat Protoc 2:3270–3277Google Scholar
  63. Melton DA et al (1984) Efficient invitro synthesis of biologically-active RNA and RNA hybridization probes from plasmids containing a bacteriophage-SP6 promoter. Nucleic Acids Res 12:7035–7056Google Scholar
  64. Milligan JF, Uhlenbeck OC (1989) Synthesis of small RNAs using T7 RNA-polymerase. Methods Enzymol 180:51–62Google Scholar
  65. Milligan JF, Groebe DR, Witherell GW, Uhlenbeck OC (1987) Oligoribonucleotide synthesis using T7 RNA-polymerase and synthetic DNA templates. Nucleic Acids Res 15:8783–8798Google Scholar
  66. Miyazaki Y et al (2010) Structure of a conserved retroviral RNA packaging element by NMR spectroscopy and cryo-electron tomography. J Mol Biol 404:751–772Google Scholar
  67. Nelissen FH et al. (2008) Multiple segmental and selective isotope labeling of large RNA for NMR structural studies. Nucleic Acids Res 36:e89Google Scholar
  68. Nikonowicz EP et al (1992) Preparation of C-13 and N-15 labeled Rnas for heteronuclear multidimensional NMR-studies. Nucleic Acids Res 20:4507–4513Google Scholar
  69. Ohgi T et al (2005) A new RNA synthetic method with a 2′-O-(2-cyanoethoxymethyl) protecting group. Org Lett 7:3477–3480Google Scholar
  70. Olsen GL et al (2005) Monitoring tat peptide binding to TAR RNA by solid-state P-31-F-19 REDOR NMR. Nucleic Acids Res 33:3447–3454Google Scholar
  71. Olsen GL et al (2008) Solid-state deuterium NMR studies reveal mu s-ns motions in the HIV-1 transactivation response RNA recognition site. J Am Chem Soc 130:2896–2897Google Scholar
  72. Olsen GL, Bardaro MF, Echodu DC, Drobny GP, Varani G (2010) Intermediate rate atomic trajectories of RNA by solid-state NMR spectroscopy. J Am Chem Soc 132:303–308Google Scholar
  73. Parkin DW, Schramm VL (1987) Catalytic and allosteric mechanism of amp nucleosidase from primary, beta-secondary, and multiple heavy-atom kinetic isotope effects. Biochemistry 26:913–920Google Scholar
  74. Pervushin K, Riek R, Wider G, Wuethrich K (1997) Attenuated T-2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift anisotropy indicates an avenue to NMR structures of very large biological macromolecules in solution. Proc Natl Acad Sci USA 94:12366–12371ADSGoogle Scholar
  75. Pleiss JA, Derrick ML, Uhlenbeck OC (1998) T7 RNA polymerase produces 5′ end heterogeneity during in vitro transcription from certain templates. RNA 4:1313–1317Google Scholar
  76. Ponchon L, Dardel F (2007) Recombinant RNA technology: the tRNA scaffold. Nat Methods 4:571Google Scholar
  77. Ponchon L, Dardel F (2011) Large scale expression and purification of recombinant RNA in Escherichia coli. Methods 54:267–273Google Scholar
  78. Ponchon L, Beauvais G, Nonin-Lecomte S, Dardel F (2009) A generic protocol for the expression and purification of recombinant RNA in Escherichia coli using a tRNA scaffold. Nat Protoc 4:947–959Google Scholar
  79. Quinn CM, Polenova T (2017) Structural biology of supramolecular assemblies by magic-angle spinning NMR spectroscopy. Q Rev Biophys 50:e1Google Scholar
  80. Reese CB (2005) Oligo- and poly-nucleotides: 50 years of chemical synthesis. Org Biomol Chem 3:3851–3868Google Scholar
  81. Religa TL, Sprangers R, Kay LE (2010) Dynamic regulation of archaeal proteasome gate opening as studied by TROSY NMR. Science 328:98–102ADSGoogle Scholar
  82. Ren AM et al (2016) Pistol ribozyme adopts a pseudoknot fold facilitating site-specific in-line cleavage. Nat Chem Biol 12:702–708Google Scholar
  83. Riedel K, Leppert J, Ohlenschläger O, Görlach M, Ramachandran R (2005a) Characterisation of hydrogen bonding networks in RNAs via magic angle spinning solid state NMR spectroscopy. J Biomol NMR 31:331–336Google Scholar
  84. Riedel K, Leppert J, Ohlenschläger O, Görlach M, Ramachandran R (2005b) TEDOR with adiabatic inversion pulses: resonance assignments of C-13/N-15 labelled RNAs. J Biomol NMR 31:49–57Google Scholar
  85. Riedel K et al (2006) Constraints on the structure of (CUG)(97) RNA from magic-angle-spinning solid-state NMR spectroscopy. Angew Chem Int Ed 45:5620–5623Google Scholar
  86. Riek R, Wider G, Pervushin K, Wüthrich K (1999) Polarization transfer by cross-correlated relaxation in solution NMR with very large molecules. Proc Natl Acad Sci USA 96:4918–4923ADSGoogle Scholar
  87. Santoro SW, Joyce GF (1997) A general purpose RNA-cleaving DNA enzyme. Proc Natl Acad Sci USA 94:4262–4266ADSGoogle Scholar
  88. Santoro SW, Joyce GF (1998) Mechanism and utility of an RNA-cleaving DNA enzyme. Biochemistry 37:13330–13342Google Scholar
  89. Scott LG, Geierstanger BH, Williamson JR, Hennig M (2004) Enzymatic synthesis and F-19 NMR studies of 2-fluoroadenine-substituted RNA. J Am Chem Soc 126:11776–11777Google Scholar
  90. Shiba Y et al (2007) Chemical synthesis of a very long oligoribonucleotide with 2-cyanoethoxymethyl (CEM) as the 2′-O-protecting group: structural identification and biological activity of a synthetic 110mer precursor-microRNA candidate. Nucleic Acids Res 35:3287–3296Google Scholar
  91. Shields TP, Mollova E, Marie LS, Hansen MR, Pardi A (1999) High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme. RNA 5:1259–1267Google Scholar
  92. Sich C, Ohlenschläger O, Ramachandran R, Görlach M, Brown LR (1997) Structure of an RNA hairpin loop with a 5′-CGUUUCG-3′ loop motif by heteronuclear NMR spectroscopy and distance geometry. Biochemistry 36:13989–14002Google Scholar
  93. Sprangers R, Kay LE (2007) Quantitative dynamics and binding studies of the 20S proteasome by NMR. Nature 445:618–622Google Scholar
  94. Szeverenyi NM, Sullivan MJ, Maciel GE (1982) Observation of spin exchange by two-dimensional Fourier-transform C-13 cross polarization-magic-angle spinning. J Magn Reson 47:462–475ADSGoogle Scholar
  95. Thakur CS, Luo Y, Chen B, Eldho NV, Dayie TK (2012) Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR. J Biomol NMR 52:103–114Google Scholar
  96. Tolbert TJ, Williamson JR (1996) Preparation of specifically deuterated RNA for NMR studies using a combination of chemical and enzymatic synthesis. J Am Chem Soc 118:7929–7940Google Scholar
  97. Tolbert TJ, Williamson JR (1997) Preparation of specifically deuterated and C-13-labeled RNA for NMR studies using enzymatic synthesis. J Am Chem Soc 119:12100–12108Google Scholar
  98. Tugarinov V, Hwang PM, Ollerenshaw JE, Kay LE (2003) Cross-correlated relaxation enhanced H-1-C-13 NMR spectroscopy of methyl groups in very high molecular weight proteins and protein complexes. J Am Chem Soc 125:10420–10428Google Scholar
  99. Tzakos AG, Easton LE, Lukavsky PJ (2007) Preparation of large RNA oligonucleotides with complementary isotope-labeled segments for NMR structural studies. Nat Protoc 2:2139–2147Google Scholar
  100. Varani G, Aboul-Ela F, Allain FHT (1996) NMR investigation of RNA structure. Prog Nucl Magn Reson Spectrosc 29:51–127Google Scholar
  101. Wang X et al (2015) N6-methyladenosine modulates messenger RNA translation efficiency. Cell 161:1388–1399Google Scholar
  102. Wolter AC et al (2017) A stably protonated adenine nucleotide with a highly shifted pK(a) value stabilizes the tertiary structure of a GTP-binding RNA Aptamer. Angew Chem Int Ed 56:401–404Google Scholar
  103. Wunderlich CH et al (2012) Synthesis of (6-C-13)Pyrimidine nucleotides as spin-labels for RNA dynamics. J Am Chem Soc 134:7558–7569Google Scholar
  104. Wyatt JR, Chastain M, Puglisi JD (1991) Synthesis and purification of large amounts of RNA oligonucleotides. Biotechniques 11:764–769Google Scholar
  105. Xu J, Lapham J, Crothers DM (1996) Determining RNA solution structure by segmental isotopic labeling and NMR: application to caenorhabditis elegans spliced leader RNA 1. Proc Natl Acad Sci USA 93:44–48ADSGoogle Scholar
  106. Yang DW, Kay LE (1999) TROSY triple-resonance four-dimensional NMR spectroscopy of a 46 ns tumbling protein. J Am Chem Soc 121:2571–2575Google Scholar
  107. Yang Y et al (2017) Proton-detected solid-state NMR Detects the inter-nucleotide correlations and architecture of dimeric RNA in microcrystals. Chem Commun 53:12886–12889Google Scholar

Copyright information

© Springer Science+Business Media B.V., part of Springer Nature 2018

Authors and Affiliations

  1. 1.Centre for Biomolecular Drug Research (BMWZ) and Institute of Organic ChemistryLeibniz University HannoverHanoverGermany
  2. 2.Organic ChemistryUniversity of Innsbruck (CCB)InnsbruckAustria
  3. 3.Helmholtz Centre for Infection Research, Group of NMR-based Structural ChemistryBrunswickGermany

Personalised recommendations