Journal of Biomolecular NMR

, Volume 63, Issue 1, pp 97–109 | Cite as

Dynamic nuclear polarization of nucleic acid with endogenously bound manganese

  • Patricia Wenk
  • Monu Kaushik
  • Diane Richter
  • Marc Vogel
  • Beatrix Suess
  • Björn Corzilius


We report the direct dynamic nuclear polarization (DNP) of 13C nuclei of a uniformly [13C,15N]-labeled, paramagnetic full-length hammerhead ribozyme (HHRz) complex with Mn2+ where the enhanced polarization is fully provided by the endogenously bound metal ion and no exogenous polarizing agent is added. A 13C enhancement factor of ε = 8 was observed by intra-complex DNP at 9.4 T. In contrast, “conventional” indirect and direct DNP experiments were performed using AMUPol as polarizing agent where we obtained a 1H enhancement factor of ε ≈ 250. Comparison with the diamagnetic (Mg2+) HHRz complex shows that the presence of Mn2+ only marginally influences the (DNP-enhanced) NMR properties of the RNA. Furthermore two-dimensional correlation spectra (15N–13C and 13C–13C) reveal structural inhomogeneity in the frozen, amorphous state indicating the coexistence of several conformational states. These demonstrations of intra-complex DNP using an endogenous metal ion as well as DNP-enhanced MAS NMR of RNA in general yield important information for the development of new methods in structural biology.


Dynamic nuclear polarization (DNP) Solid-state NMR EPR RNA Transition metal Polarizing agent 



This work was supported by the Deutsche Forschungsgemeinschaft (DFG) via Emmy Noether Grant CO802/2-1 issued to B.C. as well as DFG collaborative research center (Sonderforschungsbereich) 902, and by the Center for Biomolecular Magnetic Resonance (BMRZ). We thank J. Becker-Baldus and T. Gutmann for access to DNP spectrometers and technical assistance. Help from D. Akhmetzyanov during 180 GHz EPR experiments is gratefully acknowledged. We thank T. Prisner for proposing HHRz as the target system of this study, as well as H. Schwalbe and J. Wöhnert for helpful discussions. The sweepable MAS DNP NMR spectrometer was Granted to G. Buntkowsky (Darmstadt) via DFG Grant BU911/20-1.


Deutsche Forschungsgemeinschaft (DFG) Grants CO802/2-1, SFB902, and BU911/20-1; Center for Biomolecular Magnetic Resonance (BMRZ).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

10858_2015_9972_MOESM1_ESM.pdf (2.9 mb)
Supplementary material 1 (PDF 2987 kb)


  1. Abragam A, Proctor WG (1958) Une nouvelle méthode de polarisation dynamique des noyaux atomiques dans les solides. CR Hebd Acad Sci 246:2253–2256Google Scholar
  2. Abramov G, Goldbourt A (2014) Nucleotide-type chemical shift assignment of the encapsulated 40 kbp dsDNA in intact bacteriophage T7 by MAS solid-state NMR. J Biomol NMR 59:219–230CrossRefGoogle Scholar
  3. Akbey Ü, Franks WT, Linden A, Lange S, Griffin RG, van Rossum BJ, Oschkinat H (2010) Dynamic nuclear polarization of deuterated proteins. Angew Chem Int Ed 49:7803–7806CrossRefGoogle Scholar
  4. Bassi GS, Murchie AI, Lilley DM (1996) The ion-induced folding of the hammerhead ribozyme: core sequence changes that perturb folding into the active conformation. RNA 2:756–768Google Scholar
  5. Bayro MJ, Maly T, Birkett NR, MacPhee CE, Dobson CM, Griffin RG (2010) High-resolution MAS NMR analysis of PI3-SH3 amyloid fibrils: backbone conformation and implications for protofilament assembly and structure. Biochemistry 49:7474–7484CrossRefGoogle Scholar
  6. Becerra LR, Gerfen GJ, Temkin RJ, Singel DJ, Griffin RG (1993) Dynamic nuclear polarization with a cyclotron resonance maser at 5T. Phys Rev Lett 71:3561–3564CrossRefADSGoogle Scholar
  7. Beilstein K, Wittmann A, Grez M, Suess B (2015) Conditional control of mammalian gene expression by tetracycline-dependent hammerhead ribozymes. ACS Synth Biol 4:526–534CrossRefGoogle Scholar
  8. Bloembergen N (1949) On the interaction of nuclear spins in a crystalline lattice. Physica 15:386–426CrossRefADSGoogle Scholar
  9. Bothe JR, Nikolova EN, Eichhorn CD, Chugh J, Hansen AL, Al-Hashimi HM (2011) Characterizing RNA dynamics at atomic resolution using solution-state NMR spectroscopy. Nat Methods 8:919–931CrossRefGoogle Scholar
  10. Castellani F, van Rossum B, Diehl A, Schubert M, Rehbein K, Oschkinat H (2002) Structure of a protein determined by solid-state magic-angle-spinning NMR spectroscopy. Nature 420:98–102CrossRefADSGoogle Scholar
  11. Cherepanov AV, Glaubitz C, Schwalbe H (2010) High-resolution studies of uniformly 13C,15N-labeled RNA by solid-state NMR spectroscopy. Angew Chem Int Ed 49:4747–4750CrossRefGoogle Scholar
  12. Chi Y-I, Martick M, Lares M, Kim R, Scott WG, Kim S-H (2008) Capturing hammerhead ribozyme structures in action by modulating general base catalysis. PLoS Biol 6:2060–2068Google Scholar
  13. Corzilius B, Smith AA, Barnes AB, Luchinat C, Bertini I, Griffin RG (2011) High-Field dynamic nuclear polarization with high-spin transition metal ions. J Am Chem Soc 133:5648–5651CrossRefGoogle Scholar
  14. Corzilius B, Smith AA, Griffin RG (2012) Solid Effect in magic angle spinning dynamic nuclear polarization. J Chem Phys 137:054201CrossRefADSGoogle Scholar
  15. Corzilius B, Andreas LB, Smith AA, Ni QZ, Griffin RG (2014) Paramagnet-induced signal quenching in MAS-DNP experiments on frozen homogeneous solutions. J Magn Reson 240:113–123CrossRefADSGoogle Scholar
  16. Dahm SC, Uhlenbeck OC (1991) Role of divalent metal ions in the hammerhead RNA cleavage reaction. Biochemistry 30:9464–9469CrossRefGoogle Scholar
  17. De la Peña M, Gago S, Flores R (2003) Peripheral regions of natural hammerhead ribozymes greatly increase their self-cleavage activity. EMBO J 22:5561–5570CrossRefGoogle Scholar
  18. Fernández-de-Alba C et al (2015) Matrix-free DNP-enhanced NMR spectroscopy of liposomes using a lipid-anchored biradical, chemistry. Eur J 21:4512–4517CrossRefGoogle Scholar
  19. Forster AC, Symons RH (1987) Self-cleavage of virusoid RNA is performed by the proposed 55-nucleotide active site. Cell 50:9–16CrossRefGoogle Scholar
  20. Franks WT, Kloepper K, Wylie B, Rienstra C (2007) Four-dimensional heteronuclear correlation experiments for chemical shift assignment of solid proteins. J Biomol NMR 39:107–131CrossRefGoogle Scholar
  21. Fürtig B, Richter C, Wöhnert J, Schwalbe H (2003) NMR spectroscopy of RNA. ChemBioChem 4:936–962CrossRefGoogle Scholar
  22. Fürtig B, Richter C, Schell P, Wenter P, Pitsch S, Schwalbe H (2008) NMR-spectroscopic characterisation of phosphodiester bond cleavage catalyzed by the minimal hammerhead ribozyme. RNA Biol 5:41–48CrossRefGoogle Scholar
  23. Glowinkowski S, Jurga S, Suchanski W, Szczesniak E (1997) Local and global dynamics in the glass-forming di-isobutyl phthalate as studied by 1H NMR. Solid State Nucl Magn Reson 7:313–317CrossRefGoogle Scholar
  24. Gutsche P, Rinsdorf M, Zimmermann H, Schmitt H, Haeberlen U (2004) The shape and information content of high-field solid-state proton NMR spectra of methyl groups. Solid State Nucl Magn Reson 25:227–240CrossRefGoogle Scholar
  25. Hall DA, Maus DC, Gerfen GJ, Inati SJ, Becerra LR, Dahlquist FW, Griffin RG (1997) Polarization-enhanced NMR spectroscopy of biomolecules in frozen solution. Science 276:930–932CrossRefGoogle Scholar
  26. Hammann C, Norman DG, Lilley DMJ (2001) Dissection of the ion-induced folding of the hammerhead ribozyme using 19F NMR. Proc Natl Acad Sci U S A 98:5503–5508CrossRefADSGoogle Scholar
  27. Hing AW, Vega S, Schaefer J (1992) Transferred-echo double-resonance NMR. J Magn Reson 96:205–209ADSGoogle Scholar
  28. Horton TE, Clardy DR, DeRose VJ (1998) Electron paramagnetic resonance spectroscopic measurement of Mn2+ binding affinities to the hammerhead ribozyme and correlation with cleavage activity. Biochemistry 37:18094–18101CrossRefGoogle Scholar
  29. Hovav Y, Feintuch A, Vega S (2010) Theoretical aspects of dynamic nuclear polarization in the solid state—the solid effect. J Magn Reson 207:176–189CrossRefADSGoogle Scholar
  30. Hovav Y, Feintuch A, Vega S (2012) Theoretical aspects of dynamic nuclear polarization in the solid state—the cross effect. J Magn Reson 214:29–41CrossRefADSGoogle Scholar
  31. Hu KN (2011) Polarizing agents and mechanisms for high-field dynamic nuclear polarization of frozen dielectric solids. Solid State Nucl Magn Reson 40:31–41CrossRefADSGoogle Scholar
  32. Hu KN, Yu HH, Swager TM, Griffin RG (2004) Dynamic nuclear polarization with biradicals. J Am Chem Soc 126:10844–10845CrossRefGoogle Scholar
  33. Hu KN, Debelouchina GT, Smith AA, Griffin RG (2011) Quantum mechanical theory of dynamic nuclear polarization in solid dielectrics. J Chem Phys 134:19Google Scholar
  34. Huang W, Bardaro MF Jr, Varani G, Drobny GP (2012) Preparation of RNA samples with narrow line widths for solid state NMR investigations. J Magn Reson 223:51–54CrossRefADSGoogle Scholar
  35. Hutchins CJ, Rathjen PD, Forster AC, Symons RH (1986) Self-cleavage of plus and minus RNA transcripts of avocado sunblotch viroid. Nucleic Acids Res 14:3627–3640CrossRefGoogle Scholar
  36. Jaroniec CP, Filip C, Griffin RG (2002) 3D TEDOR NMR experiments for the simultaneous measurement of multiple carbon-nitrogen distances in uniformly C-13, N-15-labeled solids. J Am Chem Soc 124:10728–10742CrossRefGoogle Scholar
  37. Jeffries CD (1957) Polarization of nuclei by resonance saturation in paramagnetic crystals. Phys Rev 106:164–165CrossRefADSGoogle Scholar
  38. Jeffries CD (1960) Dynamic orientation of nuclei by forbidden transitions in paramagnetic resonance. Phys Rev 117:1056–1069CrossRefADSGoogle Scholar
  39. Kessenikh AV, Lushchikov VI, Manenkov AA, Taran YV (1963) Proton polarization in irradiated polyethylenes. Sov Phys-Sol State 5:321–329Google Scholar
  40. Khvorova A, Lescoute A, Westhof E, Jayasena SD (2003) Sequence elements outside the hammerhead ribozyme catalytic core enable intracellular activity. Nat Struct Mol Biol 10:708–712CrossRefGoogle Scholar
  41. Lange S, Linden AH, Akbey Ü, Franks WT, Loening NM, van Rossum B-J, Oschkinat H (2012) The effect of biradical concentration on the performance of DNP-MAS-NMR. J Magn Reson 216:209–212CrossRefADSGoogle Scholar
  42. Li Y, Berthold DA, Frericks HL, Gennis RB, Rienstra CM (2007) Partial 13C and 15N chemical-shift assignments of the disulfide-bond-forming enzyme DsbB by 3D magic-angle spinning NMR spectroscopy. ChemBioChem 8:434–442zbMATHCrossRefGoogle Scholar
  43. Maly T, Miller AF, Griffin RG (2010) In situ high-field dynamic nuclear polarization-direct and indirect polarization of C-13 nuclei. ChemPhysChem 11:999–1001CrossRefGoogle Scholar
  44. Maly T, Cui D, Griffin RG, Miller A-F (2012) 1H dynamic nuclear polarization based on an endogenous radical. J Phys Chem B 116:7055–7065CrossRefGoogle Scholar
  45. Marchanka A, Simon B, Carlomagno T (2013) A suite of solid-state NMR experiments for RNA intranucleotide resonance assignment in a 21 kDa protein–RNA complex. Angew Chem Int Ed 52:9996–10001CrossRefGoogle Scholar
  46. Marchanka A, Simon B, Althoff-Ospelt G, Carlomagno T (2015) RNA structure determination by solid-state NMR spectroscopy. Nat Commun 6:7024CrossRefADSGoogle Scholar
  47. Martick M, Scott WG (2006) Tertiary contacts distant from the active site prime a ribozyme for catalysis. Cell 126:309–320CrossRefGoogle Scholar
  48. Martick M, Lee T-S, York DM, Scott WG (2008) Solvent structure and hammerhead ribozyme catalysis. Chem Biol 15:332–342CrossRefGoogle Scholar
  49. Morrissey SR, Horton TE, Grant CV, Hoogstraten CG, Britt RD, DeRose VJ (1999) Mn2+-nitrogen interactions in RNA probed by electron spin-echo envelope modulation spectroscopy: application to the hammerhead ribozyme. J Am Chem Soc 121:9215–9218CrossRefGoogle Scholar
  50. Morrissey SR, Horton TE, DeRose VJ (2000) Mn2+ sites in the hammerhead ribozyme investigated by EPR and continuous-wave Q-band ENDOR spectroscopies. J Am Chem Soc 122:3473–3481CrossRefGoogle Scholar
  51. Murray JB, Terwey DP, Maloney L, Karpeisky A, Usman N, Beigelman L, Scott WG (1998) The structural basis of hammerhead ribozyme self-cleavage. Cell 92:665–673CrossRefGoogle Scholar
  52. Ni QZ et al (2013) High frequency dynamic nuclear polarization. Acc Chem Res 46:1933–1941CrossRefGoogle Scholar
  53. Nikolova EN, Kim E, Wise AA, O’Brien PJ, Andricioaei I, Al-Hashimi HM (2011) Transient Hoogsteen base pairs in canonical duplex DNA. Nature 470:498–502CrossRefADSGoogle Scholar
  54. Nozirov F, Nazirov A, Jurga S, Fu R (2006) Molecular dynamics of poly(l-lactide) biopolymer studied by wide-line solid-state 1H and 2H NMR spectroscopy. Solid State Nucl Magn Reson 29:258–266CrossRefGoogle Scholar
  55. Osborne EM, Ward WL, Ruehle MZ, DeRose VJ (2009) The identity of the nucleophile substitution may influence metal interactions with the cleavage site of the minimal hammerhead ribozyme. Biochemistry 48:10654–10664CrossRefGoogle Scholar
  56. Overhauser AW (1953) Polarization of nuclei in metals. Phys Rev 92:411–415zbMATHCrossRefADSGoogle Scholar
  57. Pauli J, Baldus M, van Rossum B, de Groot H, Oschkinat H (2001) Backbone and side-chain 13C and 15N signal assignments of the α-spectrin SH3 domain by magic angle spinning solid-state NMR at 17.6 Tesla. ChemBioChem 2:272–281CrossRefGoogle Scholar
  58. Pley HW, Flaherty KM, McKay DB (1994) Three-dimensional structure of a hammerhead ribozyme. Nature 372:68–74CrossRefADSGoogle Scholar
  59. Prody GA, Bakos JT, Buzayan JM, Schneider IR, Bruening G (1986) Autolytic processing of dimeric plant virus satellite RNA. Science 231:1577–1580CrossRefADSGoogle Scholar
  60. Renault M et al (2012) Solid-state NMR spectroscopy on cellular preparations enhanced by dynamic nuclear polarization. Angew Chem Int Ed 51:2998–3001CrossRefGoogle Scholar
  61. Rinnenthal J, Buck J, Ferner J, Wacker A, Fürtig B, Schwalbe H (2011) Mapping the landscape of RNA dynamics with NMR spectroscopy. Acc Chem Res 44:1292–1301CrossRefGoogle Scholar
  62. Rohrer M, Brügmann O, Kinzer B, Prisner TF (2001) High-field/high-frequency EPR spectrometer operating in pulsed and continuous-wave mode at 180 GHz. Appl Magn Reson 21:257–274CrossRefGoogle Scholar
  63. Rossini AJ et al (2012) One hundred fold overall sensitivity enhancements for Silicon-29 NMR spectroscopy of surfaces by dynamic nuclear polarization with CPMG acquisition. Chem Sci 3:108–115CrossRefGoogle Scholar
  64. Sauvée C, Rosay M, Casano G, Aussenac F, Weber RT, Ouari O, Tordo P (2013) Highly efficient, water-soluble polarizing agents for dynamic nuclear polarization at high frequency. Angew Chem Int Ed 52:10858–10861CrossRefGoogle Scholar
  65. Schiemann O, Fritscher J, Kisseleva N, Sigurdsson ST, Prisner TF (2003) Structural investigation of a high-affinity MnII binding site in the hammerhead ribozyme by EPR spectroscopy and DFT calculations. Effects of neomycin B on metal–ion binding. ChemBioChem 4:1057–1065CrossRefGoogle Scholar
  66. Scott WG, Finch JT, Klug A (1995) The crystal structure of an AII-RNA hammerhead ribozyme: a proposed mechanism for RNA catalytic cleavage. Cell 81:991–1002CrossRefGoogle Scholar
  67. Scott WG, Murray JB, Arnold JRP, Stoddard BL, Klug A (1996) Capturing the structure of a catalytic RNA intermediate: the hammerhead ribozyme. Science 274:2065–2069CrossRefADSGoogle Scholar
  68. Shimon D, Hovav Y, Feintuch A, Goldfarb D, Vega S (2012) Dynamic nuclear polarization in the solid state: a transition between the cross effect and the solid effect. Phys Chem Chem Phys 14:5729–5743CrossRefGoogle Scholar
  69. Smith AA, Corzilius B, Barnes AB, Maly T, Griffin RG (2012) Solid effect dynamic nuclear polarization and polarization pathways. J Chem Phys 136:015101CrossRefADSGoogle Scholar
  70. Smith AN, Caporini MA, Fanucci GE, Long JR (2015) A method for dynamic nuclear polarization enhancement of membrane proteins. Angew Chem Int Ed 54:1542–1546CrossRefGoogle Scholar
  71. Sripakdeevong P et al (2014) Structure determination of noncanonical RNA motifs guided by 1H NMR chemical shifts. Nat Methods 11:413–416CrossRefGoogle Scholar
  72. Stoll S, Schweiger A (2006) EasySpin, a comprehensive software package for spectral simulation and analysis in EPR. J Magn Reson 178:42–55CrossRefADSGoogle Scholar
  73. Szeverenyi NM, Sullivan MJ, Maciel GE (1982) Observation of spin exchange by two-dimensional fourier transform 13C cross polarization-magic-angle spinning. J Magn Reson 47:462–475ADSGoogle Scholar
  74. Wang S, Karbstein K, Peracchi A, Beigelman L, Herschlag D (1999) Identification of the hammerhead ribozyme metal ion binding site responsible for rescue of the deleterious effect of a cleavage site phosphorothioate. Biochemistry 38:14363–14378CrossRefGoogle Scholar
  75. Ward WL, DeRose VJ (2012) Ground-state coordination of a catalytic metal to the scissile phosphate of a tertiary-stabilized hammerhead ribozyme. RNA 18:16–23CrossRefGoogle Scholar
  76. Wasmer C, Lange A, Van Melckebeke H, Siemer AB, Riek R, Meier BH (2008) Amyloid fibrils of the HET-s(218-289) prion form a beta solenoid with a triangular hydrophobic core. Science 319:1523–1526CrossRefADSGoogle Scholar
  77. Wenckebach WT (2008) The solid effect. Appl Magn Reson 34:227–235CrossRefGoogle Scholar
  78. Wittmann A, Suess B (2011) Selection of tetracycline inducible self-cleaving ribozymes as synthetic devices for gene regulation in yeast. Mol BioSyst 7:2419–2427CrossRefGoogle Scholar
  79. Wylie BJ, Dzikovski BG, Pawsey S, Caporini M, Rosay M, Freed JH, McDermott AE (2015) Dynamic nuclear polarization of membrane proteins: covalently bound spin-labels at protein–protein interfaces. J Biomol NMR 61:361–367CrossRefGoogle Scholar
  80. Yen L et al (2004) Exogenous control of mammalian gene expression through modulation of RNA self-cleavage. Nature 431:471–476CrossRefADSGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Patricia Wenk
    • 1
    • 3
  • Monu Kaushik
    • 1
  • Diane Richter
    • 1
  • Marc Vogel
    • 2
  • Beatrix Suess
    • 2
  • Björn Corzilius
    • 1
  1. 1.Institute of Physical und Theoretical Chemistry, Institute of Biophysical Chemistry und Center for Biomolecular Magnetic Resonance (BMRZ)Goethe UniversityFrankfurt am MainGermany
  2. 2.Department of BiologyTechnical University DarmstadtDarmstadtGermany
  3. 3.Werner Siemens Imaging Center and Department of Preclinical Imaging and RadiopharmacyUniversity of TübingenTübingenGermany

Personalised recommendations