Skip to main content
SpringerLink
Log in

Evaluation of 15N-detected H–N correlation experiments on increasingly large RNAs

  • Article
  • Published:
Journal of Biomolecular NMR Aims and scope Submit manuscript

Abstract

Recently, 15N-detected multidimensional NMR experiments have been introduced for the investigation of proteins. Utilization of the slow transverse relaxation of nitrogen nuclei in a 15N-TROSY experiment allowed recording of high quality spectra for high molecular weight proteins, even in the absence of deuteration. Here, we demonstrate the applicability of three 15N-detected H–N correlation experiments (TROSY, BEST-TROSY and HSQC) to RNA. With the newly established 15N-detected BEST-TROSY experiment, which proves to be the most sensitive 15N-detected H–N correlation experiment, spectra for five RNA molecules ranging in size from 5 to 100 kDa were recorded. These spectra yielded high resolution in the 15N-dimension even for larger RNAs since the increase in line width with molecular weight is more pronounced in the 1H- than in the 15N-dimension. Further, we could experimentally validate the difference in relaxation behavior of imino groups in AU and GC base pairs. Additionally, we showed that 15N-detected experiments theoretically should benefit from sensitivity and resolution advantages at higher static fields but that the latter is obscured by exchange dynamics within the RNAs.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  • Adrian M, Heddi B, Phan AT (2012) NMR spectroscopy of G-quadruplexes. Methods 57:11–24

    Article  Google Scholar 

  • Alexandrescu AT, Loh SN, Markley JL (1990) Chemical exchange spectroscopy based on carbon-13 NMR. Applications to enzymology and protein folding. J Magn Reson 87:523–535

    ADS  Google Scholar 

  • Alvarado LJ, Leblanc RM, Longhini AP, Keane SC, Jain N, Yildiz ZF, Tolbert BS, Souza V. M. D., Summers MF, Kreutz C, Dayie TK (2014) Regio-selective chemical-enzymatic synthesis of pyrimidine nucleotides facilitates RNA structure and dynamics studies. ChemBioChem 15:1573–1577

    Article  Google Scholar 

  • Bax A, Morris GA (1981) An improved method for heteronuclear chemical shift correlation by two-dimensional NMR. J Magn Reson 42:501–505

    ADS  Google Scholar 

  • Bermel W, Bertini I, Felli IC, Piccioli M, Pierattelli R (2006a) 13C-detected protonless NMR spectroscopy of proteins in solution. Prog Nucl Magn Reson Spectrosc 48:25–45

    Article  Google Scholar 

  • Bhattacharya PK, Cha J, Barton JK (2002) 1H NMR determination of base-pair lifetimes in oligonucleotides containing single base mismatches. Nucleic Acids Res 30:4740–4750

    Article  Google Scholar 

  • Czernek J (2001) An ab Initio study of hydrogen bonding effects on the tensors in the Watson—Crick base pairs 15N and 1H chemical shielding. J Phys Chem 105:1357–1365

    Article  Google Scholar 

  • de la Torre JG, Huertas ML, Carrasco B. HYDRONMR (2000) Prediction of NMR relaxation of globular proteins from atomic-level structures and hydrodynamic calculations. J Magn Reson 147:138–146

    Article  ADS  Google Scholar 

  • Dethoff EA, Petzold K, Chugh J, Casiano-negroni A, Al-Hashimi HM (2012) Visualizing transient low-populated structures of RNA. Nature 491:724–728

    ADS  Google Scholar 

  • Dingley AJ, Grzesiek S (1998) Direct observation of hydrogen bonds in nucleic acid base pairs by internucleotide 2JNN couplings. J Am Chem Soc 7863:714–718

    Google Scholar 

  • Dittmer J, Kim C-H, Bodenhausen G (2003) Similarities between intra- and intermolecular hydrogen bonds in RNA kissing complexes found by means of cross-correlated relaxation. J Biomol NMR 26:259–275

    Article  Google Scholar 

  • Duchardt-Ferner E, Gottstein-Schmidtke SR, Weigand JE, Ohlenschläger O, Wurm J-P, Hammann C, Suess B, Wöhnert J (2016) What a difference an OH makes: conformational dynamics as the basis for the ligand specificity of the neomycin-sensing riboswitch. Angew Chem Int Ed 55:1527–1530

    Article  Google Scholar 

  • Fares C, Amata I, Carlomagno T (2007) 13C-detection in RNA bases: revealing structure - chemical shift relationships. J Am Chem Soc 129:15814–15823

    Article  Google Scholar 

  • Favier A, Brutscher B (2011) Recovering lost magnetization: polarization enhancement in biomolecular NMR. J Biomol NMR 49:9–15

    Article  Google Scholar 

  • Felli IC, Pierattelli R (2014) Novel methods based on proteins 13C detection to study intrinsically disordered proteins. J Magn Reson 241:115–125

    Article  ADS  Google Scholar 

  • Fiala R, Sklenár V (2007) 13C-detected NMR experiments for measuring chemical shifts and coupling constants in nucleic acid bases. J Biomol NMR 39:153–163

    Article  Google Scholar 

  • Fürtig B, Richter C, Bermel W, Schwalbe H (2004) New NMR experiments for RNA nucleobase resonance assignment and chemical shift analysis of an RNA UUCG tetraloop. J Biomol NMR 28:69–79

    Article  Google Scholar 

  • Fürtig B, Schnieders R, Richter C, Zetzsche H, Keyhani S, Helmling C, Kovacs H, Schwalbe H (2016) Direct 13C-detected NMR experiments for mapping and characterization of hydrogen bonds in RNA. J Biomol NMR 64:207–221

    Article  Google Scholar 

  • Gal M, Edmonds KA, Milbradt AG, Takeuchi K, Wagner G (2011) Speeding up direct 15N detection: hCaN 2D NMR experiment. J Biomol NMR 51:497–504

    Article  Google Scholar 

  • Geen H, Freeman R (1991) Band-selective radiofrequency pulses. J Magn Reson 93:93–141

    ADS  Google Scholar 

  • Grishaev A, Yao L, Ying J, Pardi A, Bax A (2009) Chemical shift anisotropy of imino 15N nuclei in Watson—Crick base pairs from magic angle spinning liquid crystal NMR and nuclear spin relaxation. J Am Chem Soc 131:9490–9491

    Article  Google Scholar 

  • Grzesiek S, Bax A (1993) The Importance of not saturating H2O in protein NMR. Application to sensitivity enhancement and NOE measurements. J Am Chem Soc 115:12593–12594

    Article  Google Scholar 

  • Guillerez J, Lopez PJ, Proux F, Launay H, Dreyfus M (2005) A mutation in T7 RNA polymerase that facilitates promoter clearance. Proc Natl Acad Sci USA 102:5958–5963

    Article  ADS  Google Scholar 

  • Helmling C, Keyhani S, Sochor F, Fürtig B, Hengesbach M, Schwalbe H (2015) Rapid NMR screening of RNA secondary structure and binding. 63:67–76

  • Imai S, Kumar P, Hellen CUT., Souza VMD., Wagner G (2016) An accurately preorganized IRES RNA structure enables eIF4G capture for initiation of viral translation. Nat Struct Mol Biol 23:859–864

    Article  Google Scholar 

  • Kovacs H, Moskau D, Spraul M (2005) Cryogenically cooled probes—a leap in NMR technology. Prog Nucl Magn Reson Spectrosc 46:131–155

    Article  Google Scholar 

  • Leppert J, Urbinati CR, Häfner S, Ohlenschläger O, Swanson MS, Görlach M, Ramachandran R (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 32:1177–1183

    Article  Google Scholar 

  • Lu K, Heng X, Garyu L, Monti S, Garcia EL, Kharytonchyk S, Dorjsuren B, Kulandaivel G, Jones S, Hiremath A, Divakaruni SS, Lacotti C, Barton S, Tummillo D, Hosic A, Edme K, Albrecht S, Telesnitsky A, Summers MF (2011) NMR detection of structures in the HIV-1 5′-leader RNA that regulate genome packaging. Science 334:242–245

    Article  ADS  Google Scholar 

  • Noeske J, Richter C, Grundl MA, Nasiri HR, Schwalbe H, Wöhnert J (2005) An intermolecular base triple as the basis of ligand specificity and affinity in the guanine- and adenine-sensing riboswitch RNAs. Proc Natl Acad Sci 102:1372–1377

    Article  ADS  Google Scholar 

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

    Article  Google Scholar 

  • Paige JS, Wu KY, Jaffrey SR (2011) RNA mimics of green fluorescent protein. Science 333:642–646

    Article  ADS  Google Scholar 

  • Pervushin K, Eletsky A (2003) A new strategy for backbone resonance assignment in large proteins using a MQ-HACACO experiment. J Biomol NMR 25:147–152

    Article  Google Scholar 

  • Pervushin K, Riek R, Wider G, Wüthrich K (1997) Attenuated T2 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 94:12366–12371

    Article  ADS  Google Scholar 

  • Redfield AG (1957) On the theory of relaxation processes. IBM J Res Dev 1:19–31

    Article  Google Scholar 

  • Reining A, Nozinovic S, Schlepckow K, Buhr F, Fürtig B, Schwalbe H (2013) Three-state mechanism couples ligand and temperature sensing in riboswitches. Nature 499:355–359

    Article  ADS  Google Scholar 

  • Richter C, Kovacs H, Buck J, Wacker A, Fürtig B, Bermel W, Schwalbe H (2010) 13C-direct detected NMR experiments for the sequential J-based resonance assignment of RNA oligonucleotides. J Biomol NMR 47:259–269

    Article  Google Scholar 

  • Rinnenthal J, Klinkert B, Narberhaus F, Schwalbe H (2010) Direct observation of the temperature-induced melting process of the Salmonella fourU RNA thermometer at base-pair resolution. Nucleic Acids Res 38:3834–3847

    Article  Google Scholar 

  • Rovnyak D, Hoch JC, Stern AS, Wagner G (2004) Resolution and sensitivity of high field nuclear magnetic resonance. J Biomol NMR 30:1–10

    Article  Google Scholar 

  • Sadek M, Brownlee RTC (1995) Study of 15N-labeled paramagnetic compunds by 15N-detected NMR experiments. J Magn Reson 109:70–75

    Article  Google Scholar 

  • Serber Z, Richter C, Moskau D, Bo J, Gerfin T, Marek D, Baselgia L, Laukien F, Stern AS, Hoch JC, Dötsch V (2000) New carbon-detected protein NMR experiments using CryoProbes. J Am Chem Soc 122:3554–3555

    Article  Google Scholar 

  • Serganov A, Yuan Y, Pikovskaya O, Polonskaia A, Malinina L, Phan AT, Hobartner C, Micura R, Breaker RR, Patel DJ (2004) Structural basis for discriminative regulation of gene expression by adenine- and guanine-sensing mRNAs. Chem Biol 11:1729–1741

    Article  Google Scholar 

  • Shaka AJ, Barker PB, Freeman R (1985) Computer-optimized decoupling scheme for wideband applications and low-level operation. J Magn Reson 64:547–552

    ADS  Google Scholar 

  • Silvers R, Keller H, Schwalbe H, Hengesbach M (2015) Differential scanning fluorimetry for monitoring RNA stability. Chembiochem 16:1109–1114

    Article  Google Scholar 

  • Solyom Z, Schwarten M, Geist L, Konrat R, Willbold D, Brutscher B (2013) BEST-TROSY experiments for time-efficient sequential resonance assignment of large disordered proteins. J Biomol NMR 55:311–321

    Article  Google Scholar 

  • Stoldt M, Wöhnert J, Görlach M, Brown LR (1998) The NMR structure of Escherichia coli ribosomal protein L25 shows homology to general stress proteins and glutaminyl-tRNA synthetases. EMBO J 17:6377–6384

    Article  Google Scholar 

  • Stoldt M, Wöhnert J, Görlach M, Brown LR (1999) The NMR structure of the 5S rRNA E-domain–protein L25 complex shows preformed and induced recognition. EMBO J 18:6508–6521

    Article  Google Scholar 

  • Takeuchi K, Heffron G, Frueh DP, Wagner G (2010) Nitrogen-detected CAN and CON experiments as alternative experiments for main chain NMR resonance assignments. J Biomol NMR 47:271–282

    Article  Google Scholar 

  • Takeuchi K, Arthanari H, Shimada I, Wagner G (2015) Nitrogen detected TROSY at high field yields high resolution and sensitivity for protein NMR. J Biomol NMR 63:1–9

    Article  Google Scholar 

  • Takeuchi K, Arthanari H, Wagner G (2016a) Perspective: revisiting the field dependence of TROSY sensitivity. J Biomol NMR 66:221–225

    Article  Google Scholar 

  • Takeuchi K, Arthanari H, Imai M, Wagner G (2016b) Nitrogen-detected TROSY yields comparable sensitivity to proton-detected TROSY for non-deuterated, large proteins under physiological salt conditions. J Biomol NMR 64:143–151

    Article  Google Scholar 

  • Tanaka Y, Ono A (2008) Nitrogen-15 NMR spectroscopy of N-metallated nucleic acids: insights into N NMR parameters and N–metal bonds. Dalton Trans 37:4965–4974. doi:10.1039/b803510p

    Article  Google Scholar 

  • Tanaka Y, Kojima C, Morita EH, Kasai Y, Yamasaki K, Ono A, Kainosho M, Taira K (2002) Identification of the metal ion binding site on an RNA motif from hammerhead ribozymes using 15 N NMR spectroscopy. J Am Chem Soc 124:4595–4601

    Article  Google Scholar 

  • Walker SC, Avis JM, Conn GL (2003) General plasmids for producing RNA in vitro transcripts with homogeneous ends. Nucleic Acids Res 31:1–6

    Article  Google Scholar 

  • Wang W, Zhao J, Han Q, Wang G, Yang G, Anthony J, Liu J, Gaffney BL, Jones RA (2009) Modulation of RNA metal binding sites by flanking bases: 15N NMR evaluation of GC, tandem GU, and tandem GA sites. Nucleosides Nucleotides Nucleic Acids 28:424–434

    Article  Google Scholar 

  • Wijmenga SS, van Buuren BNM (1998) The use of NMR methods for conformational studies of nucleic acids. Prog Nucl Magn Reson Spectrosc 32:287–387

    Article  Google Scholar 

  • Wilusz JE, Sunwoo H, Spector DL (2009) Long noncoding RNAs: functional surprises from the RNA world. Genes Dev 23:1494–1504

    Article  Google Scholar 

  • Zhao B, Hansen AL, Zhang Q (2014) Characterizing slow chemical exchange in nucleic acids by carbon CEST and low spin-lock field R1p NMR spectroscopy. J Am Chem Soc 136:20–23

    Article  Google Scholar 

  • Ziegeler M, Cevec M, Richter C, Schwalbe H (2012) NMR studies of HAR1 RNA secondary structures reveal conformational dynamics in the human RNA. ChemBioChem 13:2100–2112

    Article  Google Scholar 

Download references

Acknowledgements

Matthias Görlach from the Core Facilities and Services (CS Protein Production) of the Fritz Lipmann Institute, Jena, is gratefully acknowledged for providing the 329 nt RNA.

Funding

This work was funded by the German funding agency (DFG) in Collaborative Research Center 902: Molecular principles of RNA-based regulation and in Graduate College: CLIC (GRK1982). Financial support by European access program (iNEXT) is gratefully acknowledged. Harald Schwalbe is member of the DFG-funded Cluster of Excellence: macromolecular complexes (EXC115). Robbin Schnieders is supported by the Fonds of the Chemical Industry. Work at BMRZ is supported by the state of Hesse. We acknowledge access to the 950 MHz spectrometer of the Astbury BioStructure Laboratory BioNMR Facility (University of Leeds) which was funded by the University of Leeds.

Author information

Authors and Affiliations

  1. Institute for Organic Chemistry and Chemical Biology, Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-Universität Frankfurt, Max-von-Laue-Str. 7, 60438, Frankfurt, Germany

    Robbin Schnieders, Christian Richter, Sven Warhaut, Vanessa de Jesus, Sara Keyhani, Harald Schwalbe & Boris Fürtig

  2. Institute for Molecular Biosciences, Center for Biomolecular Magnetic Resonance (BMRZ), Johann Wolfgang Goethe-Universität Frankfurt, Max-von-Laue Str. 9, 60438, Frankfurt, Germany

    Elke Duchardt-Ferner, Heiko Keller & Jens Wöhnert

  3. Astbury Centre for Structural Molecular Biology, Faculty of Biological Sciences, University of Leeds, Leeds, LS2 9JT, UK

    Lars T. Kuhn & Alexander L. Breeze

  4. Bruker BioSpin GmbH, Silberstreifen 4, 76287, Rheinstetten, Germany

    Wolfgang Bermel

Authors
  1. Robbin Schnieders
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Christian Richter
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sven Warhaut
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vanessa de Jesus
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Sara Keyhani
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Elke Duchardt-Ferner
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Heiko Keller
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jens Wöhnert
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Lars T. Kuhn
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Alexander L. Breeze
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Wolfgang Bermel
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Harald Schwalbe
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Boris Fürtig
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Harald Schwalbe or Boris Fürtig.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary material 1 (DOCX 547 KB)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Schnieders, R., Richter, C., Warhaut, S. et al. Evaluation of 15N-detected H–N correlation experiments on increasingly large RNAs. J Biomol NMR 69, 31–44 (2017). https://doi.org/10.1007/s10858-017-0132-7

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10858-017-0132-7

Keywords

Search

Navigation