Advertisement

Journal of Biomolecular NMR

, Volume 63, Issue 1, pp 67–76 | Cite as

Rapid NMR screening of RNA secondary structure and binding

  • Christina Helmling
  • Sara Keyhani
  • Florian Sochor
  • Boris Fürtig
  • Martin Hengesbach
  • Harald Schwalbe
Article

Abstract

Determination of RNA secondary structures by NMR spectroscopy is a useful tool e.g. to elucidate RNA folding space or functional aspects of regulatory RNA elements. However, current approaches of RNA synthesis and preparation are usually time-consuming and do not provide analysis with single nucleotide precision when applied for a large number of different RNA sequences. Here, we significantly improve the yield and 3′ end homogeneity of RNA preparation by in vitro transcription. Further, by establishing a native purification procedure with increased throughput, we provide a shortcut to study several RNA constructs simultaneously. We show that this approach yields μmol quantities of RNA with purities comparable to PAGE purification, while avoiding denaturation of the RNA.

Keywords

NMR spectroscopy RNA secondary structure In vitro transcription Riboswitch RNA High throughput method 

Notes

Acknowledgments

We greatly acknowledge support for RNA purification by Elke Stirnal and Christian Richter for continuous support with the NMR-spectrometers. This work was supported by Deutsche Forschungsgemeinschaft (DFG) in the SFB902: Molecular principles of RNA-based regulation and in Graduate College: CLIC. C.H. is supported by a fellowship of the Fonds der Chemischen Industrie. Work at the Center for Biomolecular Magnetic Resonance (BMRZ) is supported by the state of Hesse. H.S. and M.H. are members of the DFG-funded Cluster of Excellence: Macromolecular Complexes (EXC115).

Supplementary material

10858_2015_9967_MOESM1_ESM.docx (1.8 mb)
Supplementary material 1 (DOCX 1879 kb)

References

  1. Azarani A, Hecker KH (2001) RNA analysis by ion-pair reversed-phase high performance liquid chromatography. Nucleic Acids Res 29:E7. doi: 10.1093/nar/29.2.e7 CrossRefGoogle Scholar
  2. Batey RT (2014) Advances in methods for native expression and purification of RNA for structural studies. Curr Opin Struct Biol 26:1–8. doi: 10.1016/j.sbi.2014.01.014 CrossRefADSGoogle Scholar
  3. Batey RT, Kieft JS (2007) Improved native affinity purification of RNA. RNA 13:1384–1389. doi: 10.1261/rna.528007 CrossRefGoogle Scholar
  4. Beckert B, Masquida B (2011) Synthesis of RNA by in vitro transcription. Methods Mol Biol 703:29–41. doi: 10.1007/978-1-59745-248-9_3 CrossRefGoogle Scholar
  5. Been MD, Perrotta AT, Rosenstein SP (1992) Secondary structure of the self-cleaving RNA of hepatitis delta virus: applications to catalytic RNA design. Biochemistry 31:11843–11852. doi: 10.1021/bi00162a024 CrossRefGoogle Scholar
  6. Birikh KR, Heaton PA, Eckstein F (1997) The structure, function and application of the hammerhead ribozyme. Eur J Biochem 245:1–16. doi: 10.1111/j.1432-1033.1997.t01-3-00001.x CrossRefGoogle Scholar
  7. Buck J, Fürtig B, Noeske J et al (2009) Time-resolved NMR spectroscopy: ligand-induced refolding of riboswitches. Methods Mol Biol 540:161–171. doi: 10.1007/978-1-59745-558-9_12 CrossRefGoogle Scholar
  8. Buck J, Noeske J, Wöhnert J, Schwalbe H (2010) Dissecting the influence of Mg2+on 3D architecture and ligand-binding of the guanine-sensing riboswitch aptamer domain. Nucleic Acids Res 38:4143–4153. doi: 10.1093/nar/gkq138 CrossRefGoogle Scholar
  9. Chen Z, Zhang Y (2005) Dimethyl sulfoxide targets phage RNA polymerases to promote transcription. Biochem Biophys Res Commun 333:664–670. doi: 10.1016/j.bbrc.2005.05.166 CrossRefGoogle Scholar
  10. Chowrira BM, Pavco PA, McSwiggen JA (1994) In vitro and in vivo comparison of hammerhead, hairpin, and hepatitis delta virus self-processing ribozyme cassettes. J Biol Chem 269:25856–25864Google Scholar
  11. Cole PE, Yang SK, Crothers DM (1972) Conformational changes of transfer ribonucleic acid. Equilibrium phase diagrams. Biochemistry 11:4358–4368. doi: 10.1021/bi00773a024 CrossRefGoogle Scholar
  12. Di Tomasso G, Lampron P, Dagenais P et al (2011) The ARiBo tag: a reliable tool for affinity purification of RNAs under native conditions. Nucleic Acids Res 39:e18. doi: 10.1093/nar/gkq1084 CrossRefGoogle Scholar
  13. Di Tomasso G, Dagenais P, Desjardins A et al (2012) Affinity purification of RNA using an ARiBo tag. Methods Mol Biol 941:137–155. doi: 10.1007/978-1-62703-113-4_11 CrossRefGoogle Scholar
  14. Di Tomasso G, Salvail-Lacoste A, Bouvette J et al (2014) Affinity purification of in vitro transcribed RNA with homogeneous ends using a 3′-ARiBo tag. Methods Enzymol 549:49–84CrossRefGoogle Scholar
  15. Draper DE, White SA, Kean JM (1988) Preparation of specific ribosomal RNA fragments. Methods Enzymol 164:221–237CrossRefGoogle Scholar
  16. Easton LE, Shibata Y, Lukavsky PJ (2010) Rapid, nondenaturing RNA purification using weak anion-exchange fast performance liquid chromatography. RNA 16:647–653. doi: 10.1261/rna.1862210 CrossRefGoogle Scholar
  17. Favier A, Brutscher B (2011) Recovering lost magnetization: polarization enhancement in biomolecular NMR. J Biomol NMR 49:9–15. doi: 10.1007/s10858-010-9461-5 CrossRefGoogle Scholar
  18. 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 24:977–978. doi: 10.1093/nar/24.5.977 CrossRefGoogle Scholar
  19. Fürtig B, Buck J, Manoharan V et al (2007) Time-resolved NMR studies of RNA folding. Biopolymers 86:360–383. doi: 10.1002/bip.20761 CrossRefGoogle Scholar
  20. Goddard TD, Kneller DG Sparky 3. University of California, San FranciscoGoogle Scholar
  21. Guillerez J, Lopez PJ, Proux F et al (2005) A mutation in T7 RNA polymerase that facilitates promoter clearance. Proc Natl Acad Sci USA 102:5958–5963. doi: 10.1073/pnas.0407141102 CrossRefADSGoogle Scholar
  22. Juang JK, Liu HJ (1987) The effect of DMSO on natural DNA conformation in enhancing transcription. Biochem Biophys Res Commun 146:1458–1464. doi: 10.1016/0006-291X(87)90813-8 CrossRefGoogle Scholar
  23. 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 5:1268–1272. doi: 10.1017/S1355838299991033 CrossRefGoogle Scholar
  24. Keel AY, Easton LE, Lukavsky PJ, Kieft JS (2009) Large-scale native preparation of in vitro transcribed RNA. Methods Enzymol 469:3–25. doi: 10.1016/S0076-6879(09)69001-7 CrossRefGoogle Scholar
  25. Kieft JS, Batey RT (2004) A general method for rapid and nondenaturing purification of RNAs. RNA 10:988–995. doi: 10.1261/rna.7040604 CrossRefGoogle Scholar
  26. Kim JN, Roth A, Breaker RR (2007) Guanine riboswitch variants from Mesoplasma florum selectively recognize 2′-deoxyguanosine. Proc Natl Acad Sci USA 104:16092–16097. doi: 10.1073/pnas.0705884104 CrossRefADSGoogle Scholar
  27. Krupp G (1988) RNA synthesis: strategies for the use of bacteriophage RNA polymerases. Gene 72:75–89CrossRefGoogle Scholar
  28. Lescop E, Kern T, Brutscher B (2010) Guidelines for the use of band-selective radiofrequency pulses in hetero-nuclear NMR: example of longitudinal-relaxation-enhanced BEST-type 1H–15N correlation experiments. J Magn Reson 203:190–198. doi: 10.1016/j.jmr.2009.12.001 CrossRefADSGoogle Scholar
  29. Lukavsky PJ, Puglisi JD (2004) Large-scale preparation and purification of polyacrylamide-free RNA oligonucleotides. RNA 10:889–893. doi: 10.1261/rna.5264804 CrossRefGoogle Scholar
  30. Milligan JF, Uhlenbeck OC (1989) Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol 180:51–62CrossRefGoogle Scholar
  31. 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. doi: 10.1093/nar/15.21.8783 CrossRefGoogle Scholar
  32. Okui S, Kawai G (2015) In NMR tube transcription for rapid screening of RNA conformation. Nucleosides Nucleotides Nucleic Acids 34:103–113. doi: 10.1080/15257770.2014.964412 CrossRefGoogle Scholar
  33. Petrov A, Wu T, Puglisi EV, Puglisi JD (2013) RNA purification by preparative polyacrylamide gel electrophoresis. Methods Enzymol 530:315–330. doi: 10.1016/B978-0-12-420037-1.00017-8 CrossRefGoogle Scholar
  34. Pikovskaya O, Polonskaia A, Patel DJ, Serganov A (2011) Structural principles of nucleoside selectivity in a 2′-deoxyguanosine riboswitch. Nat Chem Biol 7:748–755. doi: 10.1038/nchembio.631 CrossRefGoogle Scholar
  35. Piotto M, Saudek V, Sklenář V (1992) Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR 2:661–665. doi: 10.1007/BF02192855 CrossRefGoogle Scholar
  36. Pokrovskaya ID, Gurevich VV (1994) In vitro transcription: preparative RNA yields in analytical scale reactions. Anal Biochem 220:420–423. doi: 10.1006/abio.1994.1360 CrossRefGoogle Scholar
  37. Price SR, Ito N, Oubridge C et al (1995) Crystallization of RNA-protein complexes. I. Methods for the large-scale preparation of RNA suitable for crystallographic studies. J Mol Biol 249:398–408. doi: 10.1006/jmbi.1995.0305 CrossRefGoogle Scholar
  38. Salvail-Lacoste A, Di Tomasso G, Piette BL, Legault P (2013) Affinity purification of T7 RNA transcripts with homogeneous ends using ARiBo and CRISPR tags. RNA 19:1003–1014. doi: 10.1261/rna.037432.112 CrossRefGoogle Scholar
  39. Sklenář V, Bax A (1987) Spin-echo water suppression for the generation of pure-phase two-dimensional NMR spectra. J Magn Reson 74:469–479ADSGoogle Scholar
  40. Stage-Zimmermann TK, Uhlenbeck OC (1998) Hammerhead ribozyme kinetics. RNA 4:875–889CrossRefGoogle Scholar
  41. Strätling WH (1976) Stimulation of transcription on chromatin by polar organic compounds. Nucleic Acids Res 3:1203–1213. doi: 10.1093/nar/3.5.1203 CrossRefGoogle Scholar
  42. Tabor CW, Tabor H (1984) Polyamines. Annu Rev Biochem 53:749–790. doi: 10.1146/annurev.bi.53.070184.003533 CrossRefGoogle Scholar
  43. Wacker A, Buck J, Mathieu D et al (2011) Structure and dynamics of the deoxyguanosine-sensing riboswitch studied by NMR-spectroscopy. Nucleic Acids Res 39:6802–6812. doi: 10.1093/nar/gkr238 CrossRefGoogle Scholar
  44. Wickiser JK, Winkler WC, Breaker RR, Crothers DM (2005) The speed of RNA transcription and metabolite binding kinetics operate an FMN riboswitch. Mol Cell 18:49–60. doi: 10.1016/j.molcel.2005.02.032 CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2015

Authors and Affiliations

  • Christina Helmling
    • 1
  • Sara Keyhani
    • 1
  • Florian Sochor
    • 1
  • Boris Fürtig
    • 1
  • Martin Hengesbach
    • 1
  • Harald Schwalbe
    • 1
  1. 1.Institut für Organische Chemie und Chemische Biologie, Center for Biomolecular Magnetic Resonance (BMRZ)Johann Wolfgang Goethe-UniversitätFrankfurt am MainGermany

Personalised recommendations