Journal of Biomolecular NMR

, Volume 31, Issue 3, pp 231–241 | Cite as

Accurate measurement of 15N–13C residual dipolar couplings in nucleic acids

  • Christopher P. Jaroniec
  • Jérôme Boisbouvier
  • Izabela Tworowska
  • Edward P. Nikonowicz
  • Ad Bax


New 3D HCN quantitative J (QJ) pulse schemes are presented for the precise and accurate measurement of one-bond 15N1/913C1′, 15N1/913C6/8, and 15N1/913C2/4 residual dipolar couplings (RDCs) in weakly aligned nucleic acids. The methods employ 1H–13C multiple quantum (MQ) coherence or TROSY-type pulse sequences for optimal resolution and sensitivity. RDCs are obtained from the intensity ratio of H1′–C1′–N1/9 (MQ-HCN-QJ) or H6/8–C6/8–N1/9 (TROSY-HCN-QJ) correlations in two interleaved 3D NMR spectra, with dephasing intervals of zero (reference spectrum) and ∼1/(2JNC) (attenuated spectrum). The different types of 15N–13C couplings can be obtained by using either the 3D MQ-HCN-QJ or TROSY-HCN-QJ pulse scheme, with the appropriate setting of the duration of the constant-time 15N evolution period and the offset of two frequency-selective 13C pulses. The methods are demonstrated for a uniformly 13C, 15N-enriched 24-nucleotide stem-loop RNA sequence, helix-35ψ, aligned in the magnetic field using phage Pf1. For measurements of RDCs systematic errors are found to be negligible, and experiments performed on a 1.5 mM helix-35ψ sample result in an estimated precision of ca. 0.07 Hz for 1DNC, indicating the utility of the measured RDCs in structure validation and refinement. Indeed, for a complete set of 15N1/913C1′, 15N1/913C6/8, and 15N1/913C2/4 dipolar couplings obtained for the stem nucleotides, the measured RDCs are in excellent agreement with those predicted for an NMR structure of helix-35ψ, refined using independently measured observables, including 13C–1H, 13C–13C and 1H–1H dipolar couplings.


DNA heteronuclear NMR liquid crystal multiple quantum coherence RDC RNA TROSY 


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  1. Barbic, A., Zimmer, D.P., Crothers, D.M. 2003Proc. Natl. Acad. Sci. USA10023692373Google Scholar
  2. Batey, R.T., Inada, M., Kujawinski, E., Puglisi, J.D., Williamson, J.R. 1992Nucleic Acid Res.2045154523Google Scholar
  3. Bax, A., Vuister, G.W., Grzesiek, S., Delaglio, F., Wang, A.C., Tschudin, R., Zhu, G. 1994Meth. Enzymol.23979105Google Scholar
  4. Bayer, P., Varani, L., Varani, G. 1999J. Biomol. NMR14149155Google Scholar
  5. Boisbouvier, J., Brutscher, B., Pardi, A., Marion, D., Simorre, J.P. 2000J. Am. Chem. Soc.12267796780Google Scholar
  6. Boisbouvier, J., Bryce, D.L., O’Neil-Cabello, E., Nikonowicz, E.P., Bax, A. 2004J. Biomol. NMR30287301Google Scholar
  7. Boisbouvier, J., Delaglio, F., Bax, A. 2003Proc. Natl. Acad. Sci. USA1001133311338Google Scholar
  8. Bondensgaard, K., Mollova, E.T., Pardi, A. 2002Biochemistry411153211542Google Scholar
  9. Brutscher, B., Boisbouvier, J., Pardi, A., Marion, D., Simorre, J.-P. 1998J. Am. Chem. Soc.1201184511851Google Scholar
  10. Cho, C.H., Urquidi, J., Singh, S., Robinson, G.W. 1999J. Phys. Chem. B10319911994Google Scholar
  11. Chou, J.J., Delaglio, F., Bax, A. 2000J. Biomol. NMR18101105Google Scholar
  12. Clore, G.M., Starich, M.R., Gronenborn, A.M. 1998J. Am. Chem. Soc.1201057110572Google Scholar
  13. Delaglio, F., Grzesiek, S., Vuister, G.W., Zhu, G., Pfeifer, J., Bax, A. 1995J. Biomol. NMR6277293Google Scholar
  14. D’Souza, V., Dey, A., Habib, D., Summers, M.F. 2004J. Mol. Biol.337427442Google Scholar
  15. Farmer, B.T.,II, Mueller, L., Nikonowicz, E.P., Pardi, A. 1993J. Am. Chem. Soc.1151104011041Google Scholar
  16. Fiala, R., Czernek, J., Sklenar, V. 2000J. Biomol. NMR16291302Google Scholar
  17. Geen, H., Freeman, R. 1991J. Magn. Reson.9393141Google Scholar
  18. Griesinger, C., Sørensen, O.W., Ernst, R.R. 1985J. Am. Chem. Soc.10763946396Google Scholar
  19. Griffey, R.H., Redfield, A.G. 1987Q. Rev. Biophys.195182Google Scholar
  20. Grzesiek, S., Bax, A. 1995J. Biomol. NMR6335339Google Scholar
  21. Hansen, M.R., Mueller, L., Pardi, A. 1998Nat. Struct. Biol.510651074Google Scholar
  22. Hennig, M., Carlomagno, T., Williamson, J.R. 2001J. Am. Chem. Soc.12333953396Google Scholar
  23. Jaravine, V.A., Cordier, F., Grzesiek, S. 2004J. Biomol. NMR29309318Google Scholar
  24. Jaroniec, C.P., Ulmer, T.S., Bax, A. 2004J. Biomol. NMR30181194Google Scholar
  25. Kay, L.E., Keifer, P., Saarinen, T. 1992J. Am. Chem. Soc.1141066310665Google Scholar
  26. Losonczi, J.A., Andrec, M., Fischer, M.W.F., Prestegard, J.H. 1999J. Magn. Reson.138334342Google Scholar
  27. Louis, J.M., Martin, R.G., Clore, G.M., Gronenborn, A.M. 1998J. Biol. Chem.27323742378Google Scholar
  28. Lukavsky, P.J., Kim, I., Otto, G.A., Puglisi, J.D. 2003Nat. Struct. Biol.1010331038Google Scholar
  29. MacDonald, D., Lu, P. 2002Curr. Opin. Struct. Biol.12337343Google Scholar
  30. Marino, J.P., Diener, J.L., Moore, P.B., Griesinger, C. 1997J. Am. Chem. Soc.11973617366Google Scholar
  31. Masse, J.E., Bortmann, P., Dieckmann, T., Feigon, J. 1998Nucleic Acid Res.2626182624Google Scholar
  32. McCallum, S.A., Pardi, A. 2003J. Mol. Biol.32610371050Google Scholar
  33. Meier, S., Haussinger, D., Jensen, P., Rogowski, M., Grzesiek, S. 2003J. Am. Chem. Soc.1254445Google Scholar
  34. Miclet, E., O’Neil-Cabello, E., Nikonowicz, E.P., Bax, A. 2003J. Am. Chem. Soc.1251574015741Google Scholar
  35. Nikonowicz, E.P., Sirr, A., Legault, P., Jucker, F.M., Baer, L.M., Pardi, A. 1992Nucleic Acid Res.2045074513Google Scholar
  36. O’Neil-Cabello, E., Bryce, D.L., Nikonowicz, E.P., Bax, A. 2004J. Am. Chem. Soc.1266667Google Scholar
  37. Padrta, P., Stefl, R., Kralik, L., Zidek, L., Sklenar, V. 2002J. Biomol. NMR24114Google Scholar
  38. Pervushin, K., Riek, R., Wider, G., Wuthrich, K. 1998aJ. Am. Chem. Soc.12063946400Google Scholar
  39. Pervushin, K., Wider, G., Wuthrich, K. 1998bJ. Biomol. NMR12345348Google Scholar
  40. Prestegard, J.H., Al-Hashimi, H.M., Tolman, J.R. 2000Q. Rev. Biophys.33371424Google Scholar
  41. Rückert, M., Otting, G. 2000J. Am. Chem. Soc.12277937797Google Scholar
  42. Sass, J., Cordier, F., Hoffmann, A., Rogowski, M., Cousin, A., Omichinski, J.G., Lowen, H., Grzesiek, S. 1999J. Am. Chem. Soc.12120472055Google Scholar
  43. Sass, H.J., Musco, G., Stahl, S.J., Wingfield, P.T., Grzesiek, S. 2000J. Biomol. NMR18303309Google Scholar
  44. Sibille, N., Pardi, A., Simorre, J.P., Blackledge, M. 2001J. Am. Chem. Soc.1231213512146Google Scholar
  45. Silver, M.S., Joseph, R.I., Hoult, D.I. 1984J. Magn. Reson.59347351Google Scholar
  46. Sklenar, V., Peterson, R.D., Rejante, M.R., Feigon, J. 1993J. Biomol. NMR3721727Google Scholar
  47. Sørensen, M.D., Meissner, A., Sørensen, O.W. 1997J. Biomol. NMR10181186Google Scholar
  48. Stefl, R., Wu, H.H., Ravindranathan, S., Sklenar, V., Feigon, J. 2004Proc. Natl. Acad. Sci. USA10111771182Google Scholar
  49. Tjandra, N., Bax, A. 1997Science27811111114Google Scholar
  50. Tjandra, N., Omichinski, J.G., Gronenborn, A.M., Clore, G.M., Bax, A. 1997Nat. Struct. Biol.4732738Google Scholar
  51. Tjandra, N., Tate, S., Ono, A., Kainosho, M., Bax, A. 2000J. Am. Chem. Soc.12261906200Google Scholar
  52. Tolman, J.R., Flanagan, J.M., Kennedy, M.A., Prestegard, J.H. 1995Proc. Natl. Acad. Sci. USA9292799283Google Scholar
  53. Tycko, R., Blanco, F.J., Ishii, Y. 2000J. Am. Chem. Soc.12293409341Google Scholar
  54. Vermeulen, A., Zhou, H.J., Pardi, A. 2000J. Am. Chem. Soc.12296389647Google Scholar
  55. Warren, J.J., Moore, P.B. 2001J. Biomol. NMR20311323Google Scholar
  56. Weigelt, J. 1998J. Am. Chem. Soc.1201077810779Google Scholar
  57. Wijmenga, S.S., Buuren, B.N.M. 1998Prog. Nucl. Magn. Reson. Spectrosc.32287387Google Scholar
  58. Wu, Z.R., Bax, A. 2002J. Am. Chem. Soc.12496729673Google Scholar
  59. Wu, Z.G., Delaglio, F., Tjandra, N., Zhurkin, V.B., Bax, A. 2003J. Biomol. NMR26297315Google Scholar
  60. Wu, Z.R., Tjandra, N., Bax, A. 2001J. Biomol. NMR19367370Google Scholar
  61. Wüthrich, K. 1986NMR of Proteins and Nucleic AcidsWileyNew York, NYGoogle Scholar
  62. Yan, J.L., Corpora, T., Pradhan, P., Bushweller, J.H. 2002J. Biomol. NMR22920Google Scholar
  63. Zidek, L., Wu, H., Feigon, J., Sklenar, V. 2001J. Biomol. NMR21153160Google Scholar
  64. Zimmer, D.P., Crothers, D.M. 1995Proc. Natl. Acad. Sci. USA9230913095Google Scholar

Copyright information

© Kluwer Academic Publishers 2005

Authors and Affiliations

  • Christopher P. Jaroniec
    • 1
  • Jérôme Boisbouvier
    • 2
  • Izabela Tworowska
    • 3
  • Edward P. Nikonowicz
    • 3
  • Ad Bax
    • 1
  1. 1.Laboratory of Chemical PhysicsNational Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of HealthBethesdaUSA
  2. 2.Laboratoire de RMNInstitut de Biologie Structurale, Jean-Pierre Ebel, UMR 5075, CNRS-CEA-UJFGrenobleFrance
  3. 3.Department of Biochemistry and Cell BiologyRice UniversityHoustonUSA

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