Springer Protocols: Abstract: 2. RNA Structure Determination by NMR

2. RNA Structure Determination by NMR

By: Lincoln G. Scott³, Mirko Hennig⁴

Abstract

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This chapter reviews the methodologies for RNA structure determination by liquid-state nuclear magnetic resonance (NMR). The routine production of milligram quantities of isotopically labeled RNA remains critical to the success of NMR-based structure studies. The standard method for the preparation of isotopically labeled RNA for structural studies in solution is in vitro transcription from DNA oligonucleotide templates using T7 RNA polymerase and unlabeled or isotopically labeled nucleotide triphosphates (NTPs). The purification of the desired RNA can be performed by either denaturing polyacrylamide gel electrophoresis (PAGE) or anion-exchange chromatography. Our basic strategy for studying RNA in solution by NMR is outlined. The topics covered include RNA resonance assignment, restraint collection, and the structure calculation process. Selected examples of NMR spectra are given for a correctly folded 30 nucleotide-containing RNA.

Affiliation(s): (3) Cassia, LLC, San Diego, CA
(4) Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC

Book Title: Bioinformatics: Data, Sequence Analysis and Evolution

Series: Methods in Molecular Biology | Volume: 452 | Pub. Date: May-01-2008 | Page Range: 29-61 | DOI: 10.1007/978-1-60327-159-2_2

Subject: Bioinformatics

Key Words: RNA - RNA synthesis - RNA purification - NMR - resonance assignment - structure determination

References

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1.	Leulliot, N., Varani, G. (2001) Current topics in RNA-protein recognition: control of specificity and biological function through induced fit and con-formational capture. Biochemistry 40, 7947–7956.

2.	Williamson, J. R. (2000) Induced fit in RNA-protein recognition. Nat Struct Biol 7, 834–837.

3.	Milligan, J. F., Groebe, D. R., Witherell, G. W., et al. (1987) Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acids Res 15, 8783–8798.

4.	Milligan, J. F., Uhlenbeck, O. C. (1989) Synthesis of small RNAs using T7 RNA polymerase. Methods Enzymol 180, 51–62.

5.	Perez-Canadillas, J. M., Varani, G. (2001) Recent advances in RNA-protein recognition. Curr Opin Struct Biol 11, 53–58.

6.	Batey, R. T., Battiste, J. L., Williamson, J. R. (1995) Preparation of isotopically enriched RNAs for heteronuclear NMR. Methods Enzymol 261, 300–322.

7.	Batey, R. T., Cloutier, N., Mao, H., et al. (1996) Improved large scale culture of Methylophilus methylotrophus for 13C/15N labeling and random fractional deuteration of ribonucleotides. Nucleic Acids Res 24, 4836–4837.

8.	Batey, R. T., Inada, M., Kujawinski, E., et al. (1992) Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA. Nucleic Acids Res 20, 4515–4523.

9.	Nikonowicz, E. P., Sirr, A., Legault, P., et al. (1992) Preparation of 13C and 15N labelled RNAs for heteronuclear multidimensional NMR studies. Nucleic Acids Res 20, 4507–4513.

10.	Zidek, L., Stefl, R., Sklenar, V. (2001) NMR methodology for the study of nucleic acids. Curr Opin Struct Biol 11, 275–281.

11.	Cromsigt, J., van Buuren, B., Schleucher, J., et al. (2001) Resonance assignment and structure determination for RNA. Methods Enzymol 338, 371–399.

12.	Furtig, B., Richter, C., Wohnert, J., et al. (2003) NMR spectroscopy of RNA. Chem-biochem 4, 936–962.

13.	Latham, M. P., Brown, D. J., McCallum, S. A., et al. (2005) NMR methods for studying the structure and dynamics of RNA. Chembiochemistry 6, 1492–1505.

14.	Wu, H., Finger, L. D., Feigon, J. (2005) Structure determination of protein/RNA complexes by NMR. Methods Enzymol 394, 525–545.

15.	Bax, A., Kontaxis, G., Tjandra, N. (2001) Dipolar couplings in macromolecular structure determination. Methods Enzymol 339, 127–174.

16.	Hansen, M. R., Mueller, L., Pardi, A. (1998) Tunable alignment of macromol-ecules by filamentous phage yields dipolar coupling interactions. Nat Struct Biol 5, 1065–1074.

17.	Tjandra, N., Bax, A. (1997) Direct measurement of distances and angles in biomol-ecules by NMR in a dilute liquid crystalline medium. Science 278, 1111–1114.

18.	Reif, B., Hennig, M., Griesinger, C. (1997) Direct measurement of angles between bond vectors in high-resolution NMR. Science 276, 1230–1233.

19.	Schwalbe, H., Carlomagno, T., Hennig, M., et al. (2001) Cross-correlated relaxation for measurement of angles between tensorial interactions. Methods Enzymol 338, 35–81.

20.	Dingley, A. J., Grzesiek, S. (1998) Direct observation of hydrogen bonds in nucleic acid base pairs by internucleotide (2)J(NN) couplings. J Am Chem Soc 120, 8293–8297.

21.	Pervushin, K., Ono, A., Fernandez, C., et al. (1998) NMR scaler couplings across Watson-Crick base pair hydrogen bonds in DNA observed by transverse relaxation optimized spectroscopy. Proc Natl Acad Sci U S A 95, 14147–14151.

22.	Churcher, M. J., Lamont, C., Hamy, F., et al. (1993) High affinity binding of TAR RNA by the human immunodeficiency virus type-1 tat protein requires base-pairs in the RNA stem and amino acid residues flanking the basic region. J Mol Biol 230, 90–110.

23.	Long, K. S., Crothers, D. M. (1995) Interaction of human immunodeficiency virus type 1 Tat-derived peptides with TAR RNA. Biochemistry 34, 8885–8895.

24.	Tao, J., Frankel, A. D. (1992) Specific binding of arginine to TAR RNA. Proc Natl Acad Sci U S A 89, 2723–2726.

25.	Tao, J., Frankel, A. D. (1993) Electrostatic interactions modulate the RNA-binding and transactivation specificities of the human immunodeficiency virus and simian immunodeficiency virus Tat proteins. Proc Natl Acad Sci U S A 90, 1571–1575.

26.	Puglisi, J. D., Chen, L., Frankel, A. D., et al. (1993) Role of RNA structure in arginine recognition of TAR RNA. Proc Natl Acad Sci U S A 90, 3680–3684.

27.	Puglisi, J. D., Tan, R., Calnan, B. J., et al. (1992) Conformation of the TAR RNA-arginine complex by NMR spectroscopy. Science 257, 76–80.

28.	Aboul-ela, F., Karn, J., Varani, G. (1995) The structure of the human immunodeficiency virus type-1 TAR RNA reveals principles of RNA recognition by Tat protein. J Mol Biol 253, 313–332.

29.	Brodsky, A. S., Williamson, J. R. (1997) Solution structure of the HIV-2 TAR-argininamide complex. J Mol Biol 267, 624–639.

30.	Aboul-ela, F., Karn, J., Varani, G. (1996) Structure of HIV-1 TAR RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge. Nucleic Acids Res 24, 3974–3781.

31.	Long, K. S., Crothers, D. M. (1999) Characterization of the solution conformations of unbound and Tat peptide-bound forms of HIV-1 TAR RNA. Biochemistry 38, 10059–10069.

32.	Sambrook, J., Fritsch, E. F., Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

33.	Yin, Y., Carter, C. W., Jr. (1996) Incomplete factorial and response surface methods in experimental design: yield optimization of tRNA(Trp) from in vitro T7 RNA polymerase transcription. Nucleic Acids Res 24, 1279–1286.

34.	Wyatt, J. R., Chastain, M., Puglisi, J. D. (1991) Synthesis and purification of large amounts of RNA oligonucleotides. Biotechniques 11, 764–769.

35.	Anderson, A. C., Scaringe, S. A., Earp, B. E., et al. (1996) HPLC purification of RNA for crystallography and NMR. RNA 2, 110–117.

36.	Shields, T. P., Mollova, E., Ste Marie, L., et al. (1999) High-performance liquid chromatography purification of homogenous-length RNA produced by trans cleavage with a hammerhead ribozyme. RNA 5, 1259–1267.

37.	Wuthrich, K. (1986) NMR of Proteins and Nucleic Acids, Wiley, New York.

38.	Nikonowicz, E. P., Pardi, A. (1992) Three-dimensional heteronuclear NMR studies of RNA. Nature 355, 184–186.

39.	Nikonowicz, E. P., Pardi, A. (1993) An efficient procedure for assignment of the proton, carbon and nitrogen resonances in 13C/15N labeled nucleic acids. J Mol Biol 232, 1141–1156.

40.	Wijmenga, S. S., van Buuren, B. N. M. (1998) The use of NMR methods for con-formational studies of nucleic acids. Prog Nucl Magn Reson Spectrosc 32, 287–387.

41.	Zhou, H., Vermeulen, A., Jucker, F. M., et al. (1999) Incorporating residual dipolar couplings into the NMR solution structure determination of nucleic acids. Biopolymers 52, 168–180.

42.	Hansen, M. R., Hanson, P., Pardi, A. (2000) Filamentous bacteriophage for aligning RNA, DNA, and proteins for measurement of nuclear magnetic resonance dipolar coupling interactions. Methods Enzymol 317, 220–240.

43.	Meiler, J., Prompers, J. J., Peti, W., et al. (2001) Model-free approach to the dynamic interpretation of residual dipolar couplings in globular proteins. J Am Chem Soc 123, 6098–6107.

44.	Peti, W., Meiler, J., Bruschweiler, R., et al. (2002) Model-free analysis of protein backbone motion from residual dipolar couplings. J Am Chem Soc 124, 5822–5833.

45.	Tolman, J. R. (2002) A novel approach to the retrieval of structural and dynamic information from residual dipolar couplings using several oriented media in biomolecu-lar NMR spectroscopy. J Am Chem Soc 124, 12020–12030.

46.	Lippens, G., Dhalluin, C., Wieruszeski, J. M. (1995) Use of a Water Flip-Back Pulse in the Homonuclear Noesy Experiment. J Biomol Nmr 5, 327–331.

47.	Peterson, R. D., Theimer, C. A., Wu, H. H., et al. (2004) New applications of 2D filtered/edited NOESY for assignment and structure elucidation of RNA and RNA-pro-tein complexes. J Biomol Nmr 28, 59–67.

48.	Duchardt, E., Richter, C., Reif, B., et al. (2001) Measurement of 2J(H,C)- and 3J(H,C)-coupling constants by alpha/beta selective HC(C)H-TOCSY. J Biomol NMR 21, 117–126.

49.	Schwalbe, H., Marino, J. P., Glaser, S. J., et al. (1995) Measurement of H,H-Coupling Constants Associated with nu1, nu2, and nu3 in Uniformly 13C-Labeled RNA by HCC-TOCSY-CCH-E.COSY J Am Chem Soc 117, 7251–7252.

50.	Schwalbe, H., Marino, J. P., King, G. C., et al. (1994) Determination of a complete set of coupling constants in 13C-labeled oligonu-cleotides. J Biomol NMR 4, 631–644.

51.	Hines, J. V., Varani, G., Landry, S. M., et al. (1993) The stereospecific assignment of H5′ and H5″ in RNA using the sign of two-bond carbon-proton scalar coupling. J Am Chem Soc 115, 11002–11003.

52.	Trantirek, L., Stefl, R., Masse, J. E., et al. (2002) Determination of the glycosidic torsion angles in uniformly C-13-labeled nucleic acids from vicinal coupling constants (3)J(C2/4-H1′) and (3)J(C6/8-H1′). J Biomol NMR 23, 1–12.

53.	Carlomagno, T., Hennig, M., Williamson, J. R. (2002) A novel PH-cT-COSY methodology for measuring JPH coupling constants in unlabeled nucleic acids. application to HIV-2 TAR RNA. J Biomol NMR 22, 65–81.

54.	Sklenar, V., Miyashiro, H., Zon, G., et al. (1986) Assignment of the 31P and 1H resonances in oligonucleotides by two-dimensional NMR spectroscopy. FEBS Lett 208, 94–98.

55.	Schwalbe, H., Samstag, W., Engels, J. W., et al. (1993) Determination of 3J(C,P) and 3J(H,P) coupling constants in nucleotide oligomers with FIDS-HSQC. J Biomol NMR 3, 479–486.

56.	Hoogstraten, C. G., Pardi, A. (1998) Measurement of carbon-phosphorus J coupling constants in RNA using spin-echo difference constant-time HCCH-COSY. J Magn Reson 133, 236–240.

57.	Legault, P., Jucker, F. M., Pardi, A. (1995) Improved measurement of 13C, 31P J coupling constants in isotopically labeled RNA. FEBS Lett 362, 156–160.

58.	Szyperski, T., Fernandez, C., Ono, A., et al. (1999) The 2D [31P] spin-echo-difference constant-time [13C, 1H]-HMQC experiment for simultaneous determination of 3J(H3′P) and 3J(C4′P) in 13C-labeled nucleic acids and their protein complexes. J Magn Reson 140, 491–494.

59.	Hu, W., Bouaziz, S., Skripkin, E., et al. (1999) Determination of 3J(H3i, Pi+1) and 3J(H5i/5i, Pi) coupling constants in 13C-labeled nucleic acids using constant-time HMQC. J Magn Reson 139, 181–185.

60.	Clore, G. M., Murphy, E. C., Gronenborn, A. M., et al. (1998) Determination of three-bond 1H3′-31P couplings in nucleic acids and protein-nucleic acid complexes by quantitative J correlation spectroscopy. J Magn Reson 134, 164–167.

61.	Gotfredsen, C. H., Meissner, A., Duus, J. O., et al. (2000) New methods for measuring 1H-31P coupling constants in nucleic acids. Magn Reson Chem 38, 692–695.

62.	Richter, C., Reif, B., Wor ner, K., et al. (1998) A new experiment for the measurement of nJ(C,P) coupling constants including 3J(C4′i,Pi) and 3J(C4′i,Pi+1) in oligonucle-otides. J Biomol NMR 12, 223–230.

63.	Legault, P., Pardi, A. (1994) 31P chemical shift as a probe of structural motifs in RNA. J Magn Reson B 103, 82–86.

64.	Richter, C., Reif, B., Griesinger, C., et al. (2000) NMR spectroscopic determination of angles and in RNA from CH-dipolar coupling, P-CSA cross-correlated relaxation. J Am Chem Soc 122, 12728– 12731.

65.	Duchardt, E., Richter, C., Ohlenschlager, O., et al. (2004) Determination of the glycosidic bond angle chi in RNA from cross-correlated relaxation of CH dipolar coupling and N chemical shift anisotropy. J Am Chem Soc 126, 1962–1970.

66.	Ravindranathan, S., Kim, C. H., Bod-enhausen, G. (2003) Cross correlations between 13C-1H dipolar interactions and 15N chemical shift anisotropy in nucleic acids. J Biomol NMR 27, 365–375.

67.	Felli, I. C., Richter, C., Griesinger, C., et al. (1999) Determination of RNA sugar pucker mode from cross-correlated relaxation in solution NMR spectroscopy. J Am Chem Soc 121, 1956–1957.

68.	Richter, C., Griesinger, C., Felli, I., et al. (1999) Determination of sugar conformation in large RNA oligonucleotides from analysis of dipole-dipole cross correlated relaxation by solution NMR spectroscopy. J Biomol NMR 15, 241–250.

69.	Andersson, P., Weigelt, J., Otting, G. (1998) Spin-state selection filters for the measurement of heteronuclear one-bond coupling constants. J Biomol NMR 12, 435–441.

70.	Tjandra, N., Bax, A. (1997) Measurement of dipolar contributions to 1JCH splittings from magnetic-field dependence of J modulation in two-dimensional NMR spectra. J Magn Reson 124, 512–515.

71.	Tjandra, N., Grzesiek, S., Bax, A. (1996) Magnetic field dependence of nitrogen-proton J splittings in N-15-enriched human ubiquitin resulting from relaxation interference and residual dipolar coupling. J Am Chem Soc 118, 6264–6272.

72.	Hennig, M., Carlomagno, T., Williamson, J. R. (2001) Residual dipolar coupling TOCSY for direct through space correlations of base protons and phosphorus nuclei in RNA. J Am Chem Soc 123, 3395–3396.

73.	Wu, Z., Tjandra, N., Bax, A. (2001) Measurement of 1H3′-31P dipolar couplings in a DNA oligonucleotide by constant-time NOESY difference spectroscopy. J Biomol NMR 19, 367–370.

74.	Wu, Z., Tjandra, N., Bax, A. (2001) 31P chemical shift anisotropy as an aid in determining nucleic acid structure in liquid crystals. J Am Chem Soc 123, 3617–3618.

75.	Dingley, A. J., Masse, J. E., Feigon, J., et al. (2000) Characterization of the hydrogen bond network in guanosine quartets by internucleotide 3hJ(NC)′ and 2hJ(NN) scalar couplings. J Biomol NMR 16, 279–289.

76.	Dingley, A. J., Masse, J. E., Peterson, R. D., et al. (1999) Internucleotide scalar couplings across hydrogen bonds in Watson-Crickand Hoogsteen base pairs of a DNA triplex. J Am Chem Soc 121, 6019–6027.

77.	Hennig, M., Williamson, J. R. (2000) Detection of N-H…N hydrogen bonding in RNA via scalar couplings in the absence of observable imino proton resonances. Nucleic Acids Res 28, 1585–1593.

78.	Liu, A. Z., Majumdar, A., Hu, W. D., et al. (2000) NMR detection of N-H…O=C hydrogen bonds in C-13,N-15-labeled nucleic acids. J Am Chem Soc 122, 3206–3210.

79.	Majumdar, A., Kettani, A., Skripkin, E. (1999) Observation and measurement of internucleotide 2JNN coupling constants between 15N nuclei with widely separated chemical shifts. J Biomol NMR 14, 67–70.

80.	Majumdar, A., Kettani, A., Skripkin, E., et al. (1999) Observation of internucleotide NH…N hydrogen bonds in the absence of directly detectable protons. J Biomol NMR 15, 207–211.

81.	Wohnert, J., Ramachandran, R., Gorlach, M., et al. (1999) Triple-resonance experiments for correlation of H5 and exchangeable pyrimidine base hydrogens in (13)C,(15) N-labeled RNA. J Magn Reson 139, 430–433.

82.	Sklenar, V., Peterson, R. D., Rejante, M. R., et al. (1994) Correlation of nucleotide base and sugar protons in a N-15-labeled Hiv-1 RNA oligonucleotide by H-1-N-15 Hsqc experiments. J Biomol NMR 4, 117–122.

83.	Fohrer, J., Hennig, M., Carlomagno, T. (2006) Influence of the 2′-hydroxyl group conformation on the stability of A-form helices in RNA. J Mol Biol 356, 280–287.

84.	Hennig, M., Fohrer, J., Carlomagno, T. (2005) Assignment and NOE analysis of 2′-hydroxyl protons in RNA: implications for stabilization of RNA A-form duplexes. J Am Chem Soc 127, 2028–2029.

85.	Giedroc, D. P., Cornish, P. V., Hennig, M. (2003) Detection of scalar couplings involving 2′-hydroxyl protons across hydrogen bonds in a frameshifting mRNA pseudoknot. J Am Chem Soc 125, 4676–4677.

86.	Simorre, J. P., Zimmermann, G. R., Mueller, L., et al. (1996) Correlation of the guano-sine exchangeable and nonexchangeable base protons in 13C-/15N-labeled RNA with an HNC-TOCSY-CH experiment. J Biomol NMR 7, 153–156.

87.	Simorre, J. P., Zimmermann, G. R., Mueller, L., et al. (1996) Triple-resonance experiments for assignment of adenine base resonances in C-13/N-15-labeled RNA. J Am Chem Soc 118, 5316–5317.

88.	Simorre, J. P., Zimmermann, G. R., Pardi, A., et al. (1995) Triple resonance HNCCCH experiments for correlating exchangeable and nonexchangeable cyti-dine and uridine base protons in RNA. J Biomol NMR 6, 427–432.

89.	Sklenar, V., Dieckmann, T., Butcher, S. E., et al. (1996) Through-bond correlation of imino and aromatic resonances in C-13-,N-15-labeled RNA via heteronuclear TOCSY. J Biomol Nmr 7, 83–87.

90.	Wohnert, J., Gorlach, M., Schwalbe, H. (2003) Triple resonance experiments for the simultaneous correlation of H6/H5 and exchangeable protons of pyrimidine nucleotides in C-13, N-15-labeled RNA applicable to larger RNA molecules. J Biomol Nmr 26, 79–83.

91.	Fiala, R., Jiang, F., Patel, D. J. (1996) Direct correlation of exchangeable and non-exchangeable protons on purine bases in 13C,15N-labeled RNA using a HCCNH-TOCSY experiment. J Am Chem Soc 118, 689–690.

92.	Simon, B., Zanier, K., Sattler, M. (2001) A TROSY relayed HCCH-COSY experiment for correlating adenine H2/H8 resonances in uniformly 13C-labeled RNA molecules. J Biomol NMR 20, 173–176.

93.	Legault, P., Farmer, B. T., Mueller, L., et al. (1994) Through-bond correlation of ade-nine protons in a C-13-labeled ribozyme. J Am Chem Soc 116, 2203–2204.