Probing RNA hairpins with cobalt(III)hexammine and electrospray ionization mass spectrometry

  • Jason W. Kieltyka
  • Christine S. ChowEmail author
Focus: Nucleic Acids In Mass Spectrometry


In this work, electrospray ionization mass spectrometry (ESI MS) was employed to study the interactions of cobalt(III) hexammine, Co(NH3) 6 3+ , with five RNA hairpins representing the 790 loop of 16S ribosomal RNA and 1920 loop of 23S ribosomal RNA. The RNAs varied in mismatch identity (G±U versus A±C) and level of base modification (pseudouridine versus uridine). Co(NH3) 6 3+ binding was observed with the four RNA hairpins that contained a G±U wobble pair in the stem region. ESI MS revealed 1:1 and 1:2 complex formation with all RNAs. Weaker binding was observed with the fifth RNA hairpin that contained an A±C wobble pair in the stem region. The effects of pH on Co(NH3) 6 3+ binding were also examined.


  1. 1.
    Hofstadler, S. A.; Griffey, R. H. Analysis of Noncovalent Complexes of DNA and RNA by Mass Spectrometry. Chem. Rev. 2001, 101, 377–390.CrossRefGoogle Scholar
  2. 2.
    Beck, J. L.; Colgave, M. L.; Ralph, S. F.; Sheil, M. M. Electrospray Ionization Mass Spectrometry of Oligonucleotide Complexes with Drugs, Metals, and Proteins. Mass Spectrom. Rev. 2001, 20, 61–87.CrossRefGoogle Scholar
  3. 3.
    Lee, K.; Varma, S.; SantaLucia J., Jr.; Cunningham, P. R. In vivo Determination of RNA Structure—Function Relationships: Analysis of the 790 Loop in Ribosomal RNA. J. Mol. Biol. 1997, 269, 732–743.CrossRefGoogle Scholar
  4. 4.
    Kieft, J. S.; Tinoco, I., Jr. Solution Structure of a Metal-Binding Site in the Major Groove of RNA Complexed with Cobalt(III) Hexammine. Structure. 1997, 5, 713–721.CrossRefGoogle Scholar
  5. 5.
    Gdaniec, Z.; Sierzputowska-Gracz, H.; Theil, E. C. Iron Regulatory Element and Internal Loop/Bulge Structure for Ferritin mRNA Studied by Cobalt(III) Hexammine Binding, Molecular Modeling, and NMR Spectroscopy. Biochemistry. 1998, 37, 1505–1512.CrossRefGoogle Scholar
  6. 6.
    Pyle, A. M. Metal Ions in the Structure and Function of RNA. J. Biol. Inorg. Chem. 2002, 7, 679–690.CrossRefGoogle Scholar
  7. 7.
    Cowan, J. A. Inorganic Biochemistry: An Introduction 2nd ed.; Wiley-VCH: New York, NY, 1997; p 274.Google Scholar
  8. 8.
    Lippard, S. J.; Berg, J. M. Principles of Bioinorganic Chemistry; University Science Books: Mill Valley, CA, 1994; p 194.Google Scholar
  9. 9.
    Holbrook, S. R.; Sussman, J. L.; Warrant, R. W.; Church, G. M.; Kim, S.-H. RNA-Ligand Interactions: (I) Magnesium Binding Sites in Yeast tRNAPhe. Nucleic Acids Res. 1977, 4, 2811–2820.CrossRefGoogle Scholar
  10. 10.
    Cowan, J. A. Metallobiochemistry of RNA: Co(NH3)63+ as a Probe for Mg2+ (aq) Binding Sites. J. Inorg. Biochem. 1993, 49, 171–175.CrossRefGoogle Scholar
  11. 11.
    Schmitz, M.; Tinoco, I., Jr. Solution Structure and Metal-Ion Binding of the P4 Element from Bacterial RNase P RNA. RNA. 2000, 6, 1212–1225.CrossRefGoogle Scholar
  12. 12.
    Rudisser, S.; Tinoco, I., Jr. Solution Structure of Cobalt(III)Hexammine Complexed to the GAAA Tetraloop, and Metal-ion Binding to G±A Mismatches. J. Mol. Biol. 2000, 295, 1211–1223.CrossRefGoogle Scholar
  13. 13.
    Kim, S.; Cowan, J. A. Inert Cobalt Complexes as Mechanistic Probes of the Biochemistry of Magnesium Co-factors: Application to Topoisomerase I. Inorg. Chem. 1992, 31, 3495–3496.CrossRefGoogle Scholar
  14. 14.
    Gonzalez, R. L., Jr.; Tinoco, I., Jr. Solution Structure and Thermodynamics of a Divalent Metal Ion Binding Site in an RNA Pseudoknot. J. Mol. Biol. 1999, 289, 1267–1282.CrossRefGoogle Scholar
  15. 15.
    Herr, W.; Chapman, N. M.; Noller, H. F. Mechanism of Ribosomal Subunit Association: Discrimination of Specific Sites in 16 S RNA Essential for Association Activity. J. Mol. Biol. 1979, 130, 433–449.CrossRefGoogle Scholar
  16. 16.
    Tapprich, W. E.; Goss, D. J.; Dahlberg, A. E. Mutation at Position 791 in Escherichia coli 16S Ribosomal RNA Affects Processes Involved in the Initiation of Protein Synthesis. Proc. Natl. Acad. Sci. U.S.A. 1989, 86, 4927–4931.CrossRefGoogle Scholar
  17. 17.
    Moazed, D.; Noller, H. F. Transfer RNA Shields Specific Nucleotides in 16S Ribosomal RNA from Attack by Chemical Probes. Cell. 1986, 47, 985–994.CrossRefGoogle Scholar
  18. 18.
    Moazed, D.; Samaha, R. R.; Gualerzi, C.; Noller, H. F. Specific Protection of 16S rRNA by Translational Initiation Factors. J. Mol. Biol. 1995, 248, 207–210.Google Scholar
  19. 19.
    Santer, M.; Bennett-Guerrero, E.; Byahatti, S.; Czarnecki, S.; O’Connell, D.; Meyer, M.; Khoury, J.; Cheng, X.; Schwartz, I.; McLaughlin, J. Base Changes at Position 792 of Escherichia coli 16S rRNA Affect Assembly of 70S Ribosomes. Proc. Natl. Acad. Sci. U.S.A. 1990, 87, 3700–3704.CrossRefGoogle Scholar
  20. 20.
    Schuwirth, B. S.; Borovinskaya, M. A.; Hau, C. W.; Zhang, W.; Vila-Sanjurjo, A.; Holton, J. M.; Cate, J. H. D. Structures of the Bacterial Ribosome at 3.5 Å Resolution. Science. 2005, 310, 827–834.CrossRefGoogle Scholar
  21. 21.
    Yusupov, M. M.; Yusupova, G. Z.; Baucom, A.; Liebeman, K.; Earnest, T. N.; Cate, J. H.; Noller, H. F. Crystal Structure of the Ribosome at 5.5 Å Resolution. Science. 2001, 292, 883–896.CrossRefGoogle Scholar
  22. 22.
    Mitchell, P.; Osswald, M.; Brimacombe, R. Identification of Intermolecular RNA Cross-Links at the Subunit Interface of the Escherichia coli Ribosome. Biochemistry. 1992, 31, 3004–3011.CrossRefGoogle Scholar
  23. 23.
    Joeseph, S.; Weiser, B.; Noller, H. F. Mapping the Inside of the Ribosome with an RNA Helical Ruler. Science. 1997, 278, 1093–1098.CrossRefGoogle Scholar
  24. 24.
    Merryman, C.; Moazed, D.; Daubresse, G.; Noller, H. F. Nucleotides in 23S rRNA Protected by the Association of 30S and 50S Ribosomal Subunits. J. Mol. Biol. 1999, 285, 107–113.CrossRefGoogle Scholar
  25. 25.
    Ofengand, J.; Bakin, A. Mapping to Nucleotide Resolution of Pseudouridine Residues in Large Subunit Ribosomal RNAs from Representative Eukaryotes, Prokaryotes, Archaebacteria, Mitochondria, and Chloroplasts. J. Mol. Biol. 1997, 266, 246–268.CrossRefGoogle Scholar
  26. 26.
    Kowalak, J. A.; Pomerantz, S. C.; Crain, P. F.; McCloskey, J. A. A Novel Method for the Determination of Post-transcriptional Modification in RNA by Mass Spectrometry. Nucleic Acids Res. 1993, 21, 4577–4585.CrossRefGoogle Scholar
  27. 27.
    Scaringe, S. A.; Wincott, F. E.; Caruthers, M. H. Novel RNA Synthesis Method Using 5′-O-Silyl-2′-O-Orthoester Protecting Groups. J. Am. Chem. Soc. 1998, 120, 11820–11821.CrossRefGoogle Scholar
  28. 28.
    Chui, H. M.-P.; Desaulniers, J.-P.; Scaringe, S. A.; Chow, C. S. Synthesis of Helix 69 of Escherichia coli 23S rRNA Containing Its Natural Modified Nucleosides, m3-Psi and Psi. J. Org. Chem. 2002, 67, 8847–8854.CrossRefGoogle Scholar
  29. 29.
    Richards, E. G. Use of Tables in Calculating Absorption, Optical Rotary Dispersion, and Circular Dichroism of Polyribonucleotides Fasman G. D., Ed.; In Handbook of Biochemistry and Molecular Biology: Nucleic Acids; CRC Press: Cleveland, OH, 1975; pp 569–599.Google Scholar
  30. 30.
    Loo, J. A. Studying Noncovalent Protein Complexes by Electrospray Ionization Mass Spectrometry. Mass Spectrom. Rev. 1997, 16, 1–23.CrossRefGoogle Scholar
  31. 31.
    Sannes-Lowery, K. A.; Griffey, R. H.; Hofstadler, S. A. Measuring Dissociation Constants of RNA and Aminoglycoside Antibiotics by Electrospray Ionization Mass Spectrometry. Anal. Biochem. 2000, 280, 264–271.CrossRefGoogle Scholar
  32. 32.
    Bligh, S. W. A.; Haley, T.; Lowe, P. N. Measurement of Dissociation Constants of Inhibitors Binding to Src SH2 Domain Protein by Noncovalent Electrospray Ionization Mass Spectrometry. J. Mol. Recognit. 2003, 16, 139–148.CrossRefGoogle Scholar
  33. 33.
    Brown, T.; Leonard, G. A.; Booth, E. D.; Kneale, G. Influence of pH on the Conformation and Stability of Mismatch Base-Pairs in DNA. J. Mol. Biol. 1990, 212, 437–440.CrossRefGoogle Scholar
  34. 34.
    Hunter, W. N.; Brown, T.; Anand, N. N.; Kennard, O. Structure of an Adenine-Cytosine Base Pair in DNA and Its Implications for Mismatch Repair. Nature. 1986, 320, 552–555.CrossRefGoogle Scholar
  35. 35.
    Cheng, X.; Gale, D. C.; Udseth, H. R.; Smith, R. D. Charge State Reduction of Oligonucleotide Negative Ions from Electrospray Ionization. Anal. Chem. 1995, 67, 586–593.CrossRefGoogle Scholar
  36. 36.
    Guo, X.; Bruist, M. F.; Davis, D. L.; Bentzley, C. M. Secondary Structural Characterization of Oligonucleotide Strands using Electrospray Ionization Mass Spectrometry. Nucleic Acids Res. 2005, 33, 3659–3666.CrossRefGoogle Scholar
  37. 37.
    Green-Church, K. B.; Limbach, P. A. Mononucleotide Gas-Phase Proton: Affinities as Determined by the Kinetic Method. J. Am. Soc. Mass Spectrom. 2000, 11, 24–32.CrossRefGoogle Scholar
  38. 38.
    Gidden, J.; Baker, E. S.; Ferzoco, A.; Bowers, M. T. Structural Motifs of DNA Complexes in the Gas Phase. Int. J. Mass Spectrom. 2005, 240, 183–193.CrossRefGoogle Scholar
  39. 39.
    Gidden, J.; Ferzoco, A.; Baker, E. S.; Bowers, M. T. Duplex Formation and the Onset of Helicity in Poly d(CG)n Oligonucleotides in a Solvent-Free Environment. J. Am. Chem. Soc. 2004, 126, 15132–15140.CrossRefGoogle Scholar
  40. 40.
    Mundoma, C.; Greenbaum, N. L. Sequestering of Eu(III) by a GAAA RNA Tetraloop. J. Am. Chem. Soc. 2002, 124, 3525–3532.CrossRefGoogle Scholar
  41. 41.
    Xu, H.; Liang, Y.; Zhang, P.; Du, F.; Zhou, B.-R.; Wu, J.; Liu, J.-H.; Liu, Z.-G.; Ji, L.-N. Biophysical Studies of a Ruthenium(II) Polypyridyl Complex Binding to DNA and RNA Prove that Nucleic Acid Structure has Significant Effects on Binding Behaviors. J. Biol. Inorg. Chem. 2005, 10, 529–538.CrossRefGoogle Scholar
  42. 42.
    Hammann, C.; Cooper, A.; Lilley, D. M. Thermodynamics of Ion-Induced RNA Folding in the Hammerhead Ribozyme: An Isothermal Titration Calorimetric Study. Biochemistry. 2001, 40, 1423–1429.CrossRefGoogle Scholar
  43. 43.
    Urathamakul, T.; Beck, J. L.; Sheil, M. M.; Aldrich-Wright, J. R.; Ralph, S. F. A Mass Spectrometric Investigation of Noncovalent Interactions Between Ruthenium Complexes and DNA. Dalton Trans. 2004, 17, 2683–2690.CrossRefGoogle Scholar
  44. 44.
    Gutell, R. R. Collection of Small Subunit (16S- and 16S-like) Ribosomal RNA Structures: 1994. Nucleic Acids Res. 1994, 22, 3502–3507.CrossRefGoogle Scholar
  45. 45.
    Gutell, R. R.; Gray, M. W.; Schnare, M. N. A Compilation of Large Subunit (23S and 23S-like) Ribosomal RNA Structures: 1993. Nucleic Acids Res. 1993, 21, 3055–3074.CrossRefGoogle Scholar

Copyright information

© American Society for Mass Spectrometry 2006

Authors and Affiliations

  1. 1.Department of ChemistryWayne State UniversityDetroitUSA

Personalised recommendations