Skip to main content
Download PDF

Gas-phase dissociation of oligoribonucleotides and their analogs studied by electrospray ionization tandem mass spectrometry

Journal of the American Society for Mass Spectrometry volume 16, pages 1262–1268 (2005)Cite this article

Abstract

Oligoribonucleotides (RNA) and modified oligonucleotides were subjected to low-energy collision-induced dissociation in a hybrid quadrupole time-of-flight mass spectrometer to investigate their fragmentation pathways. Only very restricted data are available on gas-phase dissociation of oligoribonucleotides and their analogs and the fundamental mechanistic aspects still need to be defined to develop mass spectrometry-based protocols for sequence identification. Such methods are needed, because chemically modified oligonucleotides can not be submitted to standard sequencing protocols.

In contrast to the dissociation of DNA, dissociation of RNA was found to be independent of nucleobase loss and it is characterized by cleavage of the 5′-P-O bond, resulting in the formation of c- and their complementary y-type ions. To evaluate the influence of different 2′-substituents, several modified tetraribonucleotides were analyzed. Oligoribonucleotides incorporating a 2′-methoxy-ribose or a 2′-fluoro-ribose show fragmentation that does not exhibit any preferred dissociation pathway because all different types of fragment ions are generated with comparable abundance. To analyze the role of the nucleobases in the fragmentation of the phosphodiester backbone, an oligonucleotide lacking the nucleobase at one position has been studied. Experiments indicated that the dissociation mechanism of RNA is not influenced by the nucleobase, thus, supporting a mechanism where dissociation is initiated by formation of an intramolecular cyclic transition state with the 2′-hydroxyl proton bridged to the 5′-phosphate oxygen.

References

  1. Zamecnik, P. C.; Stephenson, M. L. Inhibition of Rous Sarcoma Virus Replication and Cell Transformation by a Specific Oligodeoxynucleotide. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 280–284.

    Article  CAS  Google Scholar 

  2. Stephenson, M. L.; Zamecnik, P. C. Inhibition of Rous sarcoma Viral RNA Translation by a Specific Oligodeoxyribonucleotide. Proc. Natl. Acad. Sci. U.S.A. 1978, 75, 285–288.

    Article  CAS  Google Scholar 

  3. Leumann, C. J. DNA Analogues: From Supramolecular Principles to Biological Properties. Bioorg. Med. Chem. 2002, 10, 841–854.

    Article  CAS  Google Scholar 

  4. Baker, B. F.; Monia, B. P. Novel Mechanisms for Antisense-Mediated Regulation of Gene Expression. Biochim. Biophys. Acta 1999, 1489, 3–18.

    Article  CAS  Google Scholar 

  5. Goodchild, J. Oligonucleotide Therapeutics: 25 Years Agrowing. Curr. Opin. Mol. Ther. 2004, 6, 120–128.

    CAS  Google Scholar 

  6. Altmann, K. H.; Dean, N. M.; Fabbro, D.; Freier, S. M.; Geiger, T.; Häner, R.; Hüsken, D.; Martin, P.; Monia, B. P.; Müller, M.; Natt, F.; Nicklin, P.; Phillips, J.; Piels, U.; Sasmor, H.; Moser, H. E. Second Generation of Antisense Oligonucleotides: From Nuclease Resistance to Biological Efficacy in Animals. Chimia. 1996, 50, 168–176.

    CAS  Google Scholar 

  7. Myers, K. J.; Dean, N. M. Sensible Use of Antisense: How to Use Oligonucleotides as Research Tools. TIPS 2000, 21, 19–23.

    CAS  Google Scholar 

  8. Campbell, J. M.; Bacon, T. A.; Wickstrom, E. Oligodeoxynucleoside Phosphorothioate Stability in Subcellular Extracts, Culture Media, Sera and Cerebrospinal Fluid. J. Biochem. Biophys. Methods 1990, 20, 259–267.

    Article  CAS  Google Scholar 

  9. Stein, C. A.; Tonkinson, J. L.; Yakubov, L. Phosphorothioate Oligodeoxynucleotides-Anti-Sense Inhibitors of Gene Expression? Pharmacol. Ther. 1991, 52, 365–384.

    Article  CAS  Google Scholar 

  10. Tang, W.; Zhu, L.; Smith, L. M. Controlling DNA Fragmentation in MALDI-MS by Chemical Modification. Anal. Chem. 1997, 69, 302–312.

    Article  CAS  Google Scholar 

  11. Sanger, F.; Nicklen, S.; Coulson, A. R. D. N. A. Sequencing with Chain-Terminating Inhibitors. Proc. Natl. Acad. Sci. U.S.A. 1977, 74, 5463–5467.

    Article  CAS  Google Scholar 

  12. Dovichi, N. J.; Zhang, J. How capillary electrophoresis sequenced the human genome. Angew. Chem. Int. Ed. 2000, 39, 4463–4468.

    Article  CAS  Google Scholar 

  13. Wan, K. X.; Gross, J.; Hillenkamp, F.; Gross, M. L. Fragmentation Mechanisms of Oligodeoxynucleotides Studied by H/D Exchange and Electrospray Ionization Tandem Mass Spectrometry. J. Am. Soc. Mass Spectrom. 2001, 12, 193–205.

    Article  CAS  Google Scholar 

  14. Wang, Z.; Wan, K. X.; Ramanathan, R; Taylor, John S.; Gross, M. L. Structure and Fragmentation Mechanisms of Isomeric T-Rich Oligodeoxynucleotides: A Comparison of Four Tandem Mass Spectrometric Methods. J. Am. Soc. Mass. Spectrom. 1998, 9, 683–691.

    Article  CAS  Google Scholar 

  15. McLuckey, S. A.; Habibi-Goudarzi, S. Decompositions of Multiply Charged Oligonucleotide Anions. J. Am. Chem. Soc. 1993, 115, 12085–12095.

    Article  CAS  Google Scholar 

  16. McLuckey, S. A.; Van Berkel, G. J.; Glish, G. L. Tandem Mass Spectrometry of Small, Multiply Charged Oligonucleotides. J. Am. Soc. Mass Spectrom. 1992, 3, 60–70.

    Article  CAS  Google Scholar 

  17. Bartlett, M. G.; McCloskey, J. A.; Manalili, S.; Griffey, R. H. The Effect of Backbone Charge on the Collision-Induced Dissociation of Oligonucleotides. J. Mass Spectrom. 1996, 31, 1277–1283.

    Article  CAS  Google Scholar 

  18. Wan, K. X.; Gross, M. L. Fragmentation Mechanisms of Oligodeoxynucleotides: Effects of Replacing Phosphates with Methylphosphonates and Thymines with Other Bases in T-Rich Sequences. J. Am. Soc. Mass Spectrom. 2001, 12, 580–589.

    Article  CAS  Google Scholar 

  19. Yacyshyn, B. R.; Chey, W. Y.; Goff, J.; Salzberg, B.; Baerg, R.; Buchman, A. L.; Tami, J.; Yu, R.; Gibiansky, E.; Shanahan, W. R. Double Blind, Placebo Controlled Trial of the Remission Inducing and Steroid Sparing Properties of an ICAM-1 Antisense Oligodeoxynucleotide, Alicaforsen (ISIS 2302), in Active Steroid Dependent Crohn’s Disease. Gut 2002, 51, 30–36.

    Article  CAS  Google Scholar 

  20. Sewell, K. L.; Geary, R. S.; Baker, B. F.; Glover, J. M.; Mant, T. G.; Yu, R. Z.; Tami, J. A.; Dorr, A. Phase I Trial of ISIS 104838, a 2′-Methoxyethyl Modified Antisense Oligonucleotide Targeting Tumor Necrosis Factor-a. JPET 2002, 303, 1334–1343.

    Article  CAS  Google Scholar 

  21. Herbst, R. S.; Frankel, S. R. Oblimersen Sodium (Genasense bcl-2 Antisense Oligonucleotide): A Rational Therapeutic to Enhance Apoptosis in Therapy of Lung Cancer. Clin. Cancer Res. 2004, 10, 4245–4248.

    Article  Google Scholar 

  22. Wang, B. H.; Hopkins, C. E.; Belenky, A. B.; Cohen, A. S. Sequencing of Modified Oligonucleotides Using In-Source Fragmentation and Delayed Pulsed Ion Extraction Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry. Int. J. Mass Spectrom. Ion Proccess 1997, 169/170, 331–350.

    Article  CAS  Google Scholar 

  23. Sannes-Lowery, K. A.; Hofstadler, S. A. Sequence Confirmation of Modified Oligonucleotides Using IRMPD in the External Ion Reservoir of an Electrospray Ionization Fourier Transform Ion Cyclotron Mass Spectrometer. J. Am. Soc. Mass Spectrom. 2003, 14, 825–833.

    Article  CAS  Google Scholar 

  24. Cerny, R. L.; Tomer, K. B.; Gross, M. L.; Grotjahn, L. Fast Atom Bombardment Combined with Tandem Mass Spectrometry for Determining Structures of Small Oligonucleotides. Anal. Biochem. 1987, 165, 175–182.

    Article  CAS  Google Scholar 

  25. Kirpekar, F.; Krogh, T. N. RNA Fragmentation Studied in a Matrix-Assisted Laser Desorption/Ionisation Tandem Quadrupole/Orthogonal Time-of-Flight Mass Spectrometer. Rapid Commun. Mass Spectrom. 2001, 15, 8–14.

    Article  CAS  Google Scholar 

  26. Schürch, S.; Bernal-Mendez, E.; Leumann, C. J. Electrospray Tandem Mass Spectrometry of Mixed-Sequence RNA/DNA Oligonucleotides. J. Am. Soc. Mass Spectrom. 2002, 13, 936–945.

    Article  Google Scholar 

  27. Nordhoff, E.; Cramer, R.; Karas, M.; Hillenkamp, F.; Kirpekar, F.; Kristiansen, K.; Roepstorff, P. Ion Stability of Nucleic Acids in Infrared Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry. Nucleic Acids Res. 1993, 21, 3347–3357.

    Article  CAS  Google Scholar 

  28. Ni, J.; Pomerantz, S. C.; Rozenski, J.; Zhang, Y.; McCloskey, J. A. Interpretation of Oligonucleotide Mass Spectra for Determination of Sequence Using Electrospray Ionization and Tandem Mass Spectrometry. Anal. Chem. 1996, 68, 1989–1999.

    Article  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Chemistry and Biochemistry, University of Bern, Freiestrasse 3, CH-3012, Bern, Switzerland

    Jan M. Tromp & Stefan Schürch

Authors
  1. Jan M. Tromp
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Stefan Schürch
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Stefan Schürch.

Additional information

Published online June 23, 2005

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Tromp, J.M., Schürch, S. Gas-phase dissociation of oligoribonucleotides and their analogs studied by electrospray ionization tandem mass spectrometry. J Am Soc Mass Spectrom 16, 1262–1268 (2005). https://doi.org/10.1016/j.jasms.2005.03.024

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1016/j.jasms.2005.03.024

Keywords

  • Hydroxyl Proton
  • Backbone Cleavage
  • Electrospray Ionization Tandem Mass Spectrometry
  • Phosphodiester Backbone
  • Phosphodiester Group