Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Protocol
  • Published:

Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances

Nature Protocols volume 2, pages 904–923 (2007)Cite this article

Abstract

In this protocol, we describe the facile synthesis of the nitroxide spin-label 2,2,5,5-tetramethyl-pyrrolin-1-oxyl-3-acetylene (TPA) and then its coupling to DNA/RNA through Sonogashira cross-coupling during automated solid-phase synthesis. Subsequently, we explain how to perform distance measurements between two such spin-labels on RNA/DNA using the pulsed electron paramagnetic resonance method pulsed electron double resonance (PELDOR). This combination of methods can be used to study global structure elements of oligonucleotides in frozen solution at RNA/DNA amounts of ∼10 nmol. We especially focus on the Sonogashira cross-coupling step, the advantages of the ACE chemistry together with the appropriate parameters for the RNA synthesizer and on the PELDOR data analysis. This procedure is applicable to RNA/DNA strands of up to ∼80 bases in length and PELDOR yields reliably spin–spin distances up to ∼6.5 nm. The synthesis of TPA takes ∼5 days and spin labeling together with purification ∼4 days. The PELDOR measurements usually take ∼16 h and data analysis from an hour up to several days depending on the extent of analysis.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Examples of EPR pulse sequences for distance measurements.
Figure 2: Schematic representation of the PELDOR data processing.
Figure 3: Reaction scheme for the synthesis of TPA 5.
Figure 4: Modified DNA cycle.
Figure 5
Figure 6: Setup as used for the Sonogashira cross-coupling on column.
Figure 7
Figure 8: As an example, this figure shows (a) the PELDOR time trace after time adjustment and normalization, (b) the Fourier transformation of a after background subtraction and (c) the Tikhonov regularization of a.

Similar content being viewed by others

References

  1. Berliner, L.J. (ed.) Biological Magnetic Resonance. Spin Labeling. The Next Millennium. Vol. 14 (Plenum Press, New York, USA, 1998).

  2. Caron, M. & Dugas, H. Specific spin-labeling of transfer ribonucleic acid molecules. Nuceic Acids Res. 3, 19–34 (1976).

    Article  CAS  Google Scholar 

  3. Luoma, G.A., Herring, F.G. & Marshall, A.G. Flexibility of end-labeled polymers from electron spin resonance line-shape analysis: 3′ terminus of transfer ribonucleic acid and 5S ribonucleic acid. Biochemistry 21, 6591–6598 (1982).

    Article  CAS  Google Scholar 

  4. Hara, H., Horiuche, T., Saneyoshi, M. & Nishimura, S. 4-Thiouridine-specific spin-labeling. Biochem. Biophys. Res. Commun. 38, 305–311 (1970).

    Article  CAS  Google Scholar 

  5. McIntosh, A.R., Caron, M. & Dugas, H. A specific spin labeling of the anticodon of E. coli tRNAGlu . Biochem. Biophys. Res. Commun. 55, 1356–1362 (1973).

    Article  CAS  Google Scholar 

  6. Nagahara, S., Murakami, A. & Makino, K. Spin-labeled oligonucleotides site specifically labeled at the internucleotide linkage. Separation of stereoisomeric probes and EPR spectroscopical detection of hybrid formation in solution. Nucleosides Nucleotides 11, 889–901 (1992).

    Article  CAS  Google Scholar 

  7. Qin, P.Z., Butcher, S.E., Feigon, J. & Hubbell, W.L. Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: a site-directed spin labeling study. Biochemistry 40, 6929–6936 (2001).

    Article  CAS  Google Scholar 

  8. Cai, Q. et al. Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe. Nucleic Acids Res. 34, 4722–4730 (2006).

    Article  CAS  Google Scholar 

  9. Edwards, T.E., Okonogi, T.M., Robinson, B.H. & Sigurdsson, S.T. Site-specific incorporation of nitroxide spin-labels into internal sites of the TAR RNA; structure-dependent dynamics of RNA by EPR spectroscopy. J. Am. Chem. Soc. 123, 1527–1528 (2001).

    Article  CAS  Google Scholar 

  10. Kim, N.-K., Murali, A. & DeRose, V.J. A distance ruler for RNA using EPR and site-directed spin labeling. Chem. Biol. 11, 939–948 (2004).

    Article  CAS  Google Scholar 

  11. Qin, P.Z., Hideg, K., Feigon, J. & Hubbell, W.L. Monitoring RNA base structure and dynamics using site-directed spin labeling. Biochemstry 42, 6772–6783 (2003).

    Article  CAS  Google Scholar 

  12. Ramos, A. & Varani, G. A new method to detect long-range protein–RNA contacts: NMR detection of electron–proton relaxation induced by nitroxide spin-labeled RNA. J. Am. Chem. Soc. 120, 10992–10993 (1998).

    Article  CAS  Google Scholar 

  13. Spaltenstein, A., Robinson, B.H. & Hopkins, P.B. A rigid and non-perturbing probe for duplex DNA motion. J. Am. Chem. Soc. 110, 1299–1301 (1988).

    Article  CAS  Google Scholar 

  14. Spaltenstein, A., Robinson, B.H. & Hopkins, P.B. Sequence- and structure-dependent DNA base dynamics: synthesis, structure, and dynamics of site and sequence specifically spin-labeled DNA. Biochemistry 28, 9484–9495 (1989).

    Article  CAS  Google Scholar 

  15. Hustedt, E.J., Kirchner, J.J., Spaltenstein, A., Hopkins, P.B. & Robinson, B.H. Monitoring DNA dynamics using spin-labels with different independent mobilities. Biochemistry 34, 4369–4375 (1995).

    Article  CAS  Google Scholar 

  16. Gannett, P.M. et al. Probing triplex formation by EPR spectroscopy using a newly synthesized spin label for oligonucleotides. Nucleic Acids Res. 30, 5328–5337 (2002).

    Article  CAS  Google Scholar 

  17. Schiemann, O., Piton, N., Mu, Y., Stock, G., Engels, J.W. & Prisner, T.F. A PELDOR-based nanometer distance ruler for oligonucleotides. J. Am. Chem. Soc. 126, 5722–5729 (2004).

    Article  CAS  Google Scholar 

  18. Piton, N., Schiemann, O., Mu, Y., Stock, G., Prisner, T.F. & Engels, J.W. Synthesis of spin-labeled RNAs for long range distance measurements by PELDOR. Nucleosides Nucleotides Nucleic Acids 24, 771–775 (2005).

    Article  CAS  Google Scholar 

  19. Piton, N., Mu, Y., Stock, G., Prisner, T.F., Schiemann, O. & Engels, J.W. Nucleic Acids Res. (in the press).

  20. Miller, T.R. et al. A probe for sequence-dependent nucleic acid dynamics. J. Am. Chem. Soc. 117, 9377–9378 (1995).

    Article  CAS  Google Scholar 

  21. Okonogi, T.M., Reese, A.W., Alley, S.C., Hopkins, P.B. & Robinson, B.H. Flexibility of duplex DNA on the sub-microsecond timescale. Biophys. J. 77, 3256–327 (1999).

    Article  CAS  Google Scholar 

  22. Barhate, N., Cekan, P., Massey, A.P. & Sigurdssson, S.T. A nucleoside that contains a rigid nitroxide spin label: a fluorophore in disguise. Angew. Chem. Int. Ed. 46, 2655–2658 (2007).

    Article  CAS  Google Scholar 

  23. Sprinzl, M., Scheit, K.H. & Cramer, F. Preparation in-vitro of a 2-thiocytidine-containing yeast transfer-RNA PHE-A73-C74-S2C75-A76 and its interaction with para-hydroxymercuribenzoate. Eur. J. Biochem. 34, 306–310 (1973).

    Article  CAS  Google Scholar 

  24. Sprinzl, M., Krämer, E. & Stehlik, D. On the structure of phenylalanine tRNA from yeast. Eur. J. Biochem. 49, 595–605 (1974).

    Article  CAS  Google Scholar 

  25. Macosko, J.C., Pio, M.S., Tinoco, J.R. & Shin, Y.-K. A novel 5′ displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA. RNA 5, 1158–1166 (1999).

    Article  CAS  Google Scholar 

  26. Bobst, A.M., Pauly, G.T., Keyes, R.S. & Bobst, E.V. Enzymatic sequence-specific spin labeling of a DNA fragment containing the recognition sequence of EcoRI endonuclease. FEBS Lett. 228, 33–36 (1988).

    Article  CAS  Google Scholar 

  27. Liang, Z., Freed, J.H., Keyes, R.S. & Bobst, A.M. An electron spin resonance study of DNA dynamics using the slowly relaxing local structure model. J. Phys. Chem. B. 104, 5372–5381 (2000).

    Article  CAS  Google Scholar 

  28. Keyes, R.S. & Bobst, A.M. Spin-labeled nucleic acids. in Biological Magnetic Resonance. Spin Labeling. The Next Millennium Vol. 14 (ed. Berliner, L.J.) 7.283–7.334 (Plenum Press, New York, 1998).

    Google Scholar 

  29. Okonogi, T.M., Alley, S.C., Reese, A.W., Hopkins, P.B. & Robinson, B.H. Sequence-dependent dynamics in duplex DNA. Biophys. J. 78, 2560–2571 (2000).

    Article  CAS  Google Scholar 

  30. van Doorslaer, S. & Jeschke, G. Dynamics by EPR: picosecond to microsecond time scales. in Fluxional Organometallic and Coordination Compounds. (eds. Gielen, M., Willem, R. & Wrackmeyer, B.) 6.219–6.242 (Wiley, Weinheim, 2004).

    Google Scholar 

  31. Jacobsen, K., Hubbell, W.L., Ernst, O.P. & Risse, T. Details of the partial unfolding of T4 lysozyme on quartz using site-directed spin labeling. Angew. Chem. Int. Ed. 45, 3874–3877 (2006).

    Article  CAS  Google Scholar 

  32. Potapenko, D.I. et al. Real-time monitoring of drug-induced changes in the stomach acidity of living rats using improved pH-sensitive nitroxides and low-field EPR techniques. J. Magn. Reson. 182, 1–11 (2006).

    Article  CAS  Google Scholar 

  33. Steinhoff, H.J. Inter- and intra-molecular distances determined by EPR spectroscopy and site-directed spin labeling reveal protein–protein and protein–oligonucleotide interaction. Biol. Chem. 385, 913–920 (2004).

    Article  CAS  Google Scholar 

  34. Halpern, H.J. et al. Diminished aqueous microviscosity of tumors in murine models measured with in vivo radiofrequency electron paramagnetic resonance. Cancer Res. 59, 5836–5841 (1999).

    CAS  PubMed  Google Scholar 

  35. Liang, B.Y., Bushweller, J.H. & Tamm, L.K. Site-directed parallel spin-labeling and paramagnetic relaxation enhancement in structure determination of membrane proteins by solution NMR spectroscopy. J. Am. Chem. Soc. 128, 4389–4397 (2006).

    Article  CAS  Google Scholar 

  36. Berliner, L.J., Eaton, S.S. & Eaton, G.R. (eds.) Biological Magnetic Resonance. Distance Measurements in Biological Systems by EPR Vol. 19 (Kluwer Academic, New York, 2000).

    Google Scholar 

  37. Jeschke, G., Bender, A., Paulsen, H., Zimmermann, H. & Godt, A. Sensitivity enhancement in pulse EPR distance measurements. J. Magn. Reson. 169, 1–12 (2004).

    Article  CAS  Google Scholar 

  38. Milov, A.D., Salikov, K.M. & Shirov, M.D. Application of the double resonance method to electron spin echo in a study of the spatial distribution of paramagnetic centers in solids. Sov. Phys. Solid State 23, 565–569 (1981).

    Google Scholar 

  39. Martin, R.E. et al. Determination of the end-to-end distances in a series of TEMPO diradicals of up to 2.8 nm length with a new four-pulse double electron electron resonance experiment. Angew. Chem. Int. Ed. 37, 2834–2837 (1998).

    CAS  Google Scholar 

  40. Milov, A.D., Maryasov, A.G. & Tsvetkov, Y.D. Pulsed electron double resonance (PELDOR) and its application in free-radicals research. Appl. Magn. Reson. 15, 107–143 (1998).

    Article  CAS  Google Scholar 

  41. Mims, W.B. in Electron Paramagnetic Resonance (ed. Geschwind, S.) 263–264 (Plenum Press, New York, 1972).

    Book  Google Scholar 

  42. Weber, A., Schiemann, O., Bode, B. & Prisner, T.F. PELDOR at S- and X-band frequencies and the separation of exchange coupling from dipolar coupling. J. Magn. Reson. 157, 277–285 (2002).

    Article  CAS  Google Scholar 

  43. Schiemann, O., Weber, A., Edwards, T.E., Prisner, T.F. & Sigurdsson, S.T. Nanometer distance measurements on RNA using PELDOR. J. Am. Chem. Soc. 125, 3434–3435 (2003).

    Article  CAS  Google Scholar 

  44. Milov, A.D. et al. The secondary structure of a membrane-modifying peptide in a supramolecular assembly studied by PELDOR and cw-ESR spectroscopy. J. Am. Chem. Soc. 123, 3784–3789 (2001).

    Article  CAS  Google Scholar 

  45. Denysenkov, V.P., Prisner, T.F., Stubbe, J. & Bennati, M. High-field pulsed electron-electron double resonance spectroscopy to determine the orientation of the tyrosyl radicals in ribonucleotide reductase. Proc. Natl. Acad. Sci. USA 103, 13386–13390 (2006).

    Article  CAS  Google Scholar 

  46. Elsässer, C., Brecht, M. & Bittl, R. Pulsed electron–electron double resonance on multinuclear metal clusters: assignment of spin projection factors based on the dipolar interaction. J. Am. Chem. Soc. 124, 12606–12611 (2002).

    Article  Google Scholar 

  47. Hara, H., Kawamori, A., Astashkin, A.V. & Ono, T. The distance from tyrosine D to redox-active components on the donor side of photosystem II determined by pulsed electron–electron double resonance. Biochim. Biophys. Acta 1276, 140–146 (1996).

    Article  Google Scholar 

  48. Raitsimring, A. “2+1” Pulse sequence as applied for distance and spatial distribution measurements of paramagnetic centers. in Biological Magnetic Resonance. Distance Measurements in Biological Systems by EPR Vol. 19 (eds. Berliner, L.J., Eaton, S.S. & Eaton, G.R.) 10.461–10.490 (Kluwer Academic, New York, 2000).

    Google Scholar 

  49. Borbat, P.P., Davis, J.H., Butcher, S.E. & Freed, J.H. Measurement of large distances in biomolecules using double quantum filtered refocused electron spin-echoes. J. Am. Chem. Soc. 126, 7746–7747 (2004).

    Article  CAS  Google Scholar 

  50. Eaton, S.S. & Eaton, G.R. Determination of distances based on T1 and Tm effects. in Biological Magnetic Resonance. Distance Measurements in Biological Systems by EPRF. Vol. 19 (eds. Berliner, L.J., Eaton, S.S. & Eaton, G.R.) 8.348–8.378 (Kluwer Academic, New York, 2000).

    Google Scholar 

  51. Lakowicz, J.R. Principles of Fluorescence Spectroscopy. 3rd edn., 13.443–13.471 (Springer, New York, 2006).

    Book  Google Scholar 

  52. Merritt, M.E., Sigurdsson, S.T. & Drobny, G.P. Long-range distance measurements to the phosphodiester backbone of solid nucleic acids using P-31-F-19 REDOR NMR. J. Am. Chem. Soc. 121, 6070–6071 (1999).

    Article  CAS  Google Scholar 

  53. Scaringe, S.A., Kitchen, D., Kaiser, R. & Marshall, W.S. Preparation of 5′-silyl-2′-orthoester ribonucleosides for use in oligoribonucleotide synthesis. Curr. Prot. Nucleics Acids Chem. 2.10.1–2.10.15 (2004).

  54. Scaringe, S.A. RNA oligonucleotide synthesis via 5′-silyl-2′-orthoester chemistry. Methods 23, 206–217 (2001).

    Article  CAS  Google Scholar 

  55. Hideg, K., Hankovszky, H.O., Lex, L. & Kulcsár, G. Nitroxyls: VI. Synthesis and reactions of 3-hydroxymethyl-2,2,5,5-tetramethyl-2,5-dihydropyrrole-1-oxyl and 3-formyl derivatives. Synthesis 911–914 (1980).

  56. Bowman, M.K., Maryasov, A.G., Kim, N.-K. & DeRose, V.J. Visualization of distance distribution from pulsed double electron–electron resonance data. Appl. Magn. Res. 26, 23–29 (2004).

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank D. Margraf for helpful advice on this manuscript. This work was supported by the DFG within the SFB 579.

Author information

Authors and Affiliations

  1. Institute of Physical and Theoretical Chemistry, Center for Biomolecular Magnetic Resonance, Frankfurt am Main, Germany

    Olav Schiemann, Jörn Plackmeyer, Bela E Bode & Thomas F Prisner

  2. Institute of Organic Chemistry and Chemical Biology, Max-von-Laue Strasse 7, J.W. Goethe-Universität, Frankfurt am Main, 60438, Germany

    Nelly Piton & Joachim W Engels

Authors
  1. Olav Schiemann
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Nelly Piton
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jörn Plackmeyer
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Bela E Bode
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Thomas F Prisner
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Joachim W Engels
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to Olav Schiemann or Joachim W Engels.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Schiemann, O., Piton, N., Plackmeyer, J. et al. Spin labeling of oligonucleotides with the nitroxide TPA and use of PELDOR, a pulse EPR method, to measure intramolecular distances. Nat Protoc 2, 904–923 (2007). https://doi.org/10.1038/nprot.2007.97

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.97

This article is cited by

Comments

By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing