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:

Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe

Nature Protocols volume 2, pages 2354–2365 (2007)Cite this article

Abstract

This protocol describes the procedures for measuring nanometer distances in nucleic acids using a nitroxide probe that can be attached to any nucleotide within a given sequence. Two nitroxides are attached to phosphorothioates that are chemically substituted at specific sites of DNA or RNA. Inter-nitroxide distances are measured using a four-pulse double electron–electron resonance technique, and the measured distances are correlated to the parent structures using a Web-accessible computer program. Four to five days are needed for sample labeling, purification and distance measurement. The procedures described herein provide a method for probing global structures and studying conformational changes of nucleic acids and protein/nucleic acid complexes.

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: Schematics of R5 labeling.
Figure 2: Pulse sequences for the 4-pulse double electron–electron resonance (DEER).
Figure 3: Sample data of R5 labeling and purification.
Figure 4

Similar content being viewed by others

References

  1. Fanucci, G.E. & Cafiso, D.S. Recent advances and applications of site-directed spin labeling. Curr. Opin. Struct. Biol. 16, 644–653 (2006).

    Article  CAS  Google Scholar 

  2. Qin, P.Z. & Dieckmann, T. Application of NMR and EPR methods to the study of RNA. Curr. Opin. Struct. Biol. 14, 350–359 (2004).

    Article  CAS  Google Scholar 

  3. Hubbell, W.L. & Altenbach, C. Investigation of structure and dynamics in membrane proteins using site-directed spin labeling. Curr. Opin. Struct. Biol. 4, 566–573 (1994).

    Article  CAS  Google Scholar 

  4. Hubbell, W.L., Cafiso, D.S. & Altenbach, C. Identifying conformational changes with site-directed spin labeling. Nat. Struct. Biol. 7, 735–739 (2000).

    Article  CAS  Google Scholar 

  5. Berliner, L.J., Eaton, G.R. & Eaton, S.S. (eds.) Distance measurements in biological systems by EPR. In: Biological Magnetic Resonance 19, 614 (Kluwer Academic, New York, 2000).

  6. Borbat, P.P., Costa-Filho, A.J., Earle, K.A., Moscicki, J.K. & Freed, J.H. Electron spin resonance in studies of membranes and proteins. Science 291, 266–269 (2001).

    Article  CAS  Google Scholar 

  7. Lakshmi, K.V. & Brudvig, G.W. Pulsed electron paramagnetic resonance methods for macromolecular structure determination. Curr. Opin. Struct. Biol. 11, 523–531 (2001).

    Article  CAS  Google Scholar 

  8. Hubbell, W.L., Altenbach, C., Hubbell, C.M. & Khorana, H.G. Rhodopsin structure, dynamics, and activation: a perspective from crystallography, site-directed spin labeling, sulfhydryl reactivity, and disulfide cross-linking. Adv. Protein. Chem. 63, 243–290 (2003).

    Article  CAS  Google Scholar 

  9. 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 

  10. Jeschke, G. EPR techniques for studying radical enzymes. Biochim. Biophys. Acta. 1707, 91–102 (2005).

    Article  CAS  Google Scholar 

  11. Jeschke, G. & Polyhach, Y. Distance measurements on spin-labelled biomacromolecules by pulsed electron paramagnetic resonance. Phys. Chem. Chem. Phys. 9, 1895–1910 (2007).

    Article  CAS  Google Scholar 

  12. Schiemann, O. 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).

    Article  CAS  Google Scholar 

  13. Rabenstein, M.D. & Shin, Y.K. Determination of the distance between two spin labels attached to a macromolecule. Proc. Natl. Acad. Sci. USA 92, 8239–8243 (1995).

    Article  CAS  Google Scholar 

  14. Altenbach, C., Oh, K.J., Trabanino, R.J., Hideg, K. & Hubbell, W.L. Estimation of inter-residue distances in spin labeled proteins at physiological temperatures: experimental strategies and practical limitations. Biochemistry, 40, 15471–15482 (2001).

    Article  CAS  Google Scholar 

  15. Kim, N., 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 

  16. Persson, M. et al. Comparison of electron paramagnetic resonance methods to determine distances between spin labels on human carbonic anhydrase II. Biophys. J. 80, 2886–2897 (2001).

    Article  CAS  Google Scholar 

  17. Borbat, P.P., McHaourab, H.S. & Freed, J.H. Protein structure determination using long-distance constraints from double-quantum coherence ESR: study of T4 lysozyme. J. Am. Chem. Soc. 124, 5304–5314 (2002).

    Article  CAS  Google Scholar 

  18. 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, 3334–3335 (2003).

    Article  Google Scholar 

  19. Schiemann, O. et al. A PELDOR based nanometer distance ruler for oligonucleotides. J. Am. Chem. Soc. 126, 5722–5729 (2004).

    Article  CAS  Google Scholar 

  20. 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 

  21. Sale, K., Song, L., Liu, Y.S., Perozo, E. & Fajer, P. Explicit treatment of spin labels in modeling of distance constraints from dipolar EPR and DEER. J. Am. Chem. Soc. 127, 9334–9335 (2005).

    Article  CAS  Google Scholar 

  22. Banham, J.E., Timmel, C.R., Abbott, R.J., Lea, S.M. & Jeschke, G. The characterization of weak protein-protein interactions: evidence from DEER for the trimerization of a von Willebrand Factor A domain in solution. Angew. Chem. Int. Ed. Engl. 45, 1058–1061 (2006).

    Article  CAS  Google Scholar 

  23. 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, 4722–4734 (2006).

    Article  CAS  Google Scholar 

  24. Piton, N. et al. Base-specific spin-labeling of RNA for structure determination. Nucleic Acids Res. 35, 3128–3143 (2007).

    Article  CAS  Google Scholar 

  25. Cai, Q. et al. Nanometer distance measurements in RNA using site-directed spin labeling. Biophys. J. 93, 2110–2117 (2007).

    Article  CAS  Google Scholar 

  26. Makinen, M.W., Mustafi, D. & Kasa, S. ENDOR of spin labels for structure determination: from small molecules to enzyme reaction intermediates. in Biological Magnetic Resonance (ed. Berliner, L.J.) 181–249 (Springer-Verlag, New York, LLC 1998).

    Google Scholar 

  27. 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 

  28. Pannier, M., Veit, S., Godt, A., Jeschke, G. & Spiess, H.W. Dead-time free measurement of dipole-dipole interactions between electron spins. J. Magn. Reson. 142, 331–340 (2000).

    Article  CAS  Google Scholar 

  29. Price, E.A., Sutch, B.T., Cai, Q., Qin, P.Z. & Haworth, I.S. Computation of nitroxide-nitroxide distances for spin-labeled DNA duplexes. Biopolymers 87, 40–50 (2007).

    Article  CAS  Google Scholar 

  30. 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 

  31. 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 

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

    Article  CAS  Google Scholar 

  33. 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 

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

    Article  CAS  Google Scholar 

  35. Milov, A.D., Salikohov, K.M. & Shirov, M.D. Applications of ENDOR in electron-spin echo for paramagnetic center space distribution in solids. Fiz. Tverd. Tela,. 23, 975–982 (1981).

    CAS  Google Scholar 

  36. Kurshev, V.V., Raitsimring, A.M. & Tsvetkov, Y.D. Selection of dipolar interaction by the “2 + 1” pulse train ESE. J. Magn. Reson. 81, 441–454 (1989).

    CAS  Google Scholar 

  37. Milov, A.D., Ponomarev, A.B. & Tsvetkov, Y.D. Electron-electron double resonance in electron spin echo: model biradical systems and the sensitized photolysis of decalin. Chem. Phys. Lett. 110, 67–72 (1984).

    Article  CAS  Google Scholar 

  38. Larsen, R.G. & Singel, D.J. Double electron—electron resonance spin—echo modulation: Spectroscopic measurement of electron spin pair separations in orientationally disordered solids. J. Chem. Phys. 98, 5134–5146 (1993).

    Article  CAS  Google Scholar 

  39. Rakowsky, M.H., More, K.M., Kulikov, A.V., Eaton, G.R. & Eaton, S.S. Time-domain electron paramagnetic resonance as a probe of electron-electron spin-spin interaction in spin-labeled low-spin iron porphyrins. J. Am. Chem. Soc. 117, 2049–2057 (1995).

    Article  CAS  Google Scholar 

  40. Saxena, S. & Freed, J.H. Double quantum two-dimensional Fourier transform electron spin resonance: distance measurements. Chem. Phys. Lett. 251, 102–110 (1996).

    Article  CAS  Google Scholar 

  41. Martin, R.E. et al. Determination of 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. Engl. 37, 2833–2837 (1998).

    Article  Google Scholar 

  42. Hinderberger, D., Schmelz, O., Rehahn, M. & Jeschke, G. Electrostatic site attachment of divalent counterions to rodlike ruthenium(II) coordination polymers characterized by EPR spectroscopy. Angew. Chem. Int. Ed. Engl. 43, 4616–4621 (2004).

    Article  CAS  Google Scholar 

  43. Pornsuwan, S., Bird, G., Schafmeister, C.E. & Saxena, S. Flexibility and lengths of bis-peptide nanostructures by electron spin resonance. J. Am. Chem. Soc. 128, 3876–3877 (2006).

    Article  CAS  Google Scholar 

  44. Jeschke, G., Wegener, C., Nietschke, M., Jung, H. & Steinhoff, H.J. Interresidual distance determination by four-pulse double electron-electron resonance in an integral membrane protein: the Na+/proline transporter PutP of Escherichia coli. Biophys. J. 86, 2551–2557 (2004).

    Article  CAS  Google Scholar 

  45. Hanson, S.M. et al. Structure and function of the visual arrestin oligomer. EMBO J. 26, 1726–1736 (2007).

    Article  CAS  Google Scholar 

  46. Oldham, W.M., Van Eps, N., Preininger, A.M., Hubbell, W.L. & Hamm, H.E. Mechanism of the receptor-catalyzed activation of heterotrimeric G proteins. Nat. Struct. Mol. Biol. 13, 756–757 (2006).

    Article  Google Scholar 

  47. Leporc, S. et al. An NMR and molecular modeling analysis of d(CTACTGCTTTAG). d(CTAAAGCAGTAG) reveals that the particular behavior of TpA steps is related to edge-to-edge contacts of their base-pairs in the major groove. Nucleic Acids Res. 27, 4759–4767 (1999).

    Article  CAS  Google Scholar 

  48. Caruthers, M.H., Beaton, G., Wu, J.V. & Wiesler, W. Chemical synthesis of deoxyoligonucleotides and deoxyoligonucleotide analogs. in: Methods in Enzymology (eds. Lilley, D.M.J. and Dahlberg, J.E.) 3–20 (Elsevier, 1992).

    Google Scholar 

  49. Usman, N. & Cedergren, R. Exploiting the chemical synthesis of RNA. Trends Biochem. Sci. 17, 334–339 (1992).

    Article  CAS  Google Scholar 

  50. Iyer, R.P., Phillips, L.R., Egan, W., Regan, J.B. & Beaucage, S.L. The automated synthesis of sulfur-containing oligodeoxyribonucleotides using 3H-1,2-benzodithiol-3-one 1,1-dioxide as a sulfur-transfer reagent. J. Org. Chem. 55, 4693–4699 (1990).

    Article  CAS  Google Scholar 

  51. Hankovszky, H.O., Hideg, K. & Lex, L. Nitroxyls; VII1. Synthesis and reactions of highly reactive 1-Oxyl-2,2,5,5-tetramethyl-2,5-dihydropyrrole-3-ylmethyl sulfonates. Synthesis 914–916 (1980).

  52. Lindgren, M. et al. Electron spin echo decay as a probe of aminoxyl environment in spin-labeled mutants of human carbonic anhydrase II. J. Chem. Soc. Perkin Trans. 2, 2549–2554 (1997).

    Article  Google Scholar 

  53. Zecevic, A., Eaton, G.R., Eaton, S.S. & Lindgren, M. Dephasing of electron spin echoes for nitroxyl radicals in glassy solvents by non-methyl and methyl protons. Mol. Phys. 95, 1255–1263 (1998).

    Article  CAS  Google Scholar 

  54. Weber, R.T. Bruker ELEXSYS E580 User's Manual (v. 2.0) 2005.

    Google Scholar 

  55. Weber, R.T. Bruker Pulsed ELDOR Option User's Manual (v. 1.0) 2006.

    Google Scholar 

  56. Jeschke, G., Panek, G., Godt, A., Bender, A. & Paulsen, H. Data analysis procedures for pulse ELDOR measurements of broad distance distributions. Appl. Magn. Reson. 26, 223–244 (2004).

    Article  CAS  Google Scholar 

  57. Bowman, M.K., Maryasov, A.G., Kim, N. & DeRose, V.J. Visulation of distance distribution from pulsed double electron-electron resonance data. Appl. Magn. Reson. 26, 23–39 (2004).

    Article  CAS  Google Scholar 

  58. Chiang, Y.W., Borbat, P.P. & Freed, J.H. The determination of pair distance distributions by pulsed ESR using Tikhonov regularization. J. Magn. Reson. 172, 279–295 (2005).

    Article  CAS  Google Scholar 

  59. Berman, H.M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).

    Article  CAS  Google Scholar 

  60. Berman, H.M. et al. The nucleic acid database. A comprehensive relational database of three-dimensional structures of nucleic acids. Biophys. J. 63, 751–759 (1992).

    Article  CAS  Google Scholar 

  61. Case, D.A. et al. AMBER8 Users' Manual (University of California, San Francisco, California, 2004).

    Google Scholar 

Download references

Acknowledgements

We thank Dr. Wayne L. Hubbell for providing access to a Bruker E-580 spectrometer; Drs. Wayne L. Hubbell, Christian Altenbach, Ned Van Eps and Balachandra G. Hegde for advice and discussions on DEER measurements; and Drs. Eric Chambers and Melina Bayramyan for early development of the NASDAC algorithm framework in which the NASNOX algorithm is based. Financial support is provided by the National Institutes of Health (R01 GM069557) and the National Science Foundation (MCB0546529). The authors also thank the William R. Wiley Environmental Molecular Sciences Laboratory for a pulse EPR instrumentation time award.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of Southern California, Los Angeles, 90089-0744, California, USA

    Peter Z Qin, Qi Cai, Gian Paola G Grant, Glenna Z Sowa, Anna Popova, Bruno Herreros & Honghang He

  2. Department of Biological Sciences, University of Southern California, Los Angeles, 90089-0744, California, USA

    Peter Z Qin & Eric A Price

  3. Department of Pharmacology and Pharmaceutical Sciences, University of Southern California, Los Angeles, 90089-9121, California, USA

    Ian S Haworth

  4. Jules Stein Eye Institute and Department of Chemistry and Biochemistry, University of California, Los Angeles, 90095, California, USA

    Ana K Kusnetzow

Authors
  1. Peter Z Qin
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Ian S Haworth
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Qi Cai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ana K Kusnetzow
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Gian Paola G Grant
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Eric A Price
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Glenna Z Sowa
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Anna Popova
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Bruno Herreros
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Honghang He
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Peter Z Qin.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Qin, P., Haworth, I., Cai, Q. et al. Measuring nanometer distances in nucleic acids using a sequence-independent nitroxide probe. Nat Protoc 2, 2354–2365 (2007). https://doi.org/10.1038/nprot.2007.308

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nprot.2007.308

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