Structural Information from Oligonucleotides

  • Richard WardEmail author
  • Olav SchiemannEmail author
Part of the Structure and Bonding book series (STRUCTURE, volume 152)


The important role of DNA in biology has been known for a long time, but during the last decade it has become apparent that also RNA has many more functions than previously believed, ranging from gene regulation to catalysis of the polypeptide bond formation in the ribosomes. Thus, biophysical methods are needed that will allow the unravelling of their structures and conformational changes as well as dynamics and complex formations. Advances in site-directed spin labelling (SDSL) of oligonucleotides and electron paramagnetic resonance (EPR) spectroscopic methods like pulsed electron–electron double resonance (PELDOR or synonymously know as DEER) offer a means to achieve this and have been applied to various oligonucleotide systems. This chapter will give an overview of recent developments and applications in this field.





Circular dichroism


Continuous wave


Double electron–electron resonance




Electron paramagnetic resonance


Förster resonance energy transfer


Nuclear magnetic resonance


Pulsed electron–electron double resonance





The BBSRC (BB/H017917/1) and the Wellcome Trust (13973) are acknowledged for funding and the Research Councils of the UK are thanked for an RCUK fellowship.


  1. 1.
    Hunsicker-Wang L, Vogt M, Derose VJ (2009) EPR methods to study specific metal-ion binding sites in RNA. Methods Enzymol 468:335–367CrossRefGoogle Scholar
  2. 2.
    Schiemann O, Fritscher J, Kisseleva N, Sigurdsson ST, Prisner TF (2003) Structural investigation of a high-affinity MnII binding site in the hammerhead ribozyme by EPR spectroscopy and DFT calculations. Effects of neomycin B on metal-ion binding. Chembiochem 4(10):1057–1065CrossRefGoogle Scholar
  3. 3.
    Kisseleva N, Khvorova A, Westhof E, Schiemann O (2005) Binding of manganese(II) to a tertiary stabilized hammerhead ribozyme as studied by electron paramagnetic resonance spectroscopy. RNA 11(1):1–6CrossRefGoogle Scholar
  4. 4.
    Kisseleva N, Kraut S, Jäschke A, Schiemann O (2007) Characterizing multiple metal ion binding sites within a ribozyme by cadmium-induced EPR silencing. HFSP J 1(2):127–136CrossRefGoogle Scholar
  5. 5.
    Clever GH, Reitmeier SJ, Carell T, Schiemann O (2010) Antiferromagnetic coupling of stacked Cu(II)-salen complexes in DNA. Angew Chem Int Ed Engl 49(29):4927–4929CrossRefGoogle Scholar
  6. 6.
    Berliner LJ (2010) From spin-labeled proteins to in vivo EPR applications. Eur Biophys J 39(4):579–588CrossRefGoogle Scholar
  7. 7.
    Zhang X, Cekan P, Sigurdsson ST, Qin PZ (2009) Studying RNA using site-directed spin-labeling and continuous-wave electron paramagnetic resonance spectroscopy. Methods Enzymol 469:303–328CrossRefGoogle Scholar
  8. 8.
    Berliner LJ, Eaton SS, Eaton GR (eds) (2000) Distance measurements in biological systems by EPR. In: Biological magnetic resonance, vol 19. Kluwer Academic/Plenum, New YorkGoogle Scholar
  9. 9.
    Schiemann O, Prisner TF (2007) Long-range distance determinations in biomacromolecules by EPR spectroscopy. Q Rev Biophys 40(1):1–53CrossRefGoogle Scholar
  10. 10.
    Margraf D, Cekan P, Prisner TF, Sigurdsson ST, Schiemann O (2009) Ferro- and antiferromagnetic exchange coupling constants in PELDOR spectra. Phys Chem Chem Phys 11(31):6708–6714CrossRefGoogle Scholar
  11. 11.
    Rabenstein MD, Shin YK (1995) Determination of the distance between two spin labels attached to a macromolecule. Proc Natl Acad Sci USA 92:8239–8243CrossRefGoogle Scholar
  12. 12.
    Marsh D, Horvath LI (1989) Spin label studies of the structure and dynamics of lipids and proteins in membranes. In: Hoff AJ (ed) Advanced EPR. Applications in biology and biochemistry. Elsevier, Amsterdam, pp 707–752Google Scholar
  13. 13.
    Robinson BH, Drobny GP (1995) Site-specific dynamics in DNA: theory and experiment. Methods Enzymol 261:451–509CrossRefGoogle Scholar
  14. 14.
    Robinson BH, Mailer C, Drobny G (1997) Site-specific dynamics in DNA: experiments. Annu Rev Biophys Biomol Struct 26:629–658CrossRefGoogle Scholar
  15. 15.
    Keyes RS, Bobst AM (1998) Spin-labeled nucleic acids. In: Berliner LJ (ed) Spin labeling. The next millennium, vol 14. Plenum, New York, pp 283–338Google Scholar
  16. 16.
    Liang ZL, Freed JH, Keyes RS, Bobst AM (2000) An electron spin resonance study of DNA dynamics using the slowly relaxing local structure model. J Phys Chem B 104:5372–5381CrossRefGoogle Scholar
  17. 17.
    Qin PZ, Butcher SE, Feigon J, Hubbell WL (2001) Quantitative analysis of the isolated GAAA tetraloop/receptor interaction in solution: a site-directed spin labeling study. Biochemistry 40:6929–6936CrossRefGoogle Scholar
  18. 18.
    Qin PZ, Dieckmann T (2004) Application of NMR and EPR methods to the study of RNA. Curr Opin Struct Biol 14:350–359CrossRefGoogle Scholar
  19. 19.
    Schiemann O, Piton N, Mu Y, Stock G, Engels JW, Prisner TF (2004) A PELDOR-based nanometer distance ruler for oligonucleotides. J Am Chem Soc 126(18):5722–5729CrossRefGoogle Scholar
  20. 20.
    Cai Q, Kusnetzow AK, Hubbell WL, Haworth IS, Gacho GP, Van Eps N, Hideg K, Chambers EJ, Qin PZ (2006) Site-directed spin labeling measurements of nanometer distances in nucleic acids using a sequence-independent nitroxide probe. Nucleic Acids Res 34(17):4722–4730CrossRefGoogle Scholar
  21. 21.
    Jeschke G, Chechik V, Ionita P, Godt A, Zimmermann H, Banham J, Timmel CR, Hilger D, Jung H (2006) DeerAnalysis2006 – a comprehensive software package for analyzing pulsed ELDOR data. Appl Magn Reson 30:473–498CrossRefGoogle Scholar
  22. 22.
    Ward R, Keeble DJ, El-Mkami H, Norman DG (2007) Distance determination in heterogeneous DNA model systems by pulsed EPR. Chembiochem 8(16):1957–1964CrossRefGoogle Scholar
  23. 23.
    Kolb HC, Finn MG, Sharpless KB (2001) Click chemistry: diverse chemical function from a few good reactions. Angew Chem Int Ed Engl 40(11):2004–2021CrossRefGoogle Scholar
  24. 24.
    Flaender M, Sicoli G, Fontecave T, Mathis G, Saint-Pierre C, Boulard Y, Gambarelli S, Gasparutto D (2008) Site-specific insertion of nitroxide-spin labels into DNA probes by click chemistry for structural analyses by ELDOR spectroscopy. Nucleic Acids Symp Ser (Oxf) 52:147–148CrossRefGoogle Scholar
  25. 25.
    Ding P, Wunnicke D, Steinhoff HJ, Seela F (2010) Site-directed spin-labeling of DNA by the azide-alkyne ‘click’ reaction: nanometer distance measurements on 7-deaza-2′-deoxyadenosine and 2′-deoxyuridine nitroxide conjugates spatially separated or linked to a ‘dA-dT’ base pair. Chemistry 16(48):14385–14396CrossRefGoogle Scholar
  26. 26.
    Flaender M, Sicoli G, Aci-Seche S, Reignier T, Maurel V, Saint-Pierre C, Boulard Y, Gambarelli S, Gasparutto D (2011) A triple spin-labeling strategy coupled with deer analysis to detect DNA modifications and enzymatic repair. Chembiochem 12:2560–2563CrossRefGoogle Scholar
  27. 27.
    Gore J, Bryant Z, Nöllmann M, Le MU, Cozzarelli NR, Bustamante C (2006) DNA overwinds when stretched. Nature 442(7104):836–839CrossRefGoogle Scholar
  28. 28.
    Mathew-Fenn RS, Das R, Harbury PA (2008) Remeasuring the double helix. Science 322(5900):446–449CrossRefGoogle Scholar
  29. 29.
    Marko A, Denysenkov V, Margraf D, Cekan P, Schiemann O, Sigurdsson ST, Prisner TF (2011) Conformational flexibility of DNA. J Am Chem Soc 133(34):13375–13379CrossRefGoogle Scholar
  30. 30.
    Schiemann O, Cekan P, Margraf D, Prisner TF, Sigurdsson ST (2009) Relative orientation of rigid nitroxides by PELDOR: beyond distance measurements in nucleic acids. Angew Chem Int Ed Engl 48:3292–3295CrossRefGoogle Scholar
  31. 31.
    Sicoli G, Mathis G, Delalande O, Boulard Y, Gasparutto D, Gambarelli S (2008) Double electron–electron resonance (DEER): a convenient method to probe DNA conformational changes. Angew Chem Int Ed Engl 47(4):735–737CrossRefGoogle Scholar
  32. 32.
    Saenger W (1984) Principles of nucleic acid structure. Springer, New YorkCrossRefGoogle Scholar
  33. 33.
    Singh V, Azarkh M, Exner TE, Hartig JS, Drescher M (2009) Human telomeric quadruplex conformations studied by pulsed EPR. Angew Chem Int Ed Engl 48(51):9728–9730CrossRefGoogle Scholar
  34. 34.
    Freeman AD, Ward R, El Mkami H, Lilley DM, Norman DG (2011) Analysis of conformational changes in the DNA junction-resolving enzyme T7 endonuclease I on binding a four-way junction using EPR. Biochemistry 50(46):9963–9972CrossRefGoogle Scholar
  35. 35.
    Bowman MK, Becker D, Sevilla MD, Zimbrick JD (2005) Track structure in DNA irradiated with heavy ions. Radiat Res 163(4):447–454CrossRefGoogle Scholar
  36. 36.
    Sicoli G, Mathis G, Aci-Sèche S, Saint-Pierre C, Boulard Y, Gasparutto D, Gambarelli S (2009) Lesion-induced DNA weak structural changes detected by pulsed EPR spectroscopy combined with site-directed spin labelling. Nucleic Acids Res 37(10):3165–3176CrossRefGoogle Scholar
  37. 37.
    Azarkh M, Okle O, Singh V, Seemann IT, Hartig JS, Dietrich DR, Drescher M (2011) Long-range distance determination in a DNA model system inside Xenopus laevis oocytes by in-cell spin-label EPR. Chembiochem 12(13):1992–1995CrossRefGoogle Scholar
  38. 38.
    Krstić I, Hänsel R, Romainczyk O, Engels JW, Dötsch V, Prisner TF (2011) Long-range distance measurements on nucleic acids in cells by pulsed EPR spectroscopy. Angew Chem Int Ed Engl 50(22):5070–5074CrossRefGoogle Scholar
  39. 39.
    Macosko JC, Pio MS, Tinoco I, Shin Y-K (1999) A novel 5′ displacement spin-labeling technique for electron paramagnetic resonance spectroscopy of RNA. RNA 5:1158–1166CrossRefGoogle Scholar
  40. 40.
    Kim N-K, Murali A, DeRose VJ (2004) A distance ruler for RNA using EPR and site-directed spin labeling. Chem Biol 11:939–948CrossRefGoogle Scholar
  41. 41.
    Edwards TE, Okonogi TM, Sigurdsson ST (2002) Investigation of RNA-protein and RNA-metal ion interactions by electron paramagnetic resonance spectroscopy. The HIV TAR-Tat motif. Chem Biol 9(6):699–706CrossRefGoogle Scholar
  42. 42.
    Schiemann O, Weber A, Edwards TE, Prisner TF, Sigurdsson ST (2003) Nanometer distance measurements on RNA using PELDOR. J Am Chem Soc 125(12):3434–3435CrossRefGoogle Scholar
  43. 43.
    Borbat PP, Davis JH, Butcher SE, Freed JH (2004) Measurement of large distances in biomolecules using double-quantum filtered refocused electron spin-echoes. J Am Chem Soc 126(25):7746–7747CrossRefGoogle Scholar
  44. 44.
    Piton N, Mu Y, Stock G, Prisner TF, Schiemann O, Engels JW (2007) Base-specific spin-labeling of RNA for structure determination. Nucleic Acids Res 35(9):3128–3143CrossRefGoogle Scholar
  45. 45.
    Romainczyk O, Endeward B, Prisner TF, Engels JW (2011) The RNA-DNA hybrid structure determined by EPR, CD and RNase H1. Mol Biosyst 7(4):1050–1052CrossRefGoogle Scholar
  46. 46.
    Cai Q, Kusnetzow AK, Hideg K, Price EA, Haworth IS, Qin PZ (2007) Nanometer distance measurements in RNA using site-directed spin labeling. Biophys J 93(6):2110–2117CrossRefGoogle Scholar
  47. 47.
    Sicoli G, Wachowius F, Bennati M, Höbartner C (2010) Probing secondary structures of spin-labeled RNA by pulsed EPR spectroscopy. Angew Chem Int Ed Engl 49(36):6443–6447CrossRefGoogle Scholar
  48. 48.
    Kim NK, Bowman MK, DeRose VJ (2010) Precise mapping of RNA tertiary structure via nanometer distance measurements with double electron-electron resonance spectroscopy. J Am Chem Soc 132(26):8882–8884CrossRefGoogle Scholar
  49. 49.
    Wunnicke D, Strohbach D, Weigand JE, Appel B, Feresin E, Suess B, Müller S, Steinhoff HJ (2011) Ligand-induced conformational capture of a synthetic tetracycline riboswitch revealed by pulse EPR. RNA 17(1):182–188CrossRefGoogle Scholar
  50. 50.
    Krstić I, Frolow O, Sezer D, Endeward B, Weigand JE, Suess B, Engels JW, Prisner TF (2010) PELDOR spectroscopy reveals preorganization of the neomycin-responsive riboswitch tertiary structure. J Am Chem Soc 132(5):1454–1455CrossRefGoogle Scholar
  51. 51.
    Reginsson GW, Hunter RI, Cruickshank PAS, Bolton DR, Sigurdsson ST, Smith GM, Schiemann O (2011) W-Band PELDOR with 1kW Microwave Power: Molecular Geometry, Flexibility and Exchange Coupling. J Mag Res, doi: 10.1016/j.jmr.2012.01.019Google Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2012

Authors and Affiliations

  1. 1.Biomedical Sciences Research Complex, Centre of Magnetic ResonanceUniversity of St AndrewsSt Andrews, FifeUK
  2. 2.Institute of Physical and Theoretical ChemistryUniversity of BonnBonnGermany

Personalised recommendations