Journal of Biomolecular NMR

, Volume 36, Issue 3, pp 137–146 | Cite as

General method of preparation of uniformly 13C, 15N-labeled DNA fragments for NMR analysis of DNA structures

  • Brigitte René
  • Grégoire Masliah
  • Loussiné Zargarian
  • Olivier Mauffret
  • Serge FermandjianEmail author


13C, 15N labeling of biomolecules allows easier assignments of NMR resonances and provides a larger number of NMR parameters, which greatly improves the quality of DNA structures. However, there is no general DNA-labeling procedure, like those employed for proteins and RNAs. Here, we describe a general and widely applicable approach designed for preparation of isotopically labeled DNA fragments that can be used for NMR studies. The procedure is based on the PCR amplification of oligonucleotides in the presence of labeled deoxynucleotides triphosphates. It allows great flexibility thanks to insertion of a short DNA sequence (linker) between two repeats of DNA sequence to study. Size and sequence of the linker are designed as to create restriction sites at the junctions with DNA of interest. DNA duplex with desired sequence and size is released upon enzymatic digestion of the PCR product. The suitability of the procedure is validated through the preparation of two biological relevant DNA fragments.


DNA isotope labeling NMR PCR structural analysis 


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This work was financially supported by a grant from the French Ministry of Research (to G.M.).


  1. Batey, R.T., Battiste, J.L., Williamson, J.R. (1995) Methods Enzymol., 261, 300–322Google Scholar
  2. Batey R.T., Inada M., Kujawinski E. et al (1992) Nucleic Acids Res 17:4515–4523Google Scholar
  3. Chen X., Mariappan S.V., Kelley J.J. III, et al. (1998) FEBS Lett 3:372–376CrossRefGoogle Scholar
  4. Cheng S., Chang S.Y., Gravitt P et al. (1994) Nature 6482:684–685CrossRefADSGoogle Scholar
  5. Clark J.M. (1988) Nucl. Acids Res. 20:9677–9686ADSGoogle Scholar
  6. Clore, G.M. and Gronenborn, A.M. (1994a) Methods Enzymol., 239, 349–363Google Scholar
  7. Clore G.M., Gronenborn A.M. (1994b) Prog. Biophys. Mol. Biol. 2:153–184CrossRefGoogle Scholar
  8. Dingley A.J., Grzesiek S. (1998) J. Am. Chem. Soc., 33:8293–8297CrossRefGoogle Scholar
  9. Ferentz A.E., Wagner G. (2000) Q. Rev. Biophys. 1:29–65CrossRefGoogle Scholar
  10. Fernandez C., Szyperski T., Ono A. et al (1998) J. Biomol. NMR 1:25–37CrossRefGoogle Scholar
  11. Fesik S.W., Zuiderweg E.R. (1990) Q. Rev. Biophys. 2:97–131CrossRefGoogle Scholar
  12. Fossé P, René B, Le Bret M, et al. (1991) Nucl. Acids Res. 11:2861–2868Google Scholar
  13. Hines J.V., Landry S.M., Varani G., et al. (1994) J. Am. Chem. Soc. 13:5823–5831CrossRefGoogle Scholar
  14. Katzman, M. and Katz, R.A. (1999) Adv. Virus Res., 52, 371–395Google Scholar
  15. Kojima, C., Ono, A. and Ono, A. et al. (2001) Methods Enzymol., 338, 261–283Google Scholar
  16. Louis J.M., Martin R.G., Clore G.M. et al. (1998) J. Biol. Chem. 4:2374–2378CrossRefGoogle Scholar
  17. Masse, J.E., Bortmann, P., Dieckmann, T. et al. (1998) Nucleic Acids Res 11:2618–2624CrossRefGoogle Scholar
  18. Michnicka M.J., Harper J.W., King G.C. (1993) Biochemistry 2:395–400CrossRefGoogle Scholar
  19. Nikonowicz E.P., Pardi A. (1992) Nature 6356:184–186CrossRefADSGoogle Scholar
  20. Ono A., Tate S. and Kainosho M. (1995) Tanpakushitsu Kakusan Koso 10:1509–1517Google Scholar
  21. Piotto M., Saudek V., Sklenar V. (1992) J. Biomol. NMR 6:661–665CrossRefGoogle Scholar
  22. Renisio J.G., Cosquer S., Cherrak I., et al. (2005) Nucl. Acids Res. 6:1970–1981CrossRefGoogle Scholar
  23. Smith D.E., Su J.Y., Jucker F.M. (1997) J. Biomol. NMR 3:245–253CrossRefGoogle Scholar
  24. Tjandra N., Marquardt J., Clore G.M. (2000) J. Magn. Reson 2:393–396CrossRefADSGoogle Scholar
  25. Varani G., Aboul-ela F., Allain F.H.T. (1996) Progress Nucl. Mag. Res. Spectr. 1–2:51–127CrossRefGoogle Scholar
  26. Werner, M.H., Gupta, V. and Lambert, L.J. et al. (2001) Methods Enzymol., 338, 283–304Google Scholar
  27. Wijmenga S.S., van Buuren B.N.M. (1998) Progress Nucl. Mag. Res. Spectr., 4:287–387CrossRefGoogle Scholar
  28. Zargarian L., Benleumi M.S., Renisio J.G. et al. (2003) J. Biol. Chem. 22:19966–19973CrossRefGoogle Scholar
  29. Zimmer D.P., Crothers D.M. (1995) Proc. Natl. Acad Sci. USA, 8:3091–3095CrossRefADSGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2006

Authors and Affiliations

  • Brigitte René
    • 1
  • Grégoire Masliah
    • 1
  • Loussiné Zargarian
    • 1
  • Olivier Mauffret
    • 1
  • Serge Fermandjian
    • 1
    Email author
  1. 1.Département de Biologie et Pharmacologie StructuralesUMR 8113 CNRS – LBPA Ecole Normale Supérieure de Cachan, Institut Gustave RoussyVillejuif CedexFrance

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