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Efficient enzymatic synthesis of 13 C,15N-labeled DNA for NMR studies

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Abstract

The power of heteronuclear NMR spectroscopy to study macromoleculesand their complexes has been amply demonstrated over the last decade. Theobstacle to routinely applying these techniques to the study of DNA has beenthe synthesis of 13C,15N-labeled DNA. Here wepresent a simple and efficient method to generate isotope-labeled DNA forNMR studies that is as easy as that for isotope labeling of RNA. The methodwas used to synthesize a uniformly13 C,15N-labeled 32-nucleotide DNA that binds tohuman basic fibroblast growth factor with high affinity and specificity.Isotope-edited experiments were applied to the13 C,15N-labeled DNA bound to unlabeled protein,and the 13 C,15N-labeled DNA was also examined incomplex with 15N-labeled protein. The NMR experiments showthat the DNA adopts a well-defined stable structure when bound to theprotein, and illustrate the potential of13 C,15N-labeled DNA for structural studies ofDNA–protein complexes.

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References

  • Avis, J.M., Allain, F.H., Howe, P.W., Varani, G., Nagai, K. and Neuhaus, D. (1996) J. Mol. Biol., 257, 398–411.

    Google Scholar 

  • Batey, R.T., Inada, M., Kujawinski, E., Puglisi, J.D. and Williamson, J.R. (1992) Nucleic Acids Res., 20, 4515–4523.

    Google Scholar 

  • Batey, R.T., Battiste, J.L. and Williamson, J.R. (1995) Methods Enzymol., {vn261}, 300–322.

    Google Scholar 

  • Battiste, J.L., Tan, R., Frankel, A.D. and Williamson, J.R. (1995) J. Biomol. NMR, 6, 375–389.

    Google Scholar 

  • Bodenhausen, G. and Ruben, D.J. (1980) Chem. Phys. Lett., 69, 185–189.

    Google Scholar 

  • Chandrasegaran, S., Kan, L.S., Sillerud, L.O., Skoglund, C. and Bothner, B.A. (1985) Nucleic Acids Res., 13, 2097–2110.

    Google Scholar 

  • Clore, G.M. and Gronenborn, A.M. (1994) Methods Enzymol., 239, 349–363.

    Google Scholar 

  • Dieckmann, T., Suzuki, E., Nakamura, G.K. and Feigon, J. (1996) RNA, 2, 628–640.

    Google Scholar 

  • Ellington, A.D. and Szostak, J.W. (1990) Nature, 346, 818–822.

    Google Scholar 

  • Farmer, B.T., Mueller, L., Nikonowicz, E.P. and Pardi, A. (1993) J. Am. Chem. Soc., 115, 11040–11041.

    Google Scholar 

  • Fesik, S.W. and Zuiderweg, E.R.P. (1990) Q. Rev. Biophys., 23, 97–131.

    Google Scholar 

  • Fujiwara, T., Sugase, K., Kainosho, M., Ono, A. and Akutsu, H. (1995) J. Am. Chem. Soc., 117, 11351–11352.

    Google Scholar 

  • Greenbaum, N.L., Radhakrishnan, I., Hirsh, D. and Patel, D.J. (1995) J. Mol. Biol., 252, 314–327.

    Google Scholar 

  • Griffey, R.H. and Redfield, A.G. (1987) Q. Rev. Biophys., 19, 51–82.

    Google Scholar 

  • Hall, K.B. (1995) Methods Enzymol., 261, 542–559.

    Google Scholar 

  • Hines, J.V., Landry, S.M., Varani, G. and Tinoco Jr., I. (1994) J. Am. Chem. Soc., 116, 5823–5831.

    Google Scholar 

  • Hoffman, D.W. and Holland, J.A. (1995) Nucleic Acids Res., 23, 3361–3362.

    Google Scholar 

  • Hore, P.J. (1983) J. Magn. Reson., 54, 539–542.

    Google Scholar 

  • Hurlbert, R.B. and Furlong, N.B. (1967) Methods Enzymol., 12, 193–202.

    Google Scholar 

  • Jellinek, D., Lynott, C.K., Rifkin, D.B. and Janjic, N. (1993) Proc. Natl. Acad. Sci. USA, 90, 11227–11231.

    Google Scholar 

  • Jucker, F.M. and Pardi, A. (1995) Biochemistry, 34, 14416–14427.

    Google Scholar 

  • Kellogg, G.W., Szewczak, A.A. and Moore, P.B. (1992) J. Am. Chem. Soc., 114, 2727–2728.

    Google Scholar 

  • Kellogg, G.W. and Schweitzer, B.I. (1993) J. Biomol. NMR, 3, 577–595.

    Google Scholar 

  • Legault, P., Farmer, B.T., Mueller, L. and Pardi, A. (1994) J. Am. Chem. Soc., 116, 2203–2204.

    Google Scholar 

  • Legault, P., Jucker, F.M. and Pardi, A. (1995) FEBS Lett., 362, 156–160.

    Google Scholar 

  • Marino, J.P., Prestegard, J.H. and Crothers, D.M. (1994) J. Am. Chem. Soc., 116, 2205–2206.

    Google Scholar 

  • Marino, J.P., Schwalbe, H., Anklin, C., Bermel, W., Crothers, D.M. and Griesinger, C. (1995) J. Biomol. NMR, 5, 87–92.

    Google Scholar 

  • Michnicka, M.J., Harper, J.W. and King, G.C. (1993) Biochemistry, {vn32}, 395–400.

    Google Scholar 

  • Mueller, L., Legault, P. and Pardi, A. (1995) J. Am. Chem. Soc., 117, 11043–11048.

    Google Scholar 

  • Nikonowicz, E.P., Sirr, A., Legault, P., Jucker, F.M., Baer, L.M. and Pardi, A. (1992) Nucleic Acids Res., 20, 4507–4513.

    Google Scholar 

  • Nikonowicz, E.P. and Pardi, A. (1993)J. Mol. Biol., 232, 1141–1156.

    Google Scholar 

  • Ono, A., Tate, S., Ishido, Y. and Kainosho, M. (1994) J. Biomol. NMR, 4, 581–586.

    Google Scholar 

  • Ott, D.G. and Hoard, D.E. (1965) J. Am. Chem. Soc., 87, 1785–1788.

    Google Scholar 

  • Otting, G. and Wüthrich, K. (1990) Q. Rev. Biophys., 23, 39–96.

    Google Scholar 

  • Pardi, A. and Nikonowicz, E.P. (1992) J. Am. Chem. Soc., 114, 9301–9302.

    Google Scholar 

  • Pardi, A. (1995) Methods Enzymol., 261, 350–380.

    Google Scholar 

  • Piotto, M., Saudek, V. and Sklenář, V. (1992) J. Biomol. NMR, 2, 661–665.

    Google Scholar 

  • Quant, S., Wechselberger, R.W., Wolter, M.A., Worner, K.H., Schell, P., Engels, J.W., Griesinger, C. and Schwalbe, H. (1994) Tetrahedron Lett., 35, 6649–6652.

    Google Scholar 

  • Robertson, D.L. and Joyce, G.F. (1990) Nature, 344, 467–468.

    Google Scholar 

  • Rosenberg, M., Wiebers, J.L. and Gilham, P.T. (1972) Biochemistry, {vn11}, 3623–3628.

    Google Scholar 

  • Sambrook, J., Fritsch, E.F. and Maniatis, T. (1989) Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, U.S.A.

    Google Scholar 

  • Schwalbe, H., Marino, J.P., King, G.C., Wechselberger, R., Bermel, W. and Griesinger, C. (1994) J. Biomol. NMR, 4, 631–644.

    Google Scholar 

  • Simon, E.S., Grabowski, S. and Whitesides, G.M. (1990) J. Org. Chem., 55, 1834–1841.

    Google Scholar 

  • Simorre, J.P., Zimmermann, G.R., Pardi, A., Farmer, B.T. and Mueller, L. (1995) J. Biomol. NMR, 6, 427–432.

    Google Scholar 

  • Simorre, J.P., Zimmermann, G.R., Mueller, L. and Pardi, A. (1996a) J. Biomol. NMR, 7, 153–156.

    Google Scholar 

  • Simorre, J.P., Zimmermann, G.R., Mueller, L. and Pardi, A. (1996b) J. Am. Chem. Soc., 118, 5316–5317.

    Google Scholar 

  • Sklenář, V., Peterson, R.D., Rejante, M.R., Wang, E. and Feigon, J. (1993) J. Am. Chem. Soc., 115, 12181–12182.

    Google Scholar 

  • Su, J.Y. and Sclafani, R.A. (1991) Nucleic Acids Res., 19, 823–827.

    Google Scholar 

  • Tate, S., Ono, A. and Kainosho, M. (1994) J. Am. Chem. Soc., 116, 5977–5978.

    Google Scholar 

  • Tate, S., Ono, A. and Kainosho, M. (1995) J. Magn. Reson., B106, 89–91.

    Google Scholar 

  • Tuerk, C. and Gold, L. (1990) Science, 249, 505–510.

    Google Scholar 

  • Van Dongen, M.J., Heus, H.A., Wijmenga, S.S., Van der Marel, G.A., Van Boom, J. and Hilbers, C.W. (1996) Biochemistry, 35, 1733–1739.

    Google Scholar 

  • Wijmenga, S.S., Heus, H.A., Leeuw, H.A., Hoppe, H., Van der Graaf, M. and Hilbers, C.W. (1995) J. Biomol. NMR, 5, 82–86.

    Google Scholar 

  • Zimmer, D.P. and Crothers, D.M. (1995) Proc. Natl. Acad. Sci. USA, {vn92}, 3091–3095.

    Google Scholar 

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  1. NeXstar Pharmaceuticals Inc., 2860 Wilderness Place, Boulder, CO, 80301, U.S.A

    Deborah E. Smith, Jin-Yuan Su & Fiona M. Jucker

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  1. Deborah E. Smith
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  2. Jin-Yuan Su
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  3. Fiona M. Jucker
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Smith, D.E., Su, JY. & Jucker, F.M. Efficient enzymatic synthesis of 13 C,15N-labeled DNA for NMR studies. J Biomol NMR 10, 245–253 (1997). https://doi.org/10.1023/A:1018358602001

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