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. 1992 Feb 25;20(4):653–657. doi: 10.1093/nar/20.4.653

Synthesis of isotope labeled oligonucleotides and their use in an NMR study of a protein-DNA complex.

E R Kellenbach 1, M L Remerowski 1, D Eib 1, R Boelens 1, G A van der Marel 1, H van den Elst 1, J H van Boom 1, R Kaptein 1
PMCID: PMC312000  PMID: 1542561

Abstract

The synthesis of an oligonucleotide labeled with 13C at the thymine methyl and 15N at the exocyclic amino groups of the cytosines is described. 13CH3I and 15NH4OH were used as sources of the labels. The labeled oligonucleotide was characterized by several NMR techniques. The duplex possesses a labeled functional group in the major groove at every base pair which makes it a very suitable probe for the study of sequence-specific protein-DNA interaction. The labeled thymine methyl group facilitates the detection of hydrophobic contacts with aliphatic side-chains of proteins. This is demonstrated in an NMR study of a complex between the glucocorticoid receptor DNA-binding domain and the labeled oligomer, which revealed a hydrophobic contact between a thymine methyl group and the methyl groups of a valine residue. There are indications for small differences between the solution structure the X-ray structure of the complex.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ashcroft J., Live D. H., Patel D. J., Cowburn D. Heteronuclear two-dimensional 15N- and 13C-NMR studies of DNA oligomers and their netropsin complexes using indirect proton detection. Biopolymers. 1991 Jan;31(1):45–55. doi: 10.1002/bip.360310105. [DOI] [PubMed] [Google Scholar]
  2. Danielsen M., Hinck L., Ringold G. M. Two amino acids within the knuckle of the first zinc finger specify DNA response element activation by the glucocorticoid receptor. Cell. 1989 Jun 30;57(7):1131–1138. doi: 10.1016/0092-8674(89)90050-0. [DOI] [PubMed] [Google Scholar]
  3. Dubendorff J. W., deHaseth P. L., Rosendahl M. S., Caruthers M. H. DNA functional groups required for formation of open complexes between Escherichia coli RNA polymerase and the lambda PR promoter. Identification via base analog substitutions. J Biol Chem. 1987 Jan 15;262(2):892–898. [PubMed] [Google Scholar]
  4. Freedman L. P., Luisi B. F., Korszun Z. R., Basavappa R., Sigler P. B., Yamamoto K. R. The function and structure of the metal coordination sites within the glucocorticoid receptor DNA binding domain. Nature. 1988 Aug 11;334(6182):543–546. doi: 10.1038/334543a0. [DOI] [PubMed] [Google Scholar]
  5. Härd T., Kellenbach E., Boelens R., Kaptein R., Dahlman K., Carlstedt-Duke J., Freedman L. P., Maler B. A., Hyde E. I., Gustafsson J. A. 1H NMR studies of the glucocorticoid receptor DNA-binding domain: sequential assignments and identification of secondary structure elements. Biochemistry. 1990 Sep 25;29(38):9015–9023. doi: 10.1021/bi00490a019. [DOI] [PubMed] [Google Scholar]
  6. Härd T., Kellenbach E., Boelens R., Maler B. A., Dahlman K., Freedman L. P., Carlstedt-Duke J., Yamamoto K. R., Gustafsson J. A., Kaptein R. Solution structure of the glucocorticoid receptor DNA-binding domain. Science. 1990 Jul 13;249(4965):157–160. doi: 10.1126/science.2115209. [DOI] [PubMed] [Google Scholar]
  7. Ivarie R. Thymine methyls and DNA-protein interactions. Nucleic Acids Res. 1987 Dec 10;15(23):9975–9983. doi: 10.1093/nar/15.23.9975. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kellenbach E., Härd T., Boelens R., Dahlman K., Carlstedt-Duke J., Gustafsson J. A., van der Marel G. A., van Boom J. H., Maler B., Yamamoto K. R. Photo-CIDNP study of the interaction between the glucocorticoid receptor DNA-binding domain and glucocorticoid response elements. J Biomol NMR. 1991 May;1(1):105–110. doi: 10.1007/BF01874574. [DOI] [PubMed] [Google Scholar]
  9. Kupferschmitt G., Schmidt J., Schmidt T., Fera B., Buck F., Rüterjans H. 15N labeling of oligodeoxynucleotides for NMR studies of DNA-ligand interactions. Nucleic Acids Res. 1987 Aug 11;15(15):6225–6241. doi: 10.1093/nar/15.15.6225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Li R., Knight J., Bream G., Stenlund A., Botchan M. Specific recognition nucleotides and their DNA context determine the affinity of E2 protein for 17 binding sites in the BPV-1 genome. Genes Dev. 1989 Apr;3(4):510–526. doi: 10.1101/gad.3.4.510. [DOI] [PubMed] [Google Scholar]
  11. Luisi B. F., Xu W. X., Otwinowski Z., Freedman L. P., Yamamoto K. R., Sigler P. B. Crystallographic analysis of the interaction of the glucocorticoid receptor with DNA. Nature. 1991 Aug 8;352(6335):497–505. doi: 10.1038/352497a0. [DOI] [PubMed] [Google Scholar]
  12. Mader S., Kumar V., de Verneuil H., Chambon P. Three amino acids of the oestrogen receptor are essential to its ability to distinguish an oestrogen from a glucocorticoid-responsive element. Nature. 1989 Mar 16;338(6212):271–274. doi: 10.1038/338271a0. [DOI] [PubMed] [Google Scholar]
  13. Martiat P., Ferrant A., Labar D., Cogneau M., Bol A., Michel C., Michaux J. L., Sokal G. In vivo measurement of carbon-11 thymidine uptake in non-Hodgkin's lymphoma using positron emission tomography. J Nucl Med. 1988 Oct;29(10):1633–1637. [PubMed] [Google Scholar]
  14. Otting G., Wüthrich K. Heteronuclear filters in two-dimensional [1H,1H]-NMR spectroscopy: combined use with isotope labelling for studies of macromolecular conformation and intermolecular interactions. Q Rev Biophys. 1990 Feb;23(1):39–96. doi: 10.1017/s0033583500005412. [DOI] [PubMed] [Google Scholar]
  15. Poupeye E., Counsell R. E., De Leenheer A., Slegers G., Goethals P. Synthesis of 11C-labelled thymidine for tumor visualization using positron emission tomography. Int J Rad Appl Instrum A. 1989;40(1):57–61. doi: 10.1016/0883-2889(89)90174-3. [DOI] [PubMed] [Google Scholar]
  16. Remerowski M. L., Kellenbach E., Boelens R., van der Marel G. A., van Boom J. H., Maler B. A., Yamamoto K. R., Kaptein R. 1H NMR studies of DNA recognition by the glucocorticoid receptor: complex of the DNA binding domain with a half-site response element. Biochemistry. 1991 Dec 17;30(50):11620–11624. doi: 10.1021/bi00114a003. [DOI] [PubMed] [Google Scholar]
  17. Steitz T. A. Structural studies of protein-nucleic acid interaction: the sources of sequence-specific binding. Q Rev Biophys. 1990 Aug;23(3):205–280. doi: 10.1017/s0033583500005552. [DOI] [PubMed] [Google Scholar]
  18. Sundoro-Wu B. M., Schmall B., Conti P. S., Dahl J. R., Drumm P., Jacobsen J. K. Selective alkylation of pyrimidyldianions: synthesis and purification of 11C labeled thymidine for tumor visualization using positron emission tomography. Int J Appl Radiat Isot. 1984 Aug;35(8):705–708. doi: 10.1016/0020-708x(84)90075-9. [DOI] [PubMed] [Google Scholar]
  19. Takeda Y., Sarai A., Rivera V. M. Analysis of the sequence-specific interactions between Cro repressor and operator DNA by systematic base substitution experiments. Proc Natl Acad Sci U S A. 1989 Jan;86(2):439–443. doi: 10.1073/pnas.86.2.439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Truss M., Chalepakis G., Beato M. Contacts between steroid hormone receptors and thymines in DNA: an interference method. Proc Natl Acad Sci U S A. 1990 Sep;87(18):7180–7184. doi: 10.1073/pnas.87.18.7180. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Tsai S. Y., Carlstedt-Duke J., Weigel N. L., Dahlman K., Gustafsson J. A., Tsai M. J., O'Malley B. W. Molecular interactions of steroid hormone receptor with its enhancer element: evidence for receptor dimer formation. Cell. 1988 Oct 21;55(2):361–369. doi: 10.1016/0092-8674(88)90059-1. [DOI] [PubMed] [Google Scholar]
  22. Umesono K., Evans R. M. Determinants of target gene specificity for steroid/thyroid hormone receptors. Cell. 1989 Jun 30;57(7):1139–1146. doi: 10.1016/0092-8674(89)90051-2. [DOI] [PubMed] [Google Scholar]

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