Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1998 Jun 1;26(11):2618–2624. doi: 10.1093/nar/26.11.2618

Simple, efficient protocol for enzymatic synthesis of uniformly 13C, 15N-labeled DNA for heteronuclear NMR studies.

J E Masse 1, P Bortmann 1, T Dieckmann 1, J Feigon 1
PMCID: PMC147604  PMID: 9592146

Abstract

The use of uniformly 13C,15N-labeled RNA has greatly facilitated structural studies of RNA oligonucleotides by NMR. Application of similar methodologies for the study of DNA has been limited, primarily due to the lack of adequate methods for sample preparation. Methods for both chemical and enzymatic synthesis of DNA oligonucleotides uniformly labeled with 13C and/or 15N have been published, but have not yet been widely used. We have developed a modified procedure for preparing uniformly 13C,15N-labeled DNA based on enzymatic synthesis using Taq DNA polymerase. The highly efficient protocol results in quantitative polymerization of the template and approximately 80% incorporation of the labeled dNTPs. Procedures for avoiding non-templated addition of nucleotides or for their removal are given. The method has been used to synthesize several DNA oligonucleotides, including two complementary 15 base strands, a 32 base DNA oligonucleotide that folds to form an intramolecular triplex and a 12 base oligonucleotide that dimerizes and folds to form a quadruplex. Heteronuclear NMR spectra of the samples illustrate the quality of the labeled DNA obtained by these procedures.

Full Text

The Full Text of this article is available as a PDF (136.3 KB).

Selected References

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

  1. Batey R. T., Inada M., Kujawinski E., Puglisi J. D., Williamson J. R. Preparation of isotopically labeled ribonucleotides for multidimensional NMR spectroscopy of RNA. Nucleic Acids Res. 1992 Sep 11;20(17):4515–4523. doi: 10.1093/nar/20.17.4515. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Brownstein M. J., Carpten J. D., Smith J. R. Modulation of non-templated nucleotide addition by Taq DNA polymerase: primer modifications that facilitate genotyping. Biotechniques. 1996 Jun;20(6):1004-6, 1008-10. doi: 10.2144/96206st01. [DOI] [PubMed] [Google Scholar]
  3. Clark J. M., Joyce C. M., Beardsley G. P. Novel blunt-end addition reactions catalyzed by DNA polymerase I of Escherichia coli. J Mol Biol. 1987 Nov 5;198(1):123–127. doi: 10.1016/0022-2836(87)90462-1. [DOI] [PubMed] [Google Scholar]
  4. Clark J. M. Novel non-templated nucleotide addition reactions catalyzed by procaryotic and eucaryotic DNA polymerases. Nucleic Acids Res. 1988 Oct 25;16(20):9677–9686. doi: 10.1093/nar/16.20.9677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Clore G. M., Gronenborn A. M. NMR structures of proteins and protein complexes beyond 20,000 M(r). Nat Struct Biol. 1997 Oct;4 (Suppl):849–853. [PubMed] [Google Scholar]
  6. Feigon J., Dieckmann T., Smith F. W. Aptamer structures from A to zeta. Chem Biol. 1996 Aug;3(8):611–617. doi: 10.1016/s1074-5521(96)90127-1. [DOI] [PubMed] [Google Scholar]
  7. Hall K. B. Uses of 13C- and 15N-labeled RNA in NMR of RNA-protein complexes. Methods Enzymol. 1995;261:542–559. doi: 10.1016/s0076-6879(95)61024-3. [DOI] [PubMed] [Google Scholar]
  8. Hite J. M., Eckert K. A., Cheng K. C. Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)n.d(G-T)n microsatellite repeats. Nucleic Acids Res. 1996 Jun 15;24(12):2429–2434. doi: 10.1093/nar/24.12.2429. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Joyce C. M., Grindley N. D. Construction of a plasmid that overproduces the large proteolytic fragment (Klenow fragment) of DNA polymerase I of Escherichia coli. Proc Natl Acad Sci U S A. 1983 Apr;80(7):1830–1834. doi: 10.1073/pnas.80.7.1830. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. LEHMAN I. R., BESSMAN M. J., SIMMS E. S., KORNBERG A. Enzymatic synthesis of deoxyribonucleic acid. I. Preparation of substrates and partial purification of an enzyme from Escherichia coli. J Biol Chem. 1958 Jul;233(1):163–170. [PubMed] [Google Scholar]
  11. Louis J. M., Martin R. G., Clore G. M., Gronenborn A. M. Preparation of uniformly isotope-labeled DNA oligonucleotides for NMR spectroscopy. J Biol Chem. 1998 Jan 23;273(4):2374–2378. doi: 10.1074/jbc.273.4.2374. [DOI] [PubMed] [Google Scholar]
  12. Macaya R., Wang E., Schultze P., Sklenár V., Feigon J. Proton nuclear magnetic resonance assignments and structural characterization of an intramolecular DNA triplex. J Mol Biol. 1992 Jun 5;225(3):755–773. doi: 10.1016/0022-2836(92)90399-5. [DOI] [PubMed] [Google Scholar]
  13. Magnuson V. L., Ally D. S., Nylund S. J., Karanjawala Z. E., Rayman J. B., Knapp J. I., Lowe A. L., Ghosh S., Collins F. S. Substrate nucleotide-determined non-templated addition of adenine by Taq DNA polymerase: implications for PCR-based genotyping and cloning. Biotechniques. 1996 Oct;21(4):700–709. doi: 10.2144/96214rr03. [DOI] [PubMed] [Google Scholar]
  14. Marion D., Driscoll P. C., Kay L. E., Wingfield P. T., Bax A., Gronenborn A. M., Clore G. M. Overcoming the overlap problem in the assignment of 1H NMR spectra of larger proteins by use of three-dimensional heteronuclear 1H-15N Hartmann-Hahn-multiple quantum coherence and nuclear Overhauser-multiple quantum coherence spectroscopy: application to interleukin 1 beta. Biochemistry. 1989 Jul 25;28(15):6150–6156. doi: 10.1021/bi00441a004. [DOI] [PubMed] [Google Scholar]
  15. Michnicka M. J., Harper J. W., King G. C. Selective isotopic enrichment of synthetic RNA: application to the HIV-1 TAR element. Biochemistry. 1993 Jan 19;32(2):395–400. doi: 10.1021/bi00053a002. [DOI] [PubMed] [Google Scholar]
  16. Nikonowicz E. P., Pardi A. An efficient procedure for assignment of the proton, carbon and nitrogen resonances in 13C/15N labeled nucleic acids. J Mol Biol. 1993 Aug 20;232(4):1141–1156. doi: 10.1006/jmbi.1993.1466. [DOI] [PubMed] [Google Scholar]
  17. Nikonowicz E. P., Sirr A., Legault P., Jucker F. M., Baer L. M., Pardi A. Preparation of 13C and 15N labelled RNAs for heteronuclear multi-dimensional NMR studies. Nucleic Acids Res. 1992 Sep 11;20(17):4507–4513. doi: 10.1093/nar/20.17.4507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Ono A., Tate S., Ishido Y., Kainosho M. Preparation and heteronuclear 2D NMR spectroscopy of a DNA dodecamer containing a thymidine residue with a uniformly 13C-labeled deoxyribose ring. J Biomol NMR. 1994 Jul;4(4):581–586. doi: 10.1007/BF00156622. [DOI] [PubMed] [Google Scholar]
  19. Pardi A. Multidimensional heteronuclear NMR experiments for structure determination of isotopically labeled RNA. Methods Enzymol. 1995;261:350–380. doi: 10.1016/s0076-6879(95)61017-0. [DOI] [PubMed] [Google Scholar]
  20. Patel D. J., Suri A. K., Jiang F., Jiang L., Fan P., Kumar R. A., Nonin S. Structure, recognition and adaptive binding in RNA aptamer complexes. J Mol Biol. 1997 Oct 10;272(5):645–664. doi: 10.1006/jmbi.1997.1281. [DOI] [PubMed] [Google Scholar]
  21. Piotto M., Saudek V., Sklenár V. Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions. J Biomol NMR. 1992 Nov;2(6):661–665. doi: 10.1007/BF02192855. [DOI] [PubMed] [Google Scholar]
  22. Ramos A., Gubser C. C., Varani G. Recent solution structures of RNA and its complexes with drugs, peptides and proteins. Curr Opin Struct Biol. 1997 Jun;7(3):317–323. doi: 10.1016/s0959-440x(97)80046-2. [DOI] [PubMed] [Google Scholar]
  23. Silveira M. H., Orgel L. E. PCR with detachable primers. Nucleic Acids Res. 1995 Mar 25;23(6):1083–1084. doi: 10.1093/nar/23.6.1083. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Simorre J. P., Zimmermann G. R., Mueller L., Pardi A. Correlation of the guanosine exchangeable and nonexchangeable base protons in 13C-/15N-labeled RNA with an HNC-TOCSY-CH experiment. J Biomol NMR. 1996 Mar;7(2):153–156. doi: 10.1007/BF00203825. [DOI] [PubMed] [Google Scholar]
  25. Sklenár V., Dieckmann T., Butcher S. E., Feigon J. Through-bond correlation of imino and aromatic resonances in 13C-, 15N-labeled RNA via heteronuclear TOCSY. J Biomol NMR. 1996 Jan;7(1):83–87. doi: 10.1007/BF00190460. [DOI] [PubMed] [Google Scholar]
  26. Sklenár V., Feigon J. Formation of a stable triplex from a single DNA strand. Nature. 1990 Jun 28;345(6278):836–838. doi: 10.1038/345836a0. [DOI] [PubMed] [Google Scholar]
  27. Sklenár V., Peterson R. D., Rejante M. R., Feigon J. Two- and three-dimensional HCN experiments for correlating base and sugar resonances in 15N,13C-labeled RNA oligonucleotides. J Biomol NMR. 1993 Nov;3(6):721–727. doi: 10.1007/BF00198375. [DOI] [PubMed] [Google Scholar]
  28. Smith D. E., Su J. Y., Jucker F. M. Efficient enzymatic synthesis of 13C,15N-labeled DNA for NMR studies. J Biomol NMR. 1997 Oct;10(3):245–253. doi: 10.1023/a:1018358602001. [DOI] [PubMed] [Google Scholar]
  29. Smith F. W., Feigon J. Quadruplex structure of Oxytricha telomeric DNA oligonucleotides. Nature. 1992 Mar 12;356(6365):164–168. doi: 10.1038/356164a0. [DOI] [PubMed] [Google Scholar]
  30. Tate S., Ono A., Kainosho M. Sequential backbone assignment in 13C-labeled DNA by the 1H, 13C, 31P triple-resonance experiment, HCP-CCH-COSY. J Magn Reson B. 1995 Jan;106(1):89–91. doi: 10.1006/jmrb.1995.1016. [DOI] [PubMed] [Google Scholar]
  31. Taussig R., Gilman A. G. Mammalian membrane-bound adenylyl cyclases. J Biol Chem. 1995 Jan 6;270(1):1–4. doi: 10.1074/jbc.270.1.1. [DOI] [PubMed] [Google Scholar]
  32. Wagner G. An account of NMR in structural biology. Nat Struct Biol. 1997 Oct;4 (Suppl):841–844. [PubMed] [Google Scholar]
  33. Walseth T. F., Graff G., Moos M. C., Jr, Goldberg N. D. Separation of 5'-ribonucleoside monophosphates by ion-pair reverse-phase high-performance liquid chromatography. Anal Biochem. 1980 Sep 1;107(1):240–245. doi: 10.1016/0003-2697(80)90516-3. [DOI] [PubMed] [Google Scholar]
  34. Zimmer D. P., Crothers D. M. NMR of enzymatically synthesized uniformly 13C15N-labeled DNA oligonucleotides. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3091–3095. doi: 10.1073/pnas.92.8.3091. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES