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. 1993 Apr 15;90(8):3745–3749. doi: 10.1073/pnas.90.8.3745

Thrombin-binding DNA aptamer forms a unimolecular quadruplex structure in solution.

R F Macaya 1, P Schultze 1, F W Smith 1, J A Roe 1, J Feigon 1
PMCID: PMC46378  PMID: 8475124

Abstract

We have used two-dimensional 1H NMR spectroscopy to study the conformation of the thrombin-binding aptamer d(GGTTGGTGTGGTTGG) in solution. This is one of a series of thrombin-binding DNA aptamers with a consensus 15-base sequence that was recently isolated and shown to inhibit thrombin-catalyzed fibrin clot formation in vitro [Bock, L. C., Griffin, L. C., Latham, J. A., Vermaas, E. H. & Toole, J. J. (1992) Nature (London) 355, 564-566]. The oligonucleotide forms a unimolecular DNA quadruplex consisting of two G-quartets connected by two TT loops and one TGT loop. A potential T.T bp is formed between the two TT loops across the diagonal of the top G-quartet. Thus, all of the invariant bases in the consensus sequence are base-paired. This aptamer structure was determined by NMR and illustrates that this molecule forms a specific folded structure. Knowledge of this structure may be used in the further development of oligonucleotide-based thrombin inhibitors.

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Selected References

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

  1. Bock L. C., Griffin L. C., Latham J. A., Vermaas E. H., Toole J. J. Selection of single-stranded DNA molecules that bind and inhibit human thrombin. Nature. 1992 Feb 6;355(6360):564–566. doi: 10.1038/355564a0. [DOI] [PubMed] [Google Scholar]
  2. Cheong C., Moore P. B. Solution structure of an unusually stable RNA tetraplex containing G- and U-quartet structures. Biochemistry. 1992 Sep 15;31(36):8406–8414. doi: 10.1021/bi00151a003. [DOI] [PubMed] [Google Scholar]
  3. Ellington A. D., Szostak J. W. In vitro selection of RNA molecules that bind specific ligands. Nature. 1990 Aug 30;346(6287):818–822. doi: 10.1038/346818a0. [DOI] [PubMed] [Google Scholar]
  4. Feigon J., Sklenár V., Wang E., Gilbert D. E., Macaya R. F., Schultze P. 1H NMR spectroscopy of DNA. Methods Enzymol. 1992;211:235–253. doi: 10.1016/0076-6879(92)11015-b. [DOI] [PubMed] [Google Scholar]
  5. Glueck R., Green D., Cohen I., Ts'ao C. H. Hematin: unique effects of hemostasis. Blood. 1983 Feb;61(2):243–249. [PubMed] [Google Scholar]
  6. Green D., Reynolds N., Klein J., Kohl H., Ts'ao C. H. The inactivation of hemostatic factors by hematin. J Lab Clin Med. 1983 Sep;102(3):361–369. [PubMed] [Google Scholar]
  7. Guschlbauer W., Chantot J. F., Thiele D. Four-stranded nucleic acid structures 25 years later: from guanosine gels to telomer DNA. J Biomol Struct Dyn. 1990 Dec;8(3):491–511. doi: 10.1080/07391102.1990.10507825. [DOI] [PubMed] [Google Scholar]
  8. Hardin C. C., Henderson E., Watson T., Prosser J. K. Monovalent cation induced structural transitions in telomeric DNAs: G-DNA folding intermediates. Biochemistry. 1991 May 7;30(18):4460–4472. doi: 10.1021/bi00232a013. [DOI] [PubMed] [Google Scholar]
  9. Innis M. A., Myambo K. B., Gelfand D. H., Brow M. A. DNA sequencing with Thermus aquaticus DNA polymerase and direct sequencing of polymerase chain reaction-amplified DNA. Proc Natl Acad Sci U S A. 1988 Dec;85(24):9436–9440. doi: 10.1073/pnas.85.24.9436. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Irvine D., Tuerk C., Gold L. SELEXION. Systematic evolution of ligands by exponential enrichment with integrated optimization by non-linear analysis. J Mol Biol. 1991 Dec 5;222(3):739–761. doi: 10.1016/0022-2836(91)90509-5. [DOI] [PubMed] [Google Scholar]
  11. Jin R. Z., Breslauer K. J., Jones R. A., Gaffney B. L. Tetraplex formation of a guanine-containing nonameric DNA fragment. Science. 1990 Oct 26;250(4980):543–546. doi: 10.1126/science.2237404. [DOI] [PubMed] [Google Scholar]
  12. Jin R., Gaffney B. L., Wang C., Jones R. A., Breslauer K. J. Thermodynamics and structure of a DNA tetraplex: a spectroscopic and calorimetric study of the tetramolecular complexes of d(TG3T) and d(TG3T2G3T). Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8832–8836. doi: 10.1073/pnas.89.18.8832. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Jones R. L. Hematin-derived anticoagulant. Generation in vitro and in vivo. J Exp Med. 1986 Mar 1;163(3):724–739. doi: 10.1084/jem.163.3.724. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kang C., Zhang X., Ratliff R., Moyzis R., Rich A. Crystal structure of four-stranded Oxytricha telomeric DNA. Nature. 1992 Mar 12;356(6365):126–131. doi: 10.1038/356126a0. [DOI] [PubMed] [Google Scholar]
  15. Kumar A., Ernst R. R., Wüthrich K. A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules. Biochem Biophys Res Commun. 1980 Jul 16;95(1):1–6. doi: 10.1016/0006-291x(80)90695-6. [DOI] [PubMed] [Google Scholar]
  16. Sen D., Gilbert W. A sodium-potassium switch in the formation of four-stranded G4-DNA. Nature. 1990 Mar 29;344(6265):410–414. doi: 10.1038/344410a0. [DOI] [PubMed] [Google Scholar]
  17. 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]
  18. Sundquist W. I., Klug A. Telomeric DNA dimerizes by formation of guanine tetrads between hairpin loops. Nature. 1989 Dec 14;342(6251):825–829. doi: 10.1038/342825a0. [DOI] [PubMed] [Google Scholar]
  19. Szostak J. W. In vitro genetics. Trends Biochem Sci. 1992 Mar;17(3):89–93. doi: 10.1016/0968-0004(92)90242-2. [DOI] [PubMed] [Google Scholar]
  20. Tuerk C., Gold L. Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase. Science. 1990 Aug 3;249(4968):505–510. doi: 10.1126/science.2200121. [DOI] [PubMed] [Google Scholar]
  21. Tuerk C., MacDougal S., Gold L. RNA pseudoknots that inhibit human immunodeficiency virus type 1 reverse transcriptase. Proc Natl Acad Sci U S A. 1992 Aug 1;89(15):6988–6992. doi: 10.1073/pnas.89.15.6988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Wang Y., Jin R., Gaffney B., Jones R. A., Breslauer K. J. Characterization by 1H NMR of glycosidic conformations in the tetramolecular complex formed by d(GGTTTTTGG). Nucleic Acids Res. 1991 Sep 11;19(17):4619–4622. doi: 10.1093/nar/19.17.4619. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Wang Y., Patel D. J. Guanine residues in d(T2AG3) and d(T2G4) form parallel-stranded potassium cation stabilized G-quadruplexes with anti glycosidic torsion angles in solution. Biochemistry. 1992 Sep 8;31(35):8112–8119. doi: 10.1021/bi00150a002. [DOI] [PubMed] [Google Scholar]
  24. Wang Y., de los Santos C., Gao X. O., Greene K., Live D., Patel D. J. Multinuclear nuclear magnetic resonance studies of Na cation-stabilized complex formed by d(G-G-T-T-T-T-C-G-G) in solution. Implications for G-tetrad structures. J Mol Biol. 1991 Dec 5;222(3):819–832. doi: 10.1016/0022-2836(91)90513-6. [DOI] [PubMed] [Google Scholar]
  25. Williamson J. R., Raghuraman M. K., Cech T. R. Monovalent cation-induced structure of telomeric DNA: the G-quartet model. Cell. 1989 Dec 1;59(5):871–880. doi: 10.1016/0092-8674(89)90610-7. [DOI] [PubMed] [Google Scholar]

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