Photoaptameric heterodimeric constructs as a new approach to enhance the efficiency of formation of photocrosslinks with a target protein

  • S. Yu. Rakhmetova
  • S. P. Radko
  • O. V. Gnedenko
  • N. V. Bodoev
  • A. S. Ivanov
  • A. I. Archakov
Proteomics and Bioinformatics

Abstract

Using DNA aptamers selectively recognizing anion-binding exosites 1 and 2 of thrombin as a model, it has been demonstrated that their conjugation by a poly-(dT)-linker (ranging from 5 to 65 nucleotides (nt) in length) to produce aptamer heterodimeric constructs results into affinity enhancement. At the linker lengths ranged from 35 to 55 nt the apparent dissociation constants (K D app ) measured using the optical biosensor Biacore-3000 for complexes of thrombin with the heterodimeric constructs reached minimum values (K D app ) = 0.2–0.4 nM), which were approximately 30-fold less than for the complexes with the initial aptamers. A photoaptamer heterodimeric construct was designed connecting photoaptamer and aptamer sequences with the poly-(dT)-linker of 35 nt long. The photoaptamer used could form photo-induced cross-links with the exosite 2 of thrombin and the aptamer could bind to the exosite 1. The (K D app value for the photoaptamer construct was approximately 40-fold less than that for the primary photoaptamer (5.3 and 190 nM, respectively). Upon exposure of the equimolar mixtures of thrombin with the photoaptamer construct to the UV radiation at 308 nm the equal yield of the crosslinked complexes was observed at concentrations, which were lower by two orders of magnitude than in the case of the primary photoaptamer. It was found that concurrently with crosslinking to thrombin a photo-induced inactivation of the photoaptamer occurs presumably due to formation of the intermolecular crosslinking.

Key words

photoaptamers heterodimeric constructs photocrosslinking affinity thrombin 

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References

  1. 1.
    Jayasena, S.D., Clin. Chem., 1999, vol. 45, pp. 1628–1650.Google Scholar
  2. 2.
    Radko, S.P., Rakhmetova, S.Yu., Bodoev, N.V., and Archakov, A.I., Biomed. Khim., 2007, vol. 53, pp. 5–24.Google Scholar
  3. 3.
    Mairal, T., Ozalp, V.C., Lozano Sánchez, P., Mir, M., Katakis, I., and O’sullivan, C.K., Anal. Bioanal. Chem., 2008, vol. 390, pp. 989–1007.CrossRefGoogle Scholar
  4. 4.
    Kulbachinskii, A.V., Usp. Biol. Khim., 2006, vol. 46, pp. 193–224.Google Scholar
  5. 5.
    Golden, M.C., Collins, B.D., Willis, M.C., and Koch, T.H., J. Biotechnol., 2000, vol. 81, pp. 167–178.CrossRefGoogle Scholar
  6. 6.
    Smith, D., Collins, B.D., Heil, J., and Koch, T.H., (2003) Mol. Cell. Proteomics, 2003, vol. 2, pp. 11–18.CrossRefGoogle Scholar
  7. 7.
    Tasset, D.M., Kubik, M.F., and Steiner, W., J. Mol. Biol., 1997, vol. 272, pp. 688–698.CrossRefGoogle Scholar
  8. 8.
    Ruckman, J., Green, L.S., Beeson, J., Waugh, S., Gillette, W.L., Henninger, D.D., Claesson-Welsh, L., and Janjic, N., J. Biol. Chem., 1998, vol. 273, pp. 20556–20567.CrossRefGoogle Scholar
  9. 9.
    Bock, C., Coleman, M., Collins, B., Davis, J., Foulds, G., Gold, L., Greef, C., Heil, J., Heilig, J.S., Hicke, B., Hurst, M.N., Husar, G.M., Miller, D., Ostroff, R., Petach, H., Schneider, D., Vant-Hull, B., Waugh, S., Weiss, A., Wilcox, S.K., and Zichi, D., Proteomics, 2004, vol. 4, pp. 609–618.CrossRefGoogle Scholar
  10. 10.
    Gander, T.R., and Brody, E.N., Expert Rev. Mol. Diagn., 2005, vol. 5, pp. 1–3.CrossRefGoogle Scholar
  11. 11.
    Ivanov, Y.D., Govorun, V.M., Bykov, V.A., and Archakov, A.I., Proteomics, 2006, vol. 6, pp. 1399–1414.CrossRefGoogle Scholar
  12. 12.
    Umehara, T., Fukuda, K., Nishikawa, F., Kohara, M., Hasegawa, T., and Nishikawa, S., J. Biochem. (Tokyo), 2005, vol. 137, pp. 339–347.Google Scholar
  13. 13.
    Müller, J., Wulffen, B., Pötzsch, B., and Mayer, G., Chembiochem., 2007, vol. 8, pp. 2223–2226.CrossRefGoogle Scholar
  14. 14.
    Kim, Y., Cao, Z., and Tan, W., Pros. Natl. Acad. Sci. USA, 2008, vol. 105, 5664–5669.CrossRefGoogle Scholar
  15. 15.
    Tian, L., and Heyduk, T., Biochemistry, 2009, vol. 48, pp. 264–275.CrossRefGoogle Scholar
  16. 16.
    Bock, L.C., Griffin, L.C., Latham, J.A., Vermass, E.H., and Toole, J.J., Nature, 1992, vol. 355, pp. 564–566.CrossRefGoogle Scholar
  17. 17.
    Macaya, R.F., Waldron, J.A., Beutel, B.A., Gao, H., Joeston, M.E., Yang, M., Patel, R., Bertelsen, A.H., and Cook, A.G., Biochemistry, 1995, vol. 34, pp. 4478–4492.CrossRefGoogle Scholar
  18. 18.
    Koch, T.H., Smith, D., Tabacman, E., and Zichi, D.A., J. Mol. Biol., 2004, vol. 336, pp. 1159–1173.CrossRefGoogle Scholar
  19. 19.
    Zeng, Y. and Wang, Y., Nucleic Acids Res., 2006, vol. 34, pp. 6521–6529.CrossRefGoogle Scholar

Copyright information

© Pleiades Publishing, Ltd. 2010

Authors and Affiliations

  • S. Yu. Rakhmetova
    • 1
  • S. P. Radko
    • 1
  • O. V. Gnedenko
    • 1
  • N. V. Bodoev
    • 1
  • A. S. Ivanov
    • 1
  • A. I. Archakov
    • 1
  1. 1.Institute of Biomedical ChemistryRussian Academy of Medical SciencesMoscowRussia

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