Advertisement

Internalization of Nucleoside Phosphates into Live Cells by Complex Formation with Different CPPs and JBS-Nucleoducin

  • Franziska Mussbach
  • Regina Pietrucha
  • Buerk Schaefer
  • Siegmund Reissmann
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 683)

Abstract

Nucleoside phosphates can bind to many functional proteins like G-proteins or other GTP-binding proteins in signal transduction or translation processes. Till now internalization of nucleoside phosphates into live cells remains a challenge. We study the internalization of a fluorescent-labelled deoxyuridine triphosphate into HeLa cells and other adhesion and suspension cells. We use different cell-penetrating peptides and a cocktail suitable for formation of non-covalent complexes with the nucleotide. Internalization is observed by fluorescence microscopy, and the uptake efficiency is quantitatively estimated by fluorescence spectroscopy. The applied concentrations of CPPs and the cocktail were checked on cell viability (MTT test) and membrane integrity (bioluminescence test with peptidyl-luciferin), indicating that the CPPs and the complexes with the nucleotide are cytotoxic above certain concentrations. These concentrations depend on CPP and cell type and are the limiting factors for the cargo uptake.

Key words

CPPs forming non-covalent complexes Internalization of nucleoside phosphates Cargo amount per cell Optimization of internalization Wash procedures Highest non-toxic concentrations of CPPs and JBS-Nucleoducin Adhesion and suspension cell lines Viability Membrane integrity 

Notes

Acknowledgments

The authors would like to thank for helpful and kind support Prof. Hans Agricola for fluorescence microscopy, Dr. Eckhard Birckner for fluorescence spectroscopy, Prof. Thorsten Heinzel, Dr. Enrico Jandt, and Sigrid Reichardt for microscopical investigations and the bioluminescence test.

References

  1. 1.
    Rubio, I., Pusch, R., and Wetzker, R. (2004) Quantification of absolute Ras-GDP/GTP levels by HPLC separation of Ras-bound [32P]-labelled nucleotides. J Biochem Biophys Methods 58, 111–117.CrossRefPubMedGoogle Scholar
  2. 2.
    Saar, K. and Langel, Ü. (2007) Toxicity Methods for Cell-Penetrating Peptides in Handbook of Cell-Penetrating Peptides (Ed. Ü. Langel), 2nd Edition, CRC-Press, Boca Raton, FL, pp. 553–565.Google Scholar
  3. 3.
    Fenton, M., Bone, N., and Sinclair, A. J. (1998) The efficient and rapid import of a peptide into primary B and T lymphocytes and a lymphoblastoid cell line. J Immunol Methods 212, 41–48.CrossRefPubMedGoogle Scholar
  4. 4.
    Wu, R. P., Youngblood, D. S., Hassinger, J. N., Lovejoy, C. E., Nelson, M. H., Iversen, P. L., and Moulton, H. M. (2007) Cell-penetrating peptides as transporters for morpholino oligomers: effects of amino acid composition on intracellular delivery and cytotoxicity. Nucleic Acids Res 35, 5182–5191.CrossRefPubMedGoogle Scholar
  5. 5.
    Niles, A. L., Moravec R. A., Hesselberth, P. E., Scurria, M. A., Daily, W. J., and Riss, T. L. (2007) A homogeneous assay to measure live and dead cells in the same sample by detecting different protease markers. Anal Biochem 366, 197–206.CrossRefPubMedGoogle Scholar
  6. 6.
    Gros, E., Deshayes, S., Morris, M. C., Aldrian-Herrada, G., Depollier, J., Heitz, F., and Divita, G. (2006) A non-covalent peptide-based strategy for protein and peptide nucleic acid transduction. Biochim Biophys Acta 1758, 384–393.CrossRefPubMedGoogle Scholar
  7. 7.
    Morris, M. C., Deshayes, S., Heitz, F., and Divita, G. (2008) Cell-penetrating peptides: from molecular mechanisms to therapeutics. Biol Cell 100, 201–217.CrossRefPubMedGoogle Scholar
  8. 8.
    Simeoni, F., Morris, M. C., Heitz, F., and Divita, G (2003) Insight into the mechanism of the peptide-based gene delivery system MPG: implication for delivery of siRNA into mammalian cells. Nucleic Acids Res 31, 2717–2724.CrossRefPubMedGoogle Scholar
  9. 9.
    Morris, M. C., Gros, E., Aldrian-Herrada, G., Choob, M., Archdeacon, J., Heitz, F, and Divita, G. (2007) A non-covalent peptide-based carrier for in vivo delivery of DNA mimics. Nucleic Acids Res 35, e49.CrossRefPubMedGoogle Scholar
  10. 10.
    Derossi, D., Joliot, A. H., Chassaing, G., and Prochiantz, A. (1994) The third helix of the Antennapedia homeodomain translocates through biological membranes. J Biol Chem 269, 10444–10450.PubMedGoogle Scholar
  11. 11.
    Ignatovich, I. A., Dishe, E. B., Pavlotskaya, A. V., Akifiev, B. N., Burov, S. V., Orlov, S. V., and Perevozchikov, A. P. (2003) Complex of plasmid DNA with basic domain 47-57 of HIV-1 Tat protein are transferred to mammalian cells by endocytosis-mediated pathways. J Biol Chem 43, 42625–42636.CrossRefGoogle Scholar
  12. 12.
    Jeang, K. T., Xiao, H., and Rich, E. A. (1999) Multifaced activities of the HIV-1 transactivator of transcription Tat. J Biol Chem 274, 28837–28840.CrossRefPubMedGoogle Scholar
  13. 13.
    Gomez, J. A., Gama, V., Yoshida, T., Sun, W., Hayes, P., Leskov, K., Boothman, D., and Matsuyama, S. (2007) Bax-inhibiting peptides derived from Ku70 and cell-penetrating pentapeptides. Biochem Soc Trans 35, 797–801.CrossRefPubMedGoogle Scholar
  14. 14.
    Aussedat, B., Sagan, S., Chassaing, G., Bolbach, G., and Burlina, F. (2006) Quantification of the efficiency of cargo delivery by peptidic and pseudo-peptidic Trojan carriers using MALDI-TOF mass spectrometry. Biochim Biophys Acta 1758, 375–383.CrossRefPubMedGoogle Scholar
  15. 15.
    Palm, C., Netzereab, S., and Hällbrink, M. (2006) Quantitatively determined uptake of cell-penetrating peptides in non-mammalian cells with an evaluation of degradation and antimicrobial effects. Peptides 27, 1710–1716.CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Franziska Mussbach
    • 1
  • Regina Pietrucha
    • 2
  • Buerk Schaefer
    • 2
  • Siegmund Reissmann
    • 3
  1. 1.Faculty of Biology and PharmacyFriedrich-Schiller-UniversityJenaGermany
  2. 2.Jena BioscienceJenaGermany
  3. 3.Faculty of Biology and Pharmacy, Institute of Biochemistry and BiophysicsFriedrich-Schiller-UniversityJenaGermany

Personalised recommendations