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