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.
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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.
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Mussbach, F., Pietrucha, R., Schaefer, B., Reissmann, S. (2011). Internalization of Nucleoside Phosphates into Live Cells by Complex Formation with Different CPPs and JBS-Nucleoducin. In: Langel, Ü. (eds) Cell-Penetrating Peptides. Methods in Molecular Biology, vol 683. Humana Press. https://doi.org/10.1007/978-1-60761-919-2_27
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DOI: https://doi.org/10.1007/978-1-60761-919-2_27
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