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.