Differences in uptake, localization, and processing of PNAs modified by COX VIII pre-sequence peptide and by triphenylphoshonium cation into mitochondria of tumor cells.

Two approaches to target PNAs (peptide nucleic acids) into mitochondria of HeLa cells were compared. In the first, PNA was modified with the lipophilic cation TPP. TPP-PNA accumulated rapidly within mitochondria driven by the membrane potential. It was found to be associated mainly with the mitochondrial membranes. In the second approach the COX VIII pre-sequence peptide was added to the PNA resulting in slow uptake of the peptide-PNA into the mitochondrial matrix. Whereas the amount of the uptake was lower, peptide-PNA was processed intramitochondrially in contrast to the TPP-PNA. Using the Chariot system to cross the cell membrane of HeLa cells, the uptake of peptide-PNA into the mitochondria was demonstrated. If a matrix localization of the free PNA is a pre-requisite for the PNA interaction with mitochondrial DNA, the coupling PNA with an appropriate peptide seems to be the better strategy.

Genetically engineered clostridial C2 toxin as a novel delivery system for living mammalian cells.

The C2 toxin of Clostridium botulinum is a binary bacterial protein toxin, comprising the enzyme component C2I and the separate binding/translocation component C2IIa. C2IIa mediates the transport of C2I into the host cell cytosol. The N-terminal domain of C2I (C2IN) is enzymatically inactive but essential for C2IIa-mediated internalization of C2I. Here, we exploit the C2IIa/C2IN system to generate a recombinant C2IN-streptavidin fusion protein allowing for the delivery of biotinylated molecules into the cytosol of mammalian cells. C2IN-streptavidin overproduced in E. coli was affinity-purified and capable of binding biotinylated proteins in a concentration-dependent manner. Real-time surface plasmon resonance confirmed the biotin-mediated interaction yielding a K(D)-value of approximately 0.75 muM. Internalization of C2IN-streptavidin into the cytosol of epithelial cells and macrophages was demonstrated by immunoblot analysis and confirmed by confocal microscopy. Cell viability studies showed no cytotoxic effects of the novel transporter. Furthermore, Vero cells treated with biotin-fluorescein or biocytin-Alexa488 as model cargo displayed a specific C2IN-streptavidin/C2IIa-dependent uptake, providing proof-of-principle for the functionality of this novel delivery system.