Synthesis, Uptake, and Intracellular Metabolism of a Hydrophobic Tetrapeptide-Peptide Nucleic Acid (PNA)–Biotin Molecule

Peptide nucleic acid (PNA) molecules are very promising tools for antigene and antisense therapies because of their remarkable refractoriness to degradation in biological fluids. However, very limited information is available on their uptake by potentially target cells and on their intracellular fate. A membrane-diffusable and fluorescence detectable PNA chimera, Phe-Leu-Phe-Leu-(Adenine)3-biotin, was obtained by solid phase peptide synthesis and characterized by combined HPLC and mass spectrometry (MS). This PNA chimera was found to permeate across the membrane of both human erythrocytes and B Namalwa cells much more extensively and rapidly than a control Gly-(Adenine)3-biotin PNA molecule. Fluorescence patterns of internalization were consistent for a diffusion process resulting in the appearance of uniform cytoplasmic distribution of the hydrophobic peptide-PNA chimera in the Namalwa cells. Degradation of the synthesized PNA chimera by cell lysates and to a much slower extent by the intact Namalwa cells was investigated by HPLC-MS analyses of the corresponding methanol extracts . It involved the progressive removal of each of the hydrophobic aminoacid residues, while the linkage with the biotin label was completely resistant to cleavage.These results hold promise for the design and synthesis of membrane-permeable PNA sequences suitable for antigene therapies.