Open AccessEnhancement of intracellular concentration and biological activity of PNA after conjugation with a cell‐penetrating synthetic model peptide
Author(s) -
Oehlke Johannes,
Wallukat Gerd,
Wolf Yvonne,
Ehrlich Angelika,
Wiesner Burkhard,
Berger Hartmut,
Bienert Michael
Publication year - 2004
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.2004.04236.x
Subject(s) - conjugate , peptide , intracellular , chemistry , peptide nucleic acid , cell penetrating peptide , microbiology and biotechnology , biochemistry , biophysics , biology , mathematical analysis , mathematics
In order to evaluate the ability of the cell‐penetrating α‐helical amphipathic model peptide KLALKLALKALKAALKLA‐NH 2 (MAP) to deliver peptide nucleic acids (PNAs) into mammalian cells, MAP was covalently linked to the 12‐mer PNA 5′‐GGAGCAGGAAAG‐3′ directed against the mRNA of the nociceptin/orphanin FQ receptor. The cellular uptake of both the naked PNA and its MAP‐conjugate was studied by means of capillary electrophoresis combined with laser‐induced fluorescence detection, confocal laser scanning microscopy and fluorescence‐activated cell sorting. Incubation with the fluorescein‐labelled PNA–peptide conjugate led to three‐ and eightfold higher intracellular concentrations in neonatal rat cardiomyocytes and CHO cells, respectively, than found after exposure of the cells to the naked PNA. Correspondingly, pretreatment of spontaneously‐beating neonatal rat cardiomyocytes with the PNA–peptide conjugate and the naked PNA slowed down the positive chronotropic effect elicited by the neuropeptide nociceptin by 10‐ and twofold, respectively. The main reasons for the higher bioavailability of the PNA–peptide conjugate were found to be a more rapid cellular uptake in combination with a lowered re‐export and resistance against influences of serum.