Sortase A is a transpeptidase that cleaves at a pentapeptide-motif and subsequently transfers the acyl component to a nucleophile containing N-terminal oligoglycines. We investigate the reaction conditions of the sortase-mediated ligation and demonstrate a useful application by the synthesis of a peptide nucleic acid-cell-penetrating peptide chimera, the reaction equilibrium of which can be shifted in favor of the product by dialyzing out the low molecular weight byproduct. The synthesized conjugate exhibits dose-dependent antisense activity.

In order to evaluate the ability of the cell-penetrating a-helical amphipathic model peptide KLALKLALKALK AALKLA-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 MAPconjugate 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.Keywords: cell-penetrating peptides; cellular uptake; PNApeptide conjugates.The wider application of peptide nucleic acids (PNAs) [1] as antisense agents appears to be limited mainly by poor cellular uptake [2,3]. Improved delivery into mammalian cells and enhanced antisense activity have been achieved after covalent coupling of PNAs to cell-penetrating peptides (CPPs), which are able to enter cells in a nonendocytic but as yet unknown mode [3][4][5][6][7][8]. The structural requirements for the delivery activity of peptides have been unclear until now. In order to contribute to an elucidation of structuredelivery activity relationships we have previously investigated the cellular uptake and biological activity of CPP-phosphorothioate oligonucleotide conjugates using the cell-penetrating amphipathic model peptide MAP (KLALKLALKALKAALKLA-NH 2 ) [9,10] as the lead compound [11]. The value of the results of this study was limited, however, by a high cell toxicity of the phosphorothioate oligonucleotide-peptide conjugates. Therefore, in the present study we evaluated the suitability of PNA to serve as the cargo molecule in MAP-based structuredelivery activity investigations. To this end we investigated cellular uptake and biological activity of a 12-mer peptide nucleic acid (5¢-GGAGCAGGAAAG-Lys-3¢; compound I; Table 1) complementary to bases 12-23 of the translated region of the nociceptin/orphanin FQ receptor, proven previously to be sensitive to antisense attacks [12,13], and of its conjugate with MAP (compound II; Table 1). For assessing the cellular uptake, we developed a protocol based on capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) providing absolute quantities of internalized PNA which was used supplementally with confocal laser scanning microscop...

Peptide nucleic acids (PNAs) have shown great promise as potential antisense drugs; however, poor cellular delivery limits their applications. Improved delivery into mammalian cells and enhanced biological activity of PNAs have been achieved by coupling to cell-penetrating peptides (CPPs). Structural requirements for the shuttling ability of these peptides as well as structural properties of the conjugates such as the linker type and peptide position remained controversial, so far. In the present study an 18mer PNA targeted to the cryptic splice site of a mutated beta-globin intron 2, which had been inserted into a luciferase reporter gene coding sequence, was coupled to various peptides. As the peptide lead we used the cell-penetrating alpha-helical amphipathic peptide KLAL KLAL KAL KAAL KLA-NH2 [model amphipathic peptide (MAP)] which was varied with respect to charge and structure-forming properties. Furthermore, the linkage and the localization of the attached peptide (C- vs N-terminal) were modified. Positive charge as well as helicity and amphipathicity of the KLA peptide was all required for efficient dose-dependent correction of aberrant splicing. The highest antisense effect was reached within 4 h without any transfection agent. Stably linked conjugates were also efficient in correction of aberrant splicing, suggesting that a cleavable disulfide bond between CPP and PNA is clearly not essential. Moreover, the placement of the attached peptide turned out to be crucial for attaining antisense activity. Coadministration of endosome disrupting agents such as chloroquine or Ca2+ significantly increased the splicing correction efficiency of some conjugates, indicating the predominant portion to be sequestered in vesicular compartments.