Cellular delivery of a double-stranded oligonucleotide NFκB decoy by hybridization to complementary PNA linked to a cell-penetrating peptide

The activation of nuclear factor Kappa B (NF Kappa B) is a key event in immune and inflammatory responses. In this study, a cell-penetrating transport peptide, transportan (TP) or its shorter analogue TP 10, was used to facilitate the cellular uptake of an NF Kappa B decoy. Peptide nucleic acid (PNA) hexamer or nonamer was linked to the transport peptide by a disulfide bond. NF Kappa B decoy oligonucleotide consisted of a double-stranded consensus sequence corresponding to the Kappa B site localized in the IL-6 gene promoter, 5'-GGGACTTTCCC-3', with a single-stranded protruding 3'-terminal sequence complementary to the PNA sequence was hybridized to the transport peptide-PNA construct. The ability of the transport peptide-PNA-NF Kappa B decoy complex to block the effect of interleukin (IL)-1 beta -induced NF Kappa B activation and IL-6 gene expression was analyzed by electrophoretic mobility shift assay and reverse transcriptase-polymerase chain reaction in rat Rinm5F insulinoma cells. Preincubation with transport peptide-PNA-NF Kappa B decoy (1 mu M, 1 h) blocked IL-1 beta -induced NF Kappa B-binding activity and significantly reduced the IL-6 mRNA expression. The same concentration of NF Kappa B decoy in the absence of transport peptide-PNA had no effect even after longer incubations. Our results showed that binding of the oligonucleotide NF Kappa B decoy to the nonamer PNA sequence resulted in a stable complex that was efficiently translocated across the plasma membrane.