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Cellular delivery of a double-stranded oligonucleotide

NFκB decoy by hybridization to complementary PNA linked to a cell-penetrating peptide

Gene Therapy volume 11, pages 1264–1272 (2004)Cite this article

Abstract

The activation of nuclear factor κB (NFκB) 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 NFκB decoy. Peptide nucleic acid (PNA) hexamer or nonamer was linked to the transport peptide by a disulfide bond. NFκB decoy oligonucleotide consisted of a double-stranded consensus sequence corresponding to the κB 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–NFκB decoy complex to block the effect of interleukin (IL)-1β-induced NFκB 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–NFκB decoy (1 μM, 1 h) blocked IL-1β-induced NFκB-binding activity and significantly reduced the IL-6 mRNA expression. The same concentration of NFκB decoy in the absence of transport peptide–PNA had no effect even after longer incubations. Our results showed that binding of the oligonucleotide NFκB decoy to the nonamer PNA sequence resulted in a stable complex that was efficiently translocated across the plasma membrane.

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Acknowledgements

This work was supported by grants from the Swedish Research Council for Medical Science and for Natural and Engineering Sciences and EC project QLK3-CT-2002-01989.

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Authors and Affiliations

  1. Department of Neurochemistry and Neurotoxicology, Stockholm University, Stockholm, Sweden

    L Fisher, U Soomets, V Cortés Toro, L Chilton, Y Jiang, Ü Langel & K Iverfeldt

  2. Department of Biochemistry, University of Tartu, Tartu, Estonia

    U Soomets

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Fisher, L., Soomets, U., Cortés Toro, V. et al. Cellular delivery of a double-stranded oligonucleotide. Gene Ther 11, 1264–1272 (2004). https://doi.org/10.1038/sj.gt.3302291

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