Cellular delivery of a double-stranded oligonucleotide NFkappaB decoy by hybridization to complementary PNA linked to a cell-penetrating peptide.
The activation of nuclear factor kappaB (NFkappaB) 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 NFkappaB decoy. Peptide nucleic acid (PNA) hexamer or nonamer was linked to the transport peptide by a disulfide bond. NFkappaB decoy oligonucleotide consisted of a double-stranded consensus sequence corresponding to the kappaB 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-NFkappaB decoy complex to block the effect of interleukin (IL)-1beta-induced NFkappaB 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-NFkappaB decoy (1 microM, 1 h) blocked IL-1beta-induced NFkappaB-binding activity and significantly reduced the IL-6 mRNA expression. The same concentration of NFkappaB decoy in the absence of transport peptide-PNA had no effect even after longer incubations. Our results showed that binding of the oligonucleotide NFkappaB decoy to the nonamer PNA sequence resulted in a stable complex that was efficiently translocated across the plasma membrane.
Department of Neurochemistry and Neurotoxicology, Stockholm University, Stockholm, Sweden.
L
L
Fisher
U
U
Soomets
V
V
Cortés Toro
L
L
Chilton
Y
Y
Jiang
U
U
Langel
K
K
Iverfeldt
D000818:Animals; D001483:Base Sequence; D004337:Drug Carriers; D024202:Electrophoretic Mobility Shift Assay / Q000379:methods; D019004:Galanin; D015870:Gene Expression; D007375:Interleukin-1 / Q000235:genetics; D015850:Interleukin-6 / Q000235:genetics; D008957:Models, Genetic; D009693:Nucleic Acid Hybridization / Q000235:genetics; D009838:Oligodeoxyribonucleotides / Q000235:genetics*; D009841:Oligonucleotides / Q000235:genetics*; D020135:Peptide Nucleic Acids / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D014862:Wasp Venoms
D004337:Drug Carriers; D007375:Interleukin-1; D015850:Interleukin-6; C468119:NF-kappaB decoy; D009838:Oligodeoxyribonucleotides; D009841:Oligonucleotides; D020135:Peptide Nucleic Acids; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014862:Wasp Venoms; C110068:transportan; D019004:Galanin