title: Cellular uptake and efficacy of antisense oligonucleotides against RNAs of two Na(+) channel isoforms.
abstract: The effects of 15-mer phosphorothioate antisense oligodeoxynucleotides (aODNs) specifically designed against the RNAs of either of two closely related Na(+) channel isoforms, hSkM1 or hH1, were tested in human myotubes. Fluorescence (3'-fluorescein isothiocyanate) labeling showed that mere incubation of cultures with aODNs did not result in aODN uptake, but liposome-mediated transfer was successful and resulted in cytoplasmic and nuclear localization of ODNs. Intracellular fluorescence was stable for at least 3 days. At 5 microM, the hH1-specific aODN was effective in suppressing ion channel function, but the hSkM1-specific aODN was not. Reverse transcription-polymerase chain reaction gave corresponding results on the mRNA level. However, in HEK-293 cells stably expressing hSkM1, the same hSkM1-specific aODN was able to reduce Na(+) currents (2.4 +/- 0.5 nA, n = 11; controls: 6.5 +/- 1.0 nA, n = 14). We conclude that cellular uptake of aODNs and intracellular access to the RNA target are necessary, but not always sufficient conditions for an effective block of mRNA translation in intact cells.
authors.affiliation: Department of General Physiology, University of Ulm, Ulm, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Keller
authors.forename: B
authors.initials: B
authors.lastname: Schu
authors.forename: R
authors.initials: R
authors.lastname: Rüdel
authors.forename: H
authors.initials: H
authors.lastname: Brinkmeier
mesh_terms: D006801:Humans; D009132:Muscles / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000493:pharmacokinetics* / Q000494:pharmacology; D020033:Protein Isoforms / Q000037:antagonists & inhibitors; D012313:RNA / Q000037:antagonists & inhibitors*; D012333:RNA, Messenger / Q000032:analysis; D026941:Sodium Channel Blockers; D015222:Sodium Channels / Q000187:drug effects / Q000235:genetics
chemical_list: D016376:Oligonucleotides, Antisense; D020033:Protein Isoforms; D012333:RNA, Messenger; D026941:Sodium Channel Blockers; D015222:Sodium Channels; D012313:RNA