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Papers In Press, published online ahead of print June 21, 2002
J. Biol. Chem, 10.1074/jbc.M203311200
Submitted on April 6, 2002
Revised on June 18, 2002
Accepted on June 21, 2002
Department of Dermatology, Ludwig-Maximilians University, München 80337
Corresponding Author: Klaus.Degitz{at}lrz.uni-muenchen.de
Selected sequences in the DNA double helix can be specifically recognized by oligonucleotides via hydrogen bonding interactions. The resulting triple-helix can modulate DNA metabolism, and especially interfere with transcription in a gene-specific manner. To explore the potential of triplex-forming oligonucleotides (TFOs) as gene repressors, a TFO was designed to target a 16 bp sequence within the third intron of the human intercellular-adhesion molecule-1 (ICAM-1) gene, which plays a key role in initiating inflammation. TFO binding to its ICAM-1 target sequence was characterized in vitro and also demonstrated in cell nuclei with the set-up of a novel magnetic capture assay, which represents a general experimental approach to the detection of specific TFO binding and to the determination of the accessibility of a given genomic DNA locus. In a human keratinocyte cell line (A431) we observed that: i) the ICAM-1 target sequence in the chromatin context within the nuclei is still available for triplex formation and ii) TFO inhibits sequence- and gene-specifically IFN-gamma induced ICAM-1 surface expression. Collectively, the data demonstrate effective and specific inhibition of ICAM-1 expression by TFO treatment and support the view that triplex-mediated gene targeting might be a valuable technique for anti-inflammatory or anti-cancer strategies.
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