Abstract
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-specific interferon-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 anticancer strategies.
Highlights
Intercellular-adhesion molecule 1 (ICAM-1,1 CD54) is a cell surface molecule that mediates adhesion processes involving leukocytes and other cell types [1]
Design of Triplex-forming Oligonucleotides Targeted to the intercellular-adhesion molecule-1 (ICAM-1) Gene—The ICAM-1 gene sequence was screened for suitable targets for triplex formation
An oligonucleotide was designed to form a triple helix at this site: TFOgt contains guanosine and thymidine nucleosides, allowing formation of T1⁄7AxT and C1⁄7GxG base triplets (with (1⁄7) standing for Watson-Crick interactions and (x) standing for reverse Hoogsteen interactions) after binding in an antiparallel orientation with respect to the purine strand of the double helical target sequence
Summary
Intercellular-adhesion molecule 1 (ICAM-1,1 CD54) is a cell surface molecule that mediates adhesion processes involving leukocytes and other cell types [1]. TFO binding to its ICAM-1 target sequence was characterized in vitro and 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.
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