title: Inhibition of gene expression by triple helix formation in hepatoma cells.
abstract: The aim of this study was to selectively inhibit human mitochondrial aldehyde dehydrogenase (ALDH2) gene expression by triple helix assembly. Eight 21-mer oligodeoxyribonucleotides were designed to bind to two purine-rich sequences in the 5'-flanking region of the human ALDH2 gene. Gel mobility shift assays showed that triplex formation is sequence-specific for the target duplex and the third strand oligonucleotide. In the presence of Mg2+, but absence of K+, triplex-forming oligonucleotides bind to their target sites with apparent dissociation constants (Kd) in the 10(-7) to 10(-9) M range. Potassium cation virtually suppressed the triplex formation of G-C-rich duplex DNA with natural oligonucleotides, but did not prevent triplex formation with phosphorothioate-modified oligonucleotides. Phosphorothioate-modified oligonucleotides were delivered into human hepatoma Hep G2 cells by cationic liposomes. The reduction in ALDH2 mRNA levels in the cells was determined by the competitive reverse transcription-polymerase chain reaction. One of the phosphorothioate-modified oligonucleotides designed to forma an antiparallel triplex with a target in the 5'-flanking region of human ALDH2 gene (-105 to -125 from the translation initiation codon ATG) reduced by 80-90% the ALDH2 mRNA levels without affecting albumin mRNA levels. Data suggest that triple-helix formation may provide a means to selectively inhibit hepatic ALDH2 gene expression for therapeutic use.
mesh_terms: D000444:Aldehyde Dehydrogenase / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D017931:DNA Primers; D015870:Gene Expression / Q000187:drug effects*; D006801:Humans; D007700:Kinetics; D008113:Liver Neoplasms; D046911:Macromolecular Substances; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology*; D016133:Polymerase Chain Reaction; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
chemical_list: D017931:DNA Primers; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D000444:Aldehyde Dehydrogenase