Inhibition of gene expression by triple helix formation in hepatoma cells.
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.
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
D017931:DNA Primers; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D000444:Aldehyde Dehydrogenase