INHIBITION OF GENE-EXPRESSION BY TRIPLE-HELIX FORMATION IN HEPATOMA-CELLS

The aim of this study was to selectively inhibit human mitochondrial a ldehyde dehydrogenase (ALDH(2)) gene expression by triple helix assemb ly. Eight 21-mer oligodeoxyribonucleotides were designed to bind to tw o purine-rich sequences in the 5'-flanking region of the human ALDH(2) gene. Gel mobility shift assays showed that tripler formation is sequ ence-specific for the target duplex and the third strand oligonucleoti de, In the presence of Mg2+, but absence of K+, tripler-forming oligon ucleotides bind to their target sites with apparent dissociation const ants (K-d) in the 10(-7) to 10(-9) M range. Potassium cation virtually suppressed the tripler formation of G-C-rich duplex DNA with natural oligonucleotides, but did not prevent tripler formation with phosphoro thioate-modified oligonucleotides. Phosphoro-thioate-modified oligonuc leotides were delivered into human hepatoma Hep G2 cells by cationic l iposomes. The reduction in ALDH(2) mRNA levels in the cells was determ ined by the competitive reverse transcription-polymerase chain reactio n. One of the phosphorothioate-modified oligonucleotides designed to f orm an antiparallel tripler with a target in the 5'-flanking region of human ALDH(2) gene (-105 to -125 from the translation initiation codo n ATG) reduced by 80-90% the ALDH(2) mRNA levels without affecting alb umin mRNA levels. Data suggest that triple-helix formation may provide a means to selectively inhibit hepatic ALDH(2) gene expression for th erapeutic use.