Modified oligonucleotides as bona fide antagonists of proteins interactingwith DNA - Hairpin antagonists of the human DNA methyltransferase

The study of the biological role of DNA methyltransferase (DNA MeTase) has been impeded by the lack of direct and specific inhibitors. This report des cribes the design of potent DNA based antagonists of DNA MeTase and their u tilization to define the interactions of DNA MeTase with its substrate and to study its biological role. We demonstrate that the size, secondary struc ture, hemimethylation, and phosphorothioate modification strongly affect th e antagonists interaction with DNA MeTase whereas base substitutions do not have a significant effect. To study whether DNA MeTase is critical for cel lular transformation, human lung non-small carcinoma cells were treated wit h the DNA MeTase antagonists. Ex vivo, hairpin inhibitors of DNA MeTase are localized to the cell nucleus in lung cancer cells. They inhibit DNA MeTas e, cell growth, and anchorage independent growth tan indicator of tumorigen esis in cell culture) in a dose-dependent manner. The inhibitors developed in this study are the first documented example of direct inhibitors of DNA MeTase in living cells and of modified oligonucleotides as bona fide antago nists of critical cellular proteins.