ERA-Chemistry: New approaches for oligonucleotide-targeted enzymatic DNA methylation

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Silencing mammalian genes by DNA methylation of selected CpG sites in the genome would be a powerful technique to analyse epigenomic information and to study the roles of DNA methylation in health and disease. The concept of targeted DNA methylation is based on covalently linking a DNA (cytosine-5) methyltransferase (MTase) to a targeting domain, which can bind to DNA with high sequence specificity, and serves to anchor the MTase in the vicinity of the addressed CpG site(s). In most studies so far zinc finger proteins (ZFP) were used as targeting domain. Triple helix-forming oligonucleotides (TFO) and peptide nucleic acids (PNA) are promising alternatives to ZFPs as targeting devices. Previous strategies used to couple the CG-specific DNA MTase M.SssI to TFO were not satisfactory because of inefficient production of the TFO-MTase conjugate or because of the necessity to use the low activity C141S mutant to protect the enzyme from maleimide inactivation in the coupling step.Here we propose new strategies that allow coupling of TFO or PNA to fully active M.SssI containing the active site Cys141 as well as to the enzymatically active catalytic domain of the human DNA MTase Dnmt3a. In the first approach genetic fusions will be constructed between the MTases and the SNAP-tag, a modified human O6-alkylguanine-DNA alkyltransferase. Coupling will be achieved by SNAP-tag-mediated enzymatic transfer of the TFO/PNA onto the DNA MTase-SNAP-tag fusion proteins. In the second approach non-covalent interactions with a cysteine-modified His-tag will be utilized to bring reactive groups on the TFO/PNA in close proximity to induce specific covalent bond formation. DNA methylation specificity of the TFO/PNA-MTase conjugates will be tested in vitro and in vivo in cultured human cells. The envisaged TFO/PNA-MTase conjugates will be useful tools for studying epigenetic regulation in a gene-specific manner and may open the way for new therapeutic approaches.