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

通过基因组中选定的CpG位点的DNA甲基化来沉默哺乳动物基因将是分析表观基因组信息和研究DNA甲基化在健康和疾病中的作用的有力技术。靶向DNA甲基化的概念是基于将DNA（胞嘧啶-5）甲基转移酶（MTase）共价连接到靶向结构域，其可以以高序列特异性结合DNA，并用于将MTase锚在所寻址的CpG位点附近。目前大多数研究都是以锌指蛋白（ZFP）作为靶向结构域。三螺旋形成寡核苷酸（TFO）和肽核酸（PNA）是ZFP作为靶向装置的有希望的替代物。先前用于将CG特异性DNA MTase M.SssI偶联至TFO的策略并不令人满意，因为TFO的生产效率低。在此，我们提出了新的策略，允许TFO或PNA偶联到含有活性位点Cys 141的完全活性的M. SssI，以及偶联到含有活性位点Cys 141的完全活性的M.SssI。人DNA MTase Dnmt 3a的酶活性催化结构域。在第一种方法中，将在MTases和SNAP-标签（一种修饰的人O 6-烷基鸟嘌呤-DNA烷基转移酶）之间构建遗传融合。通过SNAP-标签介导的TFO/PNA酶促转移到DNA MTase-SNAP-标签融合蛋白上来实现偶联。在第二种方法中，将利用与半胱氨酸修饰的His标签的非共价相互作用来使TFO/PNA上的反应性基团紧密接近以诱导特异性共价键形成。TFO/PNA-MTase偶联物的DNA甲基化特异性将在体外和体内培养的人细胞中进行测试。设想的TFO/PNA-MTase缀合物将是以基因特异性方式研究表观遗传调控的有用工具，并可能为新的治疗方法开辟道路。

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.