Creation of site-specific DNA methyltransferases

描述（由申请人提供）： 在体内（以及体外）位点特异性甲基化DNA的能力将广泛适用于基础生物医学问题的研究，以及使位点特异性DNA甲基化作为治疗癌症和涉及DNA异常低甲基化的其他疾病的治疗策略的潜力的研究成为可能。位点特异性DNA甲基转移酶将是有用的（1）作为研究DNA甲基化和甲基化模式的传播的工具，（2）作为沉默感兴趣的基因的分子生物学工具，和（3）作为选择性沉默基因的潜在基因治疗剂（例如在癌症治疗中）。目前还不存在对DNA甲基化进行真正位点特异性控制的分子工具。涉及完整甲基转移酶与DNA结合结构域融合的现有方法最多产生对靶位点处的甲基化具有适度偏好的甲基转移酶，因为甲基转移酶在不存在与其靶位点结合的DNA结合结构域的情况下保持活性。这项拟议研究的目标是设计位点特异性DNA甲基转移酶，使其具有甲基化独特位点（或位点簇）的精确特异性，这些位点以每个基因组仅一次的频率发生。这种高度的特异性将产生于甲基转移酶仅在与靶序列结合的情况下组装成活性结构的事实，所述靶序列足够长以在基因组中是独特的。一种新的组合蛋白质工程方法加上定向进化将被用来创建这些位点特异性甲基转移酶。本提案中描述的研究将验证我们的方法，并将有助于为开发位点特异性甲基转移酶的长期目标获得资金，这些甲基转移酶可以在体内基因组的独特位点改变甲基化模式，并可以改变体内单个基因的表达-产物。

DESCRIPTION (provided by applicant): The ability to site-specifically methylate DNA in vivo (as well as in vitro) would have wide applicability to the study of basic biomedical problems as well as enable studies on the potential of site-specific DNA methylation as a therapeutic strategy for the treatment of cancers and other diseases that involve abnormal hypomethylation of DNA. A site specific DNA methyltransferase would be useful (1) as a tool for studying DNA methylation and the spread of methylation patterns, (2) as a molecular biological tool for silencing genes of interest, and (3) as a potential gene-therapy agent for the selective silencing of genes (e.g. in the treatment of cancers). The molecular tools to carry out truly site-specific control of DNA methylation do not exist. Existing approaches involving the fusion of intact methyltransferases to DNA binding domains at best create methyltransferases with modest preferences for methylation at target sites because the methyltransferases remain active in the absence of the DNA binding domain binding to its target site. The goal of this proposed research is to engineer site-specific DNA methyltransferases that will have the exquisite specificity of methylating unique sites (or clusters of sites) that occur at a frequency of only once per genome. This high degree of specificity will arise from the fact that the methyltransferase will only assemble into an active structure in the context of binding to a target sequence that is long enough to be unique in a genome. A novel combinatorial protein engineering approach coupled with directed evolution will be used to created these site-specific methyltransferases. The research described in this proposal will validate our approach and will be useful in obtaining funding for the long-term goals of developing site-specific methyltransferases that can site alter the methylation pattern at unique sites in a genome in vivo and can alter the expression of a single gene-product in vivo.