Creation of site-specific DNA methyltransferases

描述(由申请人提供)： 在体内(以及在体外)对DNA进行定点甲基化的能力将广泛适用于基本生物医学问题的研究，并使研究定点DNA甲基化作为治疗癌症和其他涉及DNA异常低甲基化的疾病的治疗策略的潜力成为可能。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扩散的工具，(2)作为沉默感兴趣的基因的分子生物学工具，以及(3)作为选择性沉默基因的潜在的基因治疗剂(例如在癌症的治疗中)。目前还不存在对DNA甲基化进行真正特定部位控制的分子工具。现有的方法包括将完整的甲基转移酶融合到DNA结合结构域，最多只能产生在目标位置具有适度甲基化偏好的甲基转移酶，因为在没有DNA结合结构域与其目标位置结合的情况下，甲基转移酶仍然保持活性。这项拟议的研究的目标是设计特定于位点的DNA甲基转移酶，这种酶将具有甲基化独特位点(或位点簇)的精致专一性，这些位点在每个基因组中只出现一次。这种高度的特异性将产生于这样一个事实，即甲基转移酶只有在与足够长的、在基因组中唯一的靶序列结合的情况下才会组装成活性结构。一种新的结合定向进化的蛋白质工程方法将被用来创造这些位点特异的甲基转移酶。这项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，这种甲基转移酶可以在体内改变基因组中独特位置的甲基化模式，并可以在体内改变单一基因产物的表达。

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.