Development or order-mede type artificial restriction enzymes and artificial repressers

为了开发通过特异性调节靶基因转录的治疗策略，我们研究了人工抑制因子对转录水平的调节。此外，还设计了人工限制性内切酶作为分子生物学的有用工具。许多转录因子也可以诱导DNA弯曲，因此，蛋白质诱导的DNA弯曲有助于许多蛋白质-蛋白质和/或蛋白质-DNA相互作用的组合，这些相互作用是各种生物反应所必需的。cys_2his_2型的锌指基序是最常见的DNA结合基序之一。因此，这个锌指基序为设计新的DNA结合蛋白提供了一个有吸引力的框架。根据Cys_2His_2锌指的DNA识别模式和DNA结合的独特结构特点，研究了将该锌指与其他功能模块如DNA结合域连接生成具有长结合位点的人工嵌合肽和DNA切割模块连接产生新型序列特异性核酸酶的方法。事实上，人工嵌合DNA结合肽对扩展的嵌合DNA结合位点显示出序列特异性。我们设计的九锌指肽还以序列特异性的方式结合18或27个连续的DNA碱基对。此外，我们通过柔性聚甘氨酸连接剂连接两个DNA结合域，创造了六个锌指蛋白。新蛋白诱导DNA在两个远端结合位点的中间区域弯曲。综上所述，人工锌指蛋白在未来的基因治疗策略中具有广泛的应用前景。

In order to develop therapeutic strategiea through specific modulation of the transcription of target genes, We studied regulation of the transcription level by artificial repressors. In addition, artificial restriction enzymes were designed as useful tool of molecular biology. Many transcription factors are also known to induce DNA bending Therefore, the protein-induced DNA bending is helpful for many combination of protein-protein and/or protein-DNA interactions that are necessary for various biological reactions. The zinc finger motif of Cys_2His_2-type is one of the most common DNA binding motifs. Therefore, this zinc finger motif offers anatractive framework for the design of novel DNA binding proteins. On the basis of the DNA recognition mode and the structural features unique to the Cys_2His_2 zinc finger of DNA binding, approaches to link this zinc fingers with other functional modules such as DNA binding domains to generate artificial chimeric peptides with long binding sitea and DNA-cleavage modules to produce novel sequence specific nucleases, were performed. Indeed, the artificial chimeric DNA binding peptides displayed sequence specificity for extended, chimeric DNA binding sites. Our designed nine-zinc finger peptides also bound 18 or 27 contiguous base pairs of DNA in a sequence-specific fashion. Furthermore, we created six zinc finger proteins by connecting two DNA binding domains through flexible polyglycine linkers. The new proteins induced DNA bending at the intervening region of the two distal binding sites. In summary, artificial zinc finger proteins could find broad application in future gene therapy strategies.