以电子设计用于猪的多位点基因打靶的人工锌指核酸酶为例,探索如何电子设计优化理想的人工锌指核酸酶,以减少人工锌指核酸酶的毒性采用OPEN电子设计方法,生物信息学方法的预测分析ZFP的立体结构,设计优化合适的连接体和间隔体组合,并通过突变优化FokⅠ异源二聚体 采用OPEN法设计三指ZF序列,并连接为ZFP序列,设计出长度为6 bp且富含AT碱基的spacer,加上长度为4个氨基酸残基的inter-domain linker,连接Fok Ⅰ切割结构域双突变子异源二聚体,并通过各种软件工具对ZFN的空间结构进行模拟预测,所设计得到的ZFN的有效率超过50％.探索获得电子设计理想ZFN的方案,即特异性结合DNA的锌指蛋白、最佳的linker和spacer的长度与组合以及优化的特异性切割的FokⅠ异源二聚体切割结构域.%Taking the electronic design of zinc finger nucleases(ZFN) for the multiple locus gene targeting of pig as an example,the electronic design of an ideal optimized artificial ZFN was explored to reduce the toxicity of the ZFN. The method of the OPEN electronic design was used. The three-dimensional structure of zinc finger protein ( ZFP) was predicted hy the methods of bioinformatics. The appropriate linkers and spacers were designed and optimized. The heterodimer of Fok I was mutated and optimized. Three-finger zinc finger sequences (ZF) were designed by OPEN method and the ZFs were connected to ZFP sequence. The AT-rich spacers with 6 bp were designed. The inter-domain linkers with four amino acid residues were connected to ZFP. The cutting domain of Fok I with double mutanl heterodimers were connected to the ZFP too. The spatial structure of ZFN was simulated and predicted by a variety of software tools. The efficient of the designed ZFN was more than 50% . The programs of electronic design of ideal ZFN including ZFP specifically bind to DNA, the linkers and spacers with the optimal length and combination, and the optimal Fok I heterodimer domain with specific cutting were explored and obtained.
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