人工定向基因修饰是基因工程技术的核心步骤，对特定核酸序列的定向识别和切割依赖于具有序列特异性的核酸工具酶。现有的限制性内切酶种类有限，而现行的用于人工构建新型序列特异性核酸酶的方法主要依赖于生物技术，制备周期长、产量低，影响因素复杂。本项目拟探索发展一种全新的通过对天然酶的体外化学修饰构建序列特异性核酸工具酶的方法。方法的基本原理是利用骨架被化学修饰保护的单链DNA或RNA作为惰性模板序列，分别与天然非限制性核酸内切酶DNase I或RNase A结合形成稳定的核酸酶@惰性模板链复合物。该复合物中酶的活性只有在遇到该模板序列的互补DNA或RNA序列时才被开启，将互补序列水解，而对体系中共存的其他DNA或RNA序列基本没有影响。项目将系统研究复合物的制备方法和获得序列特异性的分子机理，并尝试应用于细胞中目标RNA的原位切割和植物基因组DNA的选择性切割，为生命科学研究提供重要的方法和技术。

Sequence specific nucleases are important enzymatic tools for processing nucleic acids in a predictable and reproducible way. Restriction enzymes are the most commonly used sequence specific nucleases. However, available restriction enzymes could only recognize and cut a limit number of specific sequences. Recombination of sequence-specific nucleases has proven successful, but depending on labor-intensive biological approaches. In this project, we propose a new strategy for regulating the sequence specificity of nucleases via chemical modification. The basic principle is to modify the native non-specific nucleases with chemically synthesized idle template strand so as to induce desired sequence specificity of the nucleases. DNase I and RNase A will be used as the native nucleases, respectively. By changing the sequences of the idle template strand, the sequence specificity of the obtained nuclease@idle-template complex can be flexibly tuned for different target strands. The mechanism for the sequence specificity of the complex will be investigated in detail. The method will be applied to cleave intracellular RNA in situ. Furthermore, the feasibility of the approach for selective cleavage of a short fragment of DNA sequence within genomic DNA will also be tested. The results of this project will offer very useful tools for biological research and genetic engineering field.