核酸剪切工具在基因调节等领域具有广阔的应用前景。诱导型的核酸剪切工具可以在时空上控制剪切的效率，使DNA剪切更加精准，有效降低了脱靶效应和遗传毒性等。其中，内源性响应的DNA剪切工具除了可以精准地控制DNA的剪切反应，还可以结合荧光等检测手段用于研究细胞中重要的生物分子变化，据此可以建立一系列活细胞内生物分子分析的新方法，为分析化学领域提供了新的发展途径。基于此，本论文发展了胞内酶控制的核酸点特异性剪切工具，并将其用于体外检测和细胞成像的研究，主要研究内容如下：1．提出了响应去甲基化酶的CRISPR-Cas12a系统。发现m6A和m1A甲基化修饰的sgRNA均可以对CRISPR-Cas12a系统产生高效的封闭作用，利用去甲基化酶去除sgRNA上的甲基化修饰可以恢复CRISPR-Cas12a的剪切活性。我们利用ALKBH5和FTO去甲基化酶激活m6A甲基化封闭的CRISPR-Cas12a系统，利用ALKBH3去甲基化酶激活m1A甲基化封闭的CRISPR-Cas12a系统。该激活系统对去甲基化酶具有优异的选择性，并且激活效果随着去甲基化酶浓度增加而增强。2．发现了髓过氧化物酶（MPO）诱导的DNA磷硫酰化位点特异性切割现象。该剪切反应对磷硫酰化位点专一性响应，在双链和单链DNA体系中均可以实现，并且不需要复杂的反应条件。我们对该剪切反应的各种影响因素进行研究，证实该剪切反应的机理是MPO将溶液中的Cl-通过H2O2氧化成HOCl/OCl-，进而破坏DNA上的磷硫酰化修饰。3．设计了两种发卡型DNA分子机器ptG4-Hairpin和ptHCR-Hairpin。利用MPO诱导的DNA剪切系统来控制磷硫酰化修饰的发卡DNA分子机器，建立了MPO的分析方法。ptG4-Hairpin分子机器的磷硫酰化位点被MPO剪切后会自发转化为G4-HRP模拟酶，结合显色反应可以实现MPO的高灵敏检测；ptHCR-Hairpin分子机器的磷硫酰化位点被MPO剪切后，可以诱发杂交链式反应（HCR），该策略成功用于活细胞中MPO及相关氧化应激通路的高灵敏成像分析。
New generation DNA cleavage tools sparks a revolution in the fields of synthetic biology and biomedical science. Inducible DNA cleavage tools can control the editing reaction in time and space simultaneously. These strategies are important for the DNA editing as reducing off-target activity and genotoxicity. The endogenous inducible DNA cleavage tools can not only precisely regulate the cleavage reaction, but also be used to determine the distribution and content of biomolecules in cells. Currently, endogenous inducible cleavage tools are mostly controlled by RNA or metabolic small molecules, and lack enzyme response in cells. Hence, this thesis aims to develop new DNA site-specific cleavage tools, which are controlled by some important endogenous enzymes.1. We developed a demethylases-inducible CRISPR-Cas12a system. We found that the m1A and m6A methylation on sgRNA could block the activity of CRISPR-Cas12a in DNA cleavage. Hence, removal of methylation modifications on sgRNA by demethylase would restore CRISPR-Cas12a activity. We used the ALKBH5 and FTO demethylases to active the m6A methylation-blocked CRISPR-Cas12a system, and used ALKBH3 demethylases to active the m1A methylation-blocked CRISPR-Cas12a system. These inducible cleavage tools have good selectivity for demethylases, and we further achieved quantitative assays of the three demethylases in vitro by demethylases inducible CRISPR-Cas12a system.2. In this part, the cleavage reaction of phosphorothioate (PT) modification by myeloperoxidase (MPO) has been proposed. This cleavage reaction performs great in both double-strand DNA and single-strand DNA. And the reaction conditions are mild and benefit for applications in living cells. We explored the contributory factors of the cleavage reaction to obtain the optimal efficiency. Accordingly, the reaction mechanism of the cleavage reaction was further studied, and MPO used its unique chlorination oxidation ability, assisted by H2O2 and chloride, to site-selectively break PT modification of DNA.3. We successfully used the cleavage reaction of PT modification to control the release of functional DNA fragments from the hairpin DNA machine. Two hairpin DNA machines, ptG4-Hairpin and ptHCR-Hairpin, were designed. After site-specific cleavage of ptG4-Hairpin hairpin DNA, the product could be converted to hemin-HRP DNAzyme, which effectively improved the oxidation capacity of solution and can be used in quantification of MPO in vitro. Besides, our design allowed implementing the HCR amplification in specific response to MPO. Therefore, we have achieved the real-time imaging of oxidative stress pathway in living cells.
