Restriction endonucleases (ENases) and DNA methyltransferases (MTases) are important enzymes in biological processes, and detection of ENases/MTases activity is significant for biological and pharmaceutical studies. However, available nonamplification methods with a versatile design, desirable sensitivity, and signal production mode of unbiased quantification toward multiple nucleases are rare. By combining deliberately designed hairpin DNA probes with the colocalized particle counting technique, we present a nonamplification, separation-free method for multiplexed detection of ENases and MTases. In the presence of target ENases, the hairpin DNA is cleaved and the resulting DNA sequence forms a sandwich structure to tie two different-colored fluorescent microbeads together to generate a colocalization signal that can be easily detected using a standard fluorescence microscope. The multiplexed assay is realized via different color combinations. For the assay of methyltransferase, methylation by MTases prevents cleavage of the hairpin by the corresponding ENase, leading to decreased colocalization events. Three ENases can be simultaneously detected with high selectivity, minimal cross-talk, and detection limits of (4.1–6.4) × 10^–4 U/mL, and the corresponding MTase activity can be measured without a change of the probe design. The potential for practical application is evaluated with human serum samples and different ENase and MTase inhibitors with satisfactory results. The proposed method is separation-free, unbiased toward multiple targets, and easy to implement, and the strategy has the potential to be extended to other targets.

中文翻译：

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通过共定位颗粒计数进行内切核酸酶和DNA甲基转移酶的非扩增多路复用测定

限制性核酸内切酶（ENases）和DNA甲基转移酶（MTases）是生物过程中的重要酶，而ENases / MTases活性的检测对于生物学和药物研究具有重要意义。但是，具有通用设计，理想灵敏度和针对多种核酸酶进行无偏定量的信号产生模式的可用非扩增方法很少见。通过将精心设计的发夹DNA探针与共定位颗粒计数技术相结合，我们提出了一种非扩增，无分离的方法，用于ENase和MTase的多重检测。在存在目标酶的情况下，发夹DNA被切割，得到的DNA序列形成一个三明治结构，将两个不同颜色的荧光微珠绑在一起，产生共定位信号，可以使用标准荧光显微镜轻松检测到。多重测定是通过不同的颜色组合来实现的。对于甲基转移酶的测定，MTase的甲基化可防止相应的ENase对发夹的切割，从而减少共定位事件。可以同时以高选择性，最小串扰和（4.1–6.4）×10的检测限检测三种ENase 导致共定位事件减少。可以同时以高选择性，最小串扰和（4.1–6.4）×10的检测限检测三种ENase 导致共定位事件减少。可以同时以高选择性，最小串扰和（4.1–6.4）×10的检测限检测三种ENase^–4 U / mL，并且无需改变探针设计即可测量相应的MTase活性。用人血清样品以及不同的ENase和MTase抑制剂评估了实际应用的潜力，结果令人满意。所提出的方法是无分离的，不偏向多个目标，并且易于实施，并且该策略具有扩展到其他目标的潜力。