Construction of an Entropy-Driven Dumbbell-Type DNAzyme Assembly Circuit for Lighting Up Uracil-DNA Glycosylase in Living Cells,Analytical Chemistry

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Construction of an Entropy-Driven Dumbbell-Type DNAzyme Assembly Circuit for Lighting Up Uracil-DNA Glycosylase in Living Cells
Analytical Chemistry ( IF 6.7 ) Pub Date : 2022-09-30 , DOI: 10.1021/acs.analchem.2c03223
Qian Zhang ₁ , Ran Zhao ₁ , Chen-Chen Li ₂ , Yan Zhang ₁ , Chunying Tang ₁ , Xiliang Luo ₂ , Fei Ma ₃ , Chun-Yang Zhang ₁

Affiliation

Sensitive monitoring of intracellular uracil-DNA glycosylase (UDG) in living cells is essential to understanding the DNA repair pathways and discovery of anticancer drugs. Herein, we demonstrate the construction of an entropy-driven dumbbell-type DNAzyme assembly circuit for lighting up UDG in living cells via the integration of entropy-driven DNA catalysis (EDC) with the DNAzyme biocatalyst. Target UDG excises the damaged uracil base, causing the breakage of detection probe and the release of trigger. The released trigger can initiate the downstream EDC reaction to form two catalytically active DNAzyme units. The resultant dual Mg²⁺-DNAzyme units serve as the signal transducers to cyclically cleave the fluorophore/quenched-modified reporters, generating an enhanced fluorescence signal. In contrast to the single-layered EDC method with a linear amplification, the proposed doublet EDC-DNAzyme strategy exhibits high signal gain and achieves a detection limit of 8.71 × 10^–6 U/mL. Notably, this assay can be performed in one-step manner at room temperature without the requirement of strict temperature control and complicated reaction procedures, and it can further screen the UDG inhibitors, measure kinetic parameters, and discriminate cancer cells from normal cells. Moreover, this strategy can monitor intracellular UDG activity with improved signal gain, and it may be exploited for sensing and imaging of other types of DNA modifying enzymes with the integration of the corresponding detection substrate, providing a facile and robust approach for biological research studies and clinical diagnosis.

中文翻译：

构建熵驱动的哑铃型 DNAzyme 组装电路，用于点亮活细胞中的尿嘧啶 DNA 糖基化酶

灵敏监测活细胞中的细胞内尿嘧啶-DNA 糖基化酶 (UDG) 对于了解 DNA 修复途径和发现抗癌药物至关重要。在此，我们展示了熵驱动的哑铃型 DNAzyme 组装电路的构建，用于通过熵驱动的 DNA 催化 (EDC) 与 DNAzyme 生物催化剂的整合来点亮活细胞中的 UDG。Target UDG切除受损的尿嘧啶碱基，导致检测探针断裂和触发释放。释放的触发器可以启动下游 EDC 反应，形成两个具有催化活性的 DNAzyme 单元。所得双 Mg ²⁺-DNAzyme 单元作为信号传感器循环切割荧光团/淬灭修饰的报告基因，产生增强的荧光信号。与具有线性放大的单层 EDC 方法相比，所提出的双峰 EDC-DNAzyme 策略表现出高信号增益并达到 8.71 × 10 ^{–6的检测限}单位/毫升。值得一提的是，该实验可以在室温下一步完成，不需要严格的温度控制和复杂的反应程序，可以进一步筛选UDG抑制剂，测量动力学参数，区分癌细胞和正常细胞。此外，该策略可以通过提高信号增益来监测细胞内 UDG 活性，并可用于其他类型 DNA 修饰酶的传感和成像，并结合相应的检测底物，为生物学研究和研究提供一种简便而可靠的方法。临床诊断。

更新日期：2022-09-30

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