Jiang Yuqian, Chen Yingying, Gong Xue, Weng Benrui, He Yuqiu, Wang Fuan
College of Chemistry and Molecular Sciences, Department of Gastroenterology, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430072, P. R. China.
Research Institute of Shenzhen, Wuhan University, Shenzhen 518057, P. R. China.
JACS Au. 2025 Sep 2;5(9):4288-4298. doi: 10.1021/jacsau.5c00672. eCollection 2025 Sep 22.
Uracil DNA glycosylase (UDG) is a crucial DNA repair enzyme involved in maintaining genomic stability. Precise monitoring of UDG facilitates in-depth elucidation of DNA repair and the onset and progression mechanisms of diseases. However, conventional UDG sensing approaches suffer from low accuracy and off-target signal leakage, limiting their applicability for precise imaging and functional studies. Here, we report a UDG-activated allosteric DNAzyme (UAZ) platform that leverages an intramolecular recognition-allosteric-amplification strategy to achieve high-fidelity UDG detection and programmable gene regulation. In this design, the UDG-responsive uracil residue is introduced to disrupt the catalytic structure of DNAzyme and make DNAzyme inactive. Upon specific uracil excision by UDG, the precise allosteric reconfiguration restores the catalytic activity of DNAzyme, enabling efficient substrate cleavage and amplified fluorescence output. This mechanism eliminates off-target activation, realizing reliability and high-contrast UDG imaging. Furthermore, the modular UAZ platform was reprogrammed into a gene-silencing variant (gsUAZ) for selectively downregulating survivin mRNA in UDG-overexpressing cancer cells, thereby inducing cancer cell apoptosis with minimal effects on normal cells, ultimately realizing targeted gene therapy. This multifunctional UAZ system thus serves as both a sensitive molecular imaging tool and a targeted gene therapy agent, offering a versatile toolbox for disease diagnosis and precision gene therapy.
尿嘧啶DNA糖基化酶（UDG）是一种参与维持基因组稳定性的关键DNA修复酶。对UDG进行精确监测有助于深入阐明DNA修复以及疾病的发生和发展机制。然而，传统的UDG传感方法存在准确性低和脱靶信号泄漏的问题，限制了它们在精确成像和功能研究中的应用。在此，我们报告了一种UDG激活的变构脱氧核酶（UAZ）平台，该平台利用分子内识别-变构-放大策略实现高保真UDG检测和可编程基因调控。在这种设计中，引入了对UDG有响应的尿嘧啶残基来破坏脱氧核酶的催化结构并使脱氧核酶失活。在UDG特异性切除尿嘧啶后，精确的变构重排恢复了脱氧核酶的催化活性，从而实现有效的底物切割和放大的荧光输出。这种机制消除了脱靶激活，实现了可靠且高对比度的UDG成像。此外，模块化的UAZ平台被重新编程为基因沉默变体（gsUAZ），用于在过表达UDG的癌细胞中选择性下调生存素mRNA，从而诱导癌细胞凋亡，同时对正常细胞的影响最小，最终实现靶向基因治疗。因此，这种多功能的UAZ系统既作为一种灵敏的分子成像工具，又作为一种靶向基因治疗剂，为疾病诊断和精确基因治疗提供了一个多功能的工具箱。
