College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan, 250014, China.
College of Chemistry and Molecular Engineering, Qingdao University of Science and Technology, Qingdao, 266042, China.
Biosens Bioelectron. 2022 Oct 1;213:114447. doi: 10.1016/j.bios.2022.114447. Epub 2022 Jun 3.
Uracil-DNA glycosylase (UDG) is essential to the maintenance of genomic integrity due to its critical role in base excision repair pathway. However, existing UDG assays suffer from laborious procedures, poor specificity, and limited sensitivity. In this research, we construct a catalytic single-molecule Föster resonance energy transfer (FRET) biosensor for in vitro and in vivo biosensing of UDG activity. Target UDG can remove uracil base from the detection probe and cause the cleavage of detection probe by apurinic/apyrimidinic endonuclease (APE1), which exposes its toehold domain and initiates catalytic assembly of two fluorescently labeled hairpin probes via toehold-meditated strand displacement reaction (SDA) to generate abundant DNA duplexes with amplified FRET signal. In this assay, target UDG signal is amplified via enzyme-free catalytic reaction and the whole reaction may be completed in one step, which greatly simplifies the assay procedure, reduces the assay time, and facilitates the cellular imaging. This biosensor enables specific and sensitive measurement of UDG down to 0.00029 U/mL, and it is suitable for analyzing kinetic parameters, screening inhibitors, and even imaging endogenous UDG in live cells. Importantly, this biosensor can visually quantify various DNA repair enzymes by rationally altering DNA substrates.
尿嘧啶-DNA 糖基化酶（UDG）在碱基切除修复途径中发挥着关键作用，对于维持基因组完整性至关重要。然而，现有的 UDG 检测方法存在操作繁琐、特异性差和灵敏度有限等问题。在本研究中，我们构建了一种催化型单分子荧光共振能量转移（FRET）生物传感器，用于体外和体内 UDG 活性的生物传感。靶标 UDG 可以从检测探针中去除尿嘧啶碱基，并通过核酸内切酶 1（APE1）引起检测探针的切割，暴露出其结合域，并通过引发两个荧光标记发夹探针的催化组装通过链置换反应（SDA）来产生大量具有放大 FRET 信号的 DNA 双链体。在该测定中，通过无酶催化反应放大靶标 UDG 信号，整个反应可以在一步中完成，这大大简化了测定程序，缩短了测定时间，并有利于细胞成像。该生物传感器可特异性和灵敏地检测低至 0.00029 U/mL 的 UDG，适用于分析动力学参数、筛选抑制剂，甚至对活细胞内的内源性 UDG 进行成像。重要的是，该生物传感器可以通过合理改变 DNA 底物来直观地定量各种 DNA 修复酶。
