Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, 214063, Jiangsu, China.
College of Light Industry and Food Engineering, Nanjing Forestry University, Nanjing, 210037, China.
Mikrochim Acta. 2019 Feb 15;186(3):181. doi: 10.1007/s00604-019-3307-y.
The authors present a fluorometric method for ultrasensitive determination of the activity of uracil-DNA glycosylase (UDG). It is based on the use of two-tailed reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and an entropy-driven reaction. The assay involves the following steps: (1) UDG-driven uracil excision repair, (2) two-tailed RT-qPCR-mediated amplification, (3) RNA polymerase-aided amplification, and (4) DNA-modified silver nanoclusters (AgNCs) as a transducer to produce a fluorescent signal. UDG enables uracil to be removed from U·A pairs in DNA1 and produces a depurinated/depyrimidinated site that is readily cleaved by endonuclease IV (Endo IV). The cleaved DNA contains the T7 RNA polymerase primer for the T7 RNA polymerase amplification which produces a large number of microRNA sequences. Subsequent two-tailed RT-qPCR leads to the formation of a prolonged DNA termed DNA3. The prolonged part of DNA3 is then hybridized with an added DNA4/DNA5 duplex, where DNA5 is labeled with gold nanoparticles (AuNPs), and DNA 4 is labeled with AgNCs. The AuNPs quench the fluorescence of the AgNCs. The duplex has a toehold to hybridize the prolong part of DNA3. This results in the formation of a DNA5/DNA3 duplex due to strand displacement (by replacing the DNA4 in the DNA4/DNA5 duplex). DNA4 is released and moves away from the AuNPs. This results in restored AgNC fluorescence, best measured at excitation/emission wavelengths of 575/635 nm. The method has a detection limit as low as 0.1 mU mL of UDG activity (3σ criterion) with a range of 0.001-0.01 U mL. It was used to measure UDG activity in cell lysates. Conceivably, it may be used to screen for UDG inhibitors such as Ugi. Graphical abstract Schematic presentation of the two-tailed RT-qPCR assay platform for ultrasensitive detection of uracil-DNA glycosylase (UDG). Two-tailed RT-qPCR-mediated amplification and RNA polymerase-aided amplification are utilized for signal amplification. DNA-modified silver nanoclusters (AgNCs) are used as a transducer to produce a fluorescent signal.
作者提出了一种用于超灵敏测定尿嘧啶-DNA 糖基化酶 (UDG) 活性的荧光法。它基于使用双向反转录定量聚合酶链反应 (RT-qPCR) 和熵驱动反应。该测定法包括以下步骤：（1）UDG 驱动的尿嘧啶切除修复，（2）双向 RT-qPCR 介导的扩增，（3）RNA 聚合酶辅助扩增，以及（4）DNA 修饰的银纳米簇 (AgNCs) 作为传感器产生荧光信号。UDG 使尿嘧啶从 DNA 中的 U·A 对中除去，并产生易于被内切核酸酶 IV (Endo IV) 切割的脱嘌呤/脱嘧啶位点。切割的 DNA 包含 T7 RNA 聚合酶引物，用于 T7 RNA 聚合酶扩增，产生大量 miRNA 序列。随后的双向 RT-qPCR 导致形成称为 DNA3 的长 DNA。然后，DNA3 的延长部分与添加的 DNA4/DNA5 双链杂交，其中 DNA5 用金纳米粒子 (AuNPs) 标记，而 DNA4 用 AgNCs 标记。AuNPs 猝灭 AgNCs 的荧光。双链体具有结合 DNA3 的延长部分的结合点。这导致由于链置换（通过取代 DNA4/DNA5 双链体中的 DNA4）形成 DNA5/DNA3 双链体。DNA4 被释放并远离 AuNPs。这导致 AgNC 荧光恢复，最佳激发/发射波长为 575/635nm。该方法的检测限低至 0.1mU mL 的 UDG 活性（3σ 标准），范围为 0.001-0.01 U mL。它用于测量细胞裂解物中的 UDG 活性。可以想象，它可以用于筛选 UDG 抑制剂，如 Ugi。
