Li Chen-Chen, Chen Hui-Yan, Hu Juan, Zhang Chun-Yang
College of Chemistry , Chemical Engineering and Materials Science , Collaborative Innovation Center of Functionalized Probes for Chemical Imaging in Universities of Shandong , Key Laboratory of Molecular and Nano Probes , Ministry of Education , Shandong Provincial Key Laboratory of Clean Production of Fine Chemicals , Shandong Normal University , Jinan 250014 , China . Email:
Chem Sci. 2020 May 18;11(22):5724-5734. doi: 10.1039/d0sc01652g. eCollection 2020 Jun 14.
DNA repair enzymes (, DNA glycosylases) play a critical role in the repair of DNA lesions, and their aberrant levels are associated with various diseases. Herein, we develop a sensitive method for simultaneous detection of multiple DNA repair enzymes based on the integration of single-molecule detection with rolling circle amplification (RCA)-driven encoding of different fluorescent molecules. We use human alkyladenine DNA glycosylase (hAAG) and uracil DNA glycosylase (UDG) as the target analytes. We design a bifunctional double-stranded DNA (dsDNA) substrate with a hypoxanthine base (I) in one strand for hAAG recognition and an uracil (U) base in the other strand for UDG recognition, whose cleavage by APE1 generates two corresponding primers. The resultant two primers can hybridize with their respective circular templates to initiate RCA, resulting in the incorporation of multiple Cy3-dCTP and Cy5-dGTP nucleotides into the amplified products. After magnetic separation and exonuclease cleavage, the Cy3 and Cy5 fluorescent molecules in the amplified products are released into the solution and subsequently quantified by total internal reflection fluorescence (TIRF)-based single-molecule detection, with Cy3 indicating the presence of hAAG and Cy5 indicating the presence of UDG. This strategy greatly increases the number of fluorescent molecules per concatemer through the introduction of RCA-driven encoding of different fluorescent molecules, without the requirement of any specially labeled detection probes for simultaneous detection. Due to the high amplification efficiency of RCA and the high signal-to-ratio of single-molecule detection, this method can achieve a detection limit of 6.10 × 10 U mL for hAAG and 1.54 × 10 U mL for UDG. It can be further applied for simultaneous detection of multiple DNA glycosylases in cancer cells at the single-cell level and the screening of DNA glycosylase inhibitors, holding great potential in early clinical diagnosis and drug discovery.
DNA修复酶（如DNA糖基化酶）在DNA损伤修复中起关键作用，其水平异常与多种疾病相关。在此，我们基于单分子检测与滚环扩增（RCA）驱动的不同荧光分子编码相结合，开发了一种同时检测多种DNA修复酶的灵敏方法。我们使用人烷基腺嘌呤DNA糖基化酶（hAAG）和尿嘧啶DNA糖基化酶（UDG）作为目标分析物。我们设计了一种双功能双链DNA（dsDNA）底物，其中一条链含有次黄嘌呤碱基（I）用于hAAG识别，另一条链含有尿嘧啶（U）碱基用于UDG识别，其被APE1切割后产生两条相应的引物。所得的两条引物可与各自的环状模板杂交以启动RCA，从而导致多个Cy3-dCTP和Cy5-dGTP核苷酸掺入扩增产物中。经过磁分离和核酸外切酶切割后，扩增产物中的Cy3和Cy5荧光分子被释放到溶液中，随后通过基于全内反射荧光（TIRF）的单分子检测进行定量，Cy3指示hAAG的存在，Cy5指示UDG的存在。该策略通过引入RCA驱动的不同荧光分子编码，大大增加了每个串联体中荧光分子的数量，无需任何专门标记的检测探针即可进行同时检测。由于RCA的高扩增效率和单分子检测的高信噪比，该方法对hAAG的检测限可达6.10×10 U/mL，对UDG的检测限可达1.54×10 U/mL。它可进一步应用于单细胞水平癌细胞中多种DNA糖基化酶的同时检测以及DNA糖基化酶抑制剂的筛选，在早期临床诊断和药物发现方面具有巨大潜力。
