Base excision repair mediated cascading triple-signal amplification for the sensitive detection of human alkyladenine DNA glycosylase
Base excision repair mediated cascading triple-signal amplification for the sensitive detection of human alkyladenine DNA glycosylase
复制标题
碱基切除修复介导的级联三信号放大用于灵敏检测人烷基腺嘌呤 DNA 糖基化酶
作者：
Huige Zhang;Lili Wang;Yi Xie;Xianwei Zuo;Hongli Chen;Xingguo Chen
DNA糖基酶(DG)在DNA损伤修复中起着重要作用，DG活性的异常与多种人类疾病有关。因此，DG活性的检测对生物医学研究和临床诊断具有重要意义。在此，我们建立了一种基于碱基切除修复(BER)介导级联三重信号放大的简便荧光方法，用于DG的灵敏检测。人烷基腺嘌呤DNA糖基酶(HAAG)的存在可以引起脱氧肌苷酶IV(Endo IV)对底物的切割，导致DNA底物的断裂。裂解的DNA底物既是一种引物，也是一种模板，启动链置换扩增(SDA)以释放引物。所释放的引物还可以进一步与环状模板结合，引发指数型引物世代滚环扩增(PG-RCA)反应，产生大量的引物。SDA和PG-RCA反应产生的引物可以诱导信号探针随后的循环裂解，导致荧光信号的产生。该方法利用了三重信号扩增的高效率和单尿嘧啶修复抑制非特异性扩增所产生的低本底信号，具有0.026 U m L−1的低检测下限和4个数量级的大动态范围。此外，该方法具有操作简单、成本低等显著优点，可以进一步定量HeLa细胞提取液中的HAAG活性，在临床诊断和生物医学研究中具有很大的潜力。
DNA glycosylase (DG) plays a significant role in repairing DNA lesions, and the dysregulation of DG activity is associated with a variety of human pathologies. Thus, the detection of DG activity is essential for biomedical research and clinical diagnosis. Herein, we develop a facile fluorometric method based on the base excision repair (BER) mediated cascading triple-signal amplification for the sensitive detection of DG. The presence of human alkyladenine DNA glycosylase (hAAG) can initiate the cleavage of the substrate at the mismatched deoxyinosine site by endonuclease IV (Endo IV), resulting in the breaking of the DNA substrate. The cleaved DNA substrate functions as both a primer and a template to initiate strand displacement amplification (SDA) to release primers. The released primers can further bind to a circular template to induce an exponential primer generation rolling circle amplification (PG-RCA) reaction, producing a large number of primers. The primers that resulted from the SDA and PG-RCA reaction can induce the subsequent recycling cleavage of signal probes, leading to the generation of a fluorescence signal. Taking advantage of the high amplification efficiency of triple-signal amplification and the low background signal resulting from single uracil repair-mediated inhibition of nonspecific amplification, this method exhibits a low detection limit of 0.026 U mL−1 and a large dynamic range of 4 orders of magnitude for hAAG. Moreover, this method has distinct advantages of simplicity and low cost, and it can further quantify the hAAG activity from HeLa cell extracts, holding great potential in clinical diagnosis and biomedical research.