College of Chemistry, Chemical Engineering and Materials Science, Shandong Normal University, Jinan 250014, China.
School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China.
Analyst. 2023 Jun 12;148(12):2732-2738. doi: 10.1039/d3an00604b.
The structure-specific endonuclease flap endonuclease 1 (FEN1) is an essential functional protein in DNA replication and genome stability, and it has been identified as a promising biomarker and drug target for multiple cancers. Herein, we develop a target-activated T7 transcription circuit-mediated multiple cycling signal amplification platform for monitoring FEN1 activity in cancer cells. In the presence of FEN1, the flapped dumbbell probe is cleaved to generate a free 5' flap single-stranded DNA (ssDNA) with the 3'-OH terminus. The ssDNA can hybridize with the T7 promoter-bearing template probe to trigger the extension with the aid of Klenow fragment (KF) DNA polymerase. Upon the addition of T7 RNA polymerase, an efficient T7 transcription amplification reaction is initiated to produce abundant single-stranded RNAs (ssRNAs). The ssRNA can hybridize with a molecular beacon to form an RNA/DNA heteroduplex that can be selectively digested by DSN to generate an enhanced fluorescence signal. This method exhibits good specificity and high sensitivity with a limit of detection (LOD) of 1.75 × 10 U μL. Moreover, it can be applied for the screening of FEN1 inhibitors and the monitoring of FEN1 activity in human cells, holding great potential in drug discovery and clinical diagnosis.
结构特异性内切酶核酸内切酶 1（FEN1）是 DNA 复制和基因组稳定性所必需的功能性蛋白，它已被确定为多种癌症有前途的生物标志物和药物靶标。在此，我们开发了一种靶激活的 T7 转录电路介导的多重循环信号放大平台，用于监测癌细胞中的 FEN1 活性。在 FEN1 的存在下，被劈开的哑铃探针被切割，生成带有 3'-OH 末端的游离 5' 发夹单链 DNA（ssDNA）。ssDNA 可以与带有 T7 启动子的模板探针杂交，在 Klenow 片段（KF）DNA 聚合酶的辅助下引发延伸。加入 T7 RNA 聚合酶后，会启动有效的 T7 转录扩增反应，产生大量的单链 RNA（ssRNA）。ssRNA 可以与分子信标杂交，形成 RNA/DNA 异源双链体，可被 DSN 选择性消化，产生增强的荧光信号。该方法具有良好的特异性和高灵敏度，检测限（LOD）为 1.75×10 U μL。此外，它还可用于筛选 FEN1 抑制剂和监测人细胞中的 FEN1 活性，在药物发现和临床诊断中具有很大的潜力。
