Detection of Cancer Marker Flap Endonuclease 1 Using One-Pot Transcription-Powered Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a Signal Expansion
Detection of Cancer Marker Flap Endonuclease 1 Using One-Pot Transcription-Powered Clustered Regularly Interspaced Short Palindromic Repeat/Cas12a Signal Expansion
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使用一锅转录驱动的簇状规则间隔短回文重复/Cas12a 信号扩展检测癌症标记物瓣核酸内切酶 1
DOI：
10.1021/acs.analchem.2c03054
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发表时间：
2022
期刊：
影响因子：
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通讯作者：
Zhuo Tang
中科院分区：
文献类型：
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作者：
Sheng Ding;Yinghua Wei;Gangyi Chen;Feng Du;Xin Cui;Xin Huang;Yi Yuan;Juan Dong;Zhuo Tang
FEN1作为DNA复制和基因组稳定的关键功能蛋白，已被认为是多种癌症的生物标志物和药物靶点。我们报道了一个转录驱动的规则间隔短回文重复序列(CRISPR)/Cas12a信号扩展平台，用于快速和灵敏地检测FEN1。在该方法中，FEN1对探针的切割产生了一个自由的5‘折叠单链DNA，它可以与单链T7启动子模板杂交并触发延伸。然后，从扩展模板转录的CRISPR引导RNA(CrRNA)激活Cas12a的侧枝DNA酶活性，从猝灭的DNA信号探针中释放荧光团，报告FEN1检测结果。通过与其他修复相关蛋白的比较，验证了FEN1的高度特异性。检测下限(LOD)可低至0.03亩，足以检测生物样品中的FEN1活性。此外，利用该平台还成功地实现了FEN1的抑制实验，证明了该平台在抑制剂筛选中的潜力。总之，本研究为FEN1活性分析提供了一种新的生物传感器，并为基于CRISPR的非核酸靶标生物传感器的发展提供了新的见解。
As a critical functional protein in DNA replication and genome stability, flap endonuclease 1 (FEN1) has been considered a promising biomarker and druggable target for multiple cancers. We report here a transcription-powered clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a signal expansion platform for rapid and sensitive detection of FEN1. In this method, the probe cleavage by FEN1 generated a free 5′ flap single-stranded DNA which could hybridize with the single-stranded T7 promoter-bearing template and trigger the extension. Then, the CRISPR guide RNA (crRNA) transcribed from the extended template activated the collateral DNase activity of Cas12a, releasing the fluorophore from the quenched DNA signal probe to report the FEN1 detection result. The high specificity for FEN1 was validated by comparing with other repair-relevant proteins. The limit of detection (LOD) could be as low as 0.03 mU, which is sensitive enough to detect the FEN1 activity in biological samples. In addition, the inhibition assay of FEN1 was also successfully achieved with this platform, proving its potential in inhibitor screening. In summary, this study provides a novel biosensor for FEN1 activity analysis and provides new insights into the development of CRISPR-based biosensors for non-nucleic acid targets.