Signal-Amplified Detection of the Tumor Biomarker FEN1 Based on Cleavage-Induced Ligation of a Dumbbell DNA Probe and Rolling Circle Amplification,Analytical Chemistry

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Signal-Amplified Detection of the Tumor Biomarker FEN1 Based on Cleavage-Induced Ligation of a Dumbbell DNA Probe and Rolling Circle Amplification
Analytical Chemistry ( IF 6.7 ) Pub Date : 2021-02-02 , DOI: 10.1021/acs.analchem.0c05275
Bingzhi Li ₁ , Anqi Xia ₁ , Siying Xie ₁ , Lei Lin ₂ , Zhirun Ji ₁ , Tiying Suo ₁ , Xing Zhang ₁ , He Huang ₁

Affiliation

Flap endonuclease 1 (FEN1), an endogenous nuclease with the ability to cleave the 5′ overhang of branched dsDNA, is of significance in DNA replication and repair. The overexpression of FEN1 is common in cancer because of the ubiquitous upregulation of DNA replication; thus, FEN1 has been recognized as a potential biomarker in oncological investigations. However, few analytical methods targeting FEN1 with high sensitivity and simplicity have been developed. This work developed a signal-amplified detection of FEN1 based on the cleavage-induced ligation of a dumbbell DNA probe and rolling circle amplification (RCA). A flapped dumbbell DNA probe (FDP) was rationally designed with a FEN1 cleavable flap at the 5′ end. The cleavage generated a nick site with juxtaposed 5′ phosphate and 3′ hydroxyl ends, which were linkable by T4 DNA ligase to form a closed dumbbell DNA probe (CDP) with a circular conformation. The CDP functioned as a template for RCA, which produced abundant DNA that could be probed using SYBR Green I. The highly sensitive detection of FEN1 with a limit of detection of 15 fM was achieved, and this method showed high specificity, which enabled the quantification of FEN1 in real samples. The inhibitory effects of chemicals on FEN1 were also evaluated. This study represents the first attempt to develop an FEN1 assay that involves signal amplification, and the novel biosensor method enriches the tools for FEN1-based diagnostics.

中文翻译：

基于切割诱导的哑铃DNA探针连接和滚环扩增的肿瘤生物标记物FEN1的信号放大检测。

瓣内切核酸酶1（FEN1）是一种内源性核酸酶，具有裂解分支dsDNA 5'突出端的能力，在DNA复制和修复中具有重要意义。由于DNA复制普遍存在上调，因此FEN1的过表达在癌症中很常见。因此，FEN1已被认为是肿瘤学研究中潜在的生物标志物。但是，很少有针对FEN1的分析方法具有高灵敏度和简便性。这项工作基于裂解诱导的哑铃形DNA探针的连接和滚环扩增（RCA），开发了FEN1的信号放大检测。拍打哑铃DNA探针（FDP）经过合理设计，在5'端带有FEN1可裂解瓣。切割产生了一个切口位点，并带有并列的5'磷酸酯和3'羟基末端，它们可通过T4 DNA连接酶连接形成具有环状构象的闭合哑铃DNA探针（CDP）。CDP用作RCA的模板，可产生大量DNA，可使用SYBR Green I进行探测。实现了FEN1的高灵敏度检测，检测限为15 fM，并且该方法显示出高特异性，可进行定量实际样本中的FEN1的百分比。还评估了化学品对FEN1的抑制作用。这项研究代表了开发涉及信号放大的FEN1检测的首次尝试，并且新颖的生物传感器方法丰富了基于FEN1的诊断工具。实现了FEN1的高灵敏度检测，检测限为15 fM，并且该方法显示出高特异性，从而可以对真实样品中的FEN1进行定量。还评估了化学品对FEN1的抑制作用。这项研究代表了开发涉及信号放大的FEN1检测的首次尝试，并且新颖的生物传感器方法丰富了基于FEN1的诊断工具。实现了FEN1的高灵敏度检测，检测限为15 fM，并且该方法显示出高特异性，从而可以对真实样品中的FEN1进行定量。还评估了化学品对FEN1的抑制作用。这项研究代表了开发涉及信号放大的FEN1检测的首次尝试，并且新颖的生物传感器方法丰富了基于FEN1的诊断工具。

更新日期：2021-02-16

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