Department of Biomedical Engineering, City University of Hong Kong, 83 Tat Chee Avenue, Hong Kong Special Administrative Region, China.
Department of Biomedical Engineering, City University of Hong Kong, 83 Tat Chee Avenue, Hong Kong Special Administrative Region, China; City University of Hong Kong Shenzhen Research Institute, Shenzhen, China.
Biosens Bioelectron. 2023 Jan 15;220:114859. doi: 10.1016/j.bios.2022.114859. Epub 2022 Oct 31.
Flap endonuclease 1 (FEN1) is an endonuclease that specially removes 5' single-stranded overhang of branched duplex DNA (5' flap). While FEN1 is essential in various DNA metabolism pathways for preventing the malignant transformation of cells, an unusual expression of FEN1 is often associated with tumor progression, making it a potential biomarker for cancer diagnosis and treatment. Here we report a multimodal detection of FEN1 activity based on CRISPR/Cas12a trans-cleavage of single-strand DNA oligonucleotides (ssDNA). A dumbbell DNA structure with a 5' flap was designed, which can be cleaved by the FEN1 and the dumbbell DNA is subsequently ligated by T4 DNA ligase. The resulting closed duplex DNA contains a specific protospacer adjacent motif (PAM) that activates trans-cleavage of ssDNA after binding to CRISPR/Cas12a-crRNA. The trans-cleavage is activated only once and is independent to length or sequence of the ssDNA, which allows efficient signal amplification and multimodal signals such as fluorescence or cleaved connection between magnetic microparticles (MMPs) and polystyrene microparticles (PMPs) that alters solution turbidity after magnetic separation. In addition, by loading the particle solution into a microfluidic chip, unconnected PMPs escaping from a magnetic separator are amassed at the particle dam, enabling a visible PMP accumulation length proportional to the FEN1 activity. This multimodal detection is selective to FEN1 and achieves a low limit of detection (LOD) with only 40 min of reaction time. Applying to cell lysates, higher FEN1 activity was detected in breast cancer cells, suggesting a great potential for cancer diagnosis.
核酸内切酶 1（FEN1）是一种专门切除分支双链 DNA 5'单链突出物（5' 发夹）的内切酶。虽然 FEN1 在各种 DNA 代谢途径中对于防止细胞恶性转化至关重要，但 FEN1 的异常表达通常与肿瘤进展相关，使其成为癌症诊断和治疗的潜在生物标志物。在这里，我们报告了一种基于 CRISPR/Cas12a 对单链 DNA 寡核苷酸（ssDNA）的转切割的 FEN1 活性的多模式检测。设计了带有 5' 发夹的哑铃 DNA 结构，该结构可被 FEN1 切割，随后由 T4 DNA 连接酶连接哑铃 DNA。所得封闭双链 DNA 包含特定的原间隔邻近基序（PAM），在与 CRISPR/Cas12a-crRNA 结合后激活 ssDNA 的转切割。转切割仅被激活一次，与 ssDNA 的长度或序列无关，这允许高效的信号放大和多模式信号，例如荧光或连接磁微珠（MMPs）和聚苯乙烯微球（PMPs）之间的切割连接，在磁性分离后改变溶液浊度。此外，通过将粒子溶液加载到微流控芯片中，从磁分离器中逃脱的未连接的 PMP 聚集在粒子坝处，使得可见的 PMP 积累长度与 FEN1 活性成正比。这种多模式检测对 FEN1 具有选择性，并且仅需 40 分钟的反应时间即可实现低检测限（LOD）。应用于细胞裂解物，在乳腺癌细胞中检测到更高的 FEN1 活性，这表明其在癌症诊断方面具有很大的潜力。
