Clinical Medical College & Affiliated Hospital, Chengdu University, Chengdu, 610041, PR China.
Natural Products Research Center, Chengdu Institute of Biology, Chinese Academy of Science, Chengdu, 610041, PR China.
Anal Chim Acta. 2024 Feb 22;1291:342212. doi: 10.1016/j.aca.2024.342212. Epub 2024 Jan 3.
As an essential protein in DNA repair, apurinic/apyrimidinic endonuclease 1 (APE1) plays multiple critical functions in maintaining homeostasis, making it a significant biomarker and therapeutic target for many disorders. Here, we describe a simple method to detect APE1 based on the Releasing-Extension-Signal amplification Test (REST) strategy that leverages the dsDNA as the activator to fully unlock the trans-cleavage activity of CRISPR/Cas12a. This assay provides a rapid and specific APE1 detection with a detection limit down to 1.05 × 10 U/mL. We also combined this method with an automated pipetting platform and a microplate reader for high-throughput screening of potential inhibitors of APE1. Besides, by changing the modification on the probe, the REST strategy was easily repurposed to detect various DNA glycosylases. Taken together, the simplicity and robustness of the method offer a new choice for APE1 detection and inhibitor screening, showing great potential in practical use. Furthermore, the REST strategy devised in this study provides a new example of applying CRISPR/Cas12a signal amplifier to non-nucleic acid biosensing and inhibitor screening, which broadens the CRISPR-Dx toolbox.
作为 DNA 修复的必需蛋白，脱嘌呤/脱嘧啶核酸内切酶 1（APE1）在维持内稳态方面发挥着多种关键作用，使其成为许多疾病的重要生物标志物和治疗靶点。在这里，我们描述了一种基于释放-扩展-信号放大检测（REST）策略的简单 APE1 检测方法，该策略利用双链 DNA 作为激活剂，充分解锁 CRISPR/Cas12a 的转切割活性。该检测方法具有快速、特异性高的特点，检测限低至 1.05×10 U/mL。我们还将该方法与自动化移液平台和微孔板读数仪相结合，用于高通量筛选 APE1 的潜在抑制剂。此外，通过改变探针的修饰，REST 策略可以很容易地重新用于检测各种 DNA 糖苷酶。总之，该方法的简单性和稳健性为 APE1 检测和抑制剂筛选提供了新的选择，在实际应用中具有很大的潜力。此外，本研究设计的 REST 策略为将 CRISPR/Cas12a 信号放大器应用于非核酸生物传感和抑制剂筛选提供了新的范例，拓宽了 CRISPR-Dx 工具包。
