School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing, 210023, China.
Department of Hepatobiliary Surgery, The Second Affiliated Hospital of Nantong University, Nantong, 226000, China.
Anal Chim Acta. 2021 Jun 22;1165:338478. doi: 10.1016/j.aca.2021.338478. Epub 2021 Apr 4.
Transcription factors (TFs) are critical proteins that regulate the expression of genes, and the abnormal change of TFs levels is directly related to physical dysfunctions. Herein, we developed a clustered regularly interspaced short palindromic repeats (CRISPR)-based biosensor for the measurement of TFs level with the assistance of exonuclease protection assay. A dsDNA (activator) with the ability to activate Cas12a was engineered to contain TFs binding domain, and the binding between TFs and the activator can protect the dsDNA from being digested by exonuclease III (Exo III). The reserved activator then triggered a CRISPR/Cas12a reporting reaction to produce fluorescent signal for detection. In the detection of nuclear factor-kappa B (NF-κB) p50 subunit, the limit of detection of 0.2 pM and limit of quantification of 0.6 pM were obtained respectively, and the performance of this biosensor has been challenged by cell nucleoprotein extracts. Additionally, this method can be applied in the screening and evaluation of TFs inhibitors, calculating the IC50 of oridonin. Integrating merits including high sensitivity, low cost, and good portability, this method may enrich the arsenal for TFs-related applications.
转录因子 (TFs) 是调控基因表达的关键蛋白，其水平的异常变化与生理功能障碍直接相关。在此，我们开发了一种基于成簇规律间隔短回文重复序列 (CRISPR) 的生物传感器，通过外切酶保护分析来测量 TFs 水平。设计了一种具有激活 Cas12a 能力的双链 DNA（激活剂），包含 TFs 结合域，TFs 与激活剂的结合可以保护 dsDNA 免受外切酶 III（Exo III）的消化。保留的激活剂随后触发 CRISPR/Cas12a 报告反应，产生荧光信号进行检测。在核因子-κB (NF-κB) p50 亚基的检测中，分别获得了 0.2 pM 的检测限和 0.6 pM 的定量限，并且该生物传感器的性能已通过细胞核蛋白提取物进行了挑战。此外，该方法可应用于 TFs 抑制剂的筛选和评价，计算冬凌草甲素的 IC50。该方法整合了高灵敏度、低成本和良好便携性等优点，可能会丰富 TFs 相关应用的工具库。
