Key Laboratory of Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044, PR China.
Key Laboratory of Biorheological Science and Technology of Ministry of Education, State and Local Joint Engineering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing, 400044, PR China; Chongqing Key Laboratory of Bio-perception & Intelligent Information Processing, School of Microelectronics and Communication Engineering, Chongqing University, Chongqing, 400044, PR China.
Anal Chim Acta. 2021 Dec 15;1188:339185. doi: 10.1016/j.aca.2021.339185. Epub 2021 Oct 19.
N methyladenine (mA) modification of the FzD5 mRNA, an important post-transcriptional regulation in eukaryotes, is closely related to the occurrence and development of breast cancer. Here, we developed an ultra-sensitive biosensor based on MazF combining with cascaded strand displacement amplification (C-SDA) and CRISPR/Cas12a to detect mA FzD5 mRNA. MazF toxin protein is a vital component of the bacterial mazEF toxin-antitoxin system that is sensitive to mA RNA. Take advantage of it, the biosensor achieved antibody-independent and gene-specific detection for mA RNA. Moreover, compared with traditional amplification methods, the more efficient C-SDA and the CRISPR/Cas12a system with trans-cleavage activity gave the fluorescent biosensor an excellent sensitivity with the detection limit of 0.64 fM. In addition, MazF, as a new antibacterial target, was detected by the biosensor based on C-SDA and CRISPR/Cas12a with the detection limit of 1.127 × 10 U mL. More importantly, the biosensor has good performance in complex samples. Therefore, the biosensor is a potential tool in detecting mA FzD5 mRNA and MazF activity.
N6-甲基腺嘌呤（mA）修饰的 FzD5 mRNA 是真核生物中一种重要的转录后调控，与乳腺癌的发生和发展密切相关。在这里，我们开发了一种基于 MazF 的超灵敏生物传感器，结合级联链置换扩增（C-SDA）和 CRISPR/Cas12a 来检测 mA FzD5 mRNA。MazF 毒素蛋白是细菌 mazEF 毒素-抗毒素系统的重要组成部分，对 mA RNA 敏感。利用这一点，该生物传感器实现了对 mA RNA 的非抗体依赖和基因特异性检测。此外，与传统的扩增方法相比，更有效的 C-SDA 和具有转录切割活性的 CRISPR/Cas12a 系统赋予了荧光生物传感器优异的灵敏度，检测限低至 0.64 fM。此外，MazF 作为一种新的抗菌靶标，也可以通过基于 C-SDA 和 CRISPR/Cas12a 的生物传感器检测，检测限为 1.127×10 U mL。更重要的是，该生物传感器在复杂样本中具有良好的性能。因此，该生物传感器是检测 mA FzD5 mRNA 和 MazF 活性的潜在工具。
