Tang Yue, Wang Xiao-Han, Xu Xu, Xin Lu-Sheng, Wang Xiu-Dan, Li Xin-Min
Institute of chronic diseases，Institute for Translational Medicine, The Affiliated Hospital of Qingdao University, School of Basic Medicine，College of Medicine, Qingdao University, Qingdao, 266073, China.
State Key Laboratory of Marine Food Processing & Safety Control, College of Food Science and Engineering, Ocean University of China, Qingdao, 266404, China.
Adv Sci (Weinh). 2025 Apr;12(13):e2406872. doi: 10.1002/advs.202406872. Epub 2025 Mar 8.
The recent article by Zhang et al. piqued the interest. An atom-modification-based strategy is reported to enhance the cleavage activity of TtAgo, improving its practicability in TtAgo-based nucleic acid testing. Specifically, the 2'-fluorine (2'F)-modified guide DNA (2'F-gDNA) shows significant enhancement in the cleavage activity of TtAgo on double-stranded (dsDNA) by increasing the melting temperature (Tm) and strengthening the binding affinity between 2'F-gDNA and the targeted dsDNA. These findings are considered important for both molecular diagnostics and gene editing. A careful review of the article, however, raises questions that merit further discussion. After comprehensively reviewing the cleavage mechanism and structure of TtAgo-gDNA-target ternary complexes, and thoroughly analyzing our results, it is believed that the increased Tm and binding affinity of 2'F-gDNA are not the primary factors that enhance cleavage activity, it is speculated that the 2'F modification at gDNA 3'-end likely influences the propagation step. The data suggest that several details need to be addressed to improve the robustness of 2'F-gDNA/TtAgo cleavage.
张等人最近的文章引起了人们的兴趣。据报道，一种基于原子修饰的策略可增强TtAgo的切割活性，提高其在基于TtAgo的核酸检测中的实用性。具体而言，2'-氟（2'F）修饰的引导DNA（2'F-gDNA）通过提高解链温度（Tm）并增强2'F-gDNA与靶向双链DNA（dsDNA）之间的结合亲和力，显著增强了TtAgo对双链DNA的切割活性。这些发现被认为对分子诊断和基因编辑都很重要。然而，仔细阅读这篇文章会引发一些值得进一步讨论的问题。在全面回顾TtAgo-gDNA-靶标三元复合物的切割机制和结构，并深入分析我们的结果后，我们认为2'F-gDNA的Tm升高和结合亲和力增加并非增强切割活性的主要因素，推测gDNA 3'-末端的2'F修饰可能影响延伸步骤。数据表明，需要解决几个细节问题以提高2'F-gDNA/TtAgo切割的稳健性。
