Comment on: “Atom‐Modified gDNA Enhances Cleavage Activity of TtAgo Enabling Ultrasensitive Nucleic Acid Testing”

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.