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Updated: May 8, 2026
Establishment of a High-throughput Setup for Screening Small Molecules That Modulate c-di-GMP Signaling in Pseudomonas aeruginosa
Published on: June 30, 2016
Integrated directed evolution and semirational design of Pyrococcus furiosus Argonaute for ultrasensitive
Yanyan Chai1, Bosheng Zhou1, Wanqi Chen2
1State Key Laboratory of Agricultural Microbiology, Huazhong Agricultural University, Wuhan 430070, China; College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Hubei Hongshan Laboratory, Wuhan 430070, China.
Abstract:
In this study, we report an integrated strategy combining directed evolution and semirational design to engineer a high-performance Pyrococcus furiosus Argonaute (PfAgo) variant. The optimized variant exhibited an 8.85-fold increase in catalytic efficiency, substantially enhanced soluble expression, and improved tolerance to elevated salt and metal ion conditions, enabling sensitive nucleic acid detection with a detection limit in the picomolar range. Mechanistic analyses suggested that these improvements may arise from reinforcement of the dimerization interface to stabilize the enzyme's active state, optimization of the catalytic center, and the introduction of charge-shielding salt bridges. Finally, a biosensing platform based on the engineered PfAgo achieved a detection limit of 10 fmol l-1 for tetracycline, demonstrating its strong potential for sensitive and robust detection. This work provides a generalizable framework for enhancing Argonaute performance and advances their application in field-deployable diagnostics across clinical, environmental, and food safety settings.
