Structure-Based Rational Design of TcAgo from Thermogladius calderae

Thermophilic prokaryotic Argonaute proteins (pAgos) have emerged as powerful tools for nucleic acid manipulation, with applications in nucleic acid detection, and DNA assembly. However, their strong dependence on high-temperature catalytic activity limits their utility under moderate conditions. TcAgo, a thermophilic Argonaute nuclease from Thermogladius calderae, exhibits efficient DNA-guided target DNA cleavage above 80 °C, yet its structural basis and catalytic mechanism remain unclear. In this study, we attempted to analyze the structure of the TcAgo ternary complex and performed rational engineering based on its structure and characteristics of cold-adapted enzymes. A mutant, mTcAgo (K574G, D577G), was obtained with enhanced activity at moderate temperatures. Compared with the wild type, mTcAgo exhibited significantly improved cleavage activity toward both DNA and RNA targets at 37 °C. It utilized multiple guide types, including 5'OH- and 5'P-modified DNA and RNA guides, with a preference for 5'P-gDNA. mTcAgo displayed optimal activity at pH 7-8, broad salt tolerance, and an extended catalytic temperature range from 37 °C to 95 °C. Notably, it retained high activity after incubation at 90 °C, with a melting temperature of ~88 °C, and efficiently cleaved GC-rich targets under low Mg²⁺ conditions. These results demonstrate that rational cold-adaptation engineering can expand the functional temperature range of thermophilic pAgos, providing a promising strategy for developing versatile nucleic acid tools.