Gao, F.; Han, C.

Argonaute-facilitated targeting, which is an enzyme-mediated, high-fidelity, and highly efficient base pairing process, can be repurposed for upgrading PCR technology. An Argonaute protein derived from thermophilic Caloramator sp (CalAgo) cleaved target dsDNA in the presence of Tte UvrD helicase at 65, suggesting dmCalAgo (a nuclease-inactive mutant) binding to target dsDNA in the same condition. Based on this, we designed an Argonaute-facilitated isothermal PCR platform (Isothermal Ago-PCR) through synergy of dmCalAgo, Tte UvrD helicase, and Bst DNA polymerase. Isothermal Ago-PCR realized amplification of template at 65. The upgrade resides in replacing primer annealing with Argonaute-facilitated targeting. Hence, Isothermal Ago-PCR not merely eliminates the reliance on complex primer design and sophisticated instruments but also achieves high-fidelity and highly efficient amplification. This platform enabled detection of lowcopy templates (as few as 3 copies per reaction) within 30 minutes, achieving comparable sensitivity to qPCR while demonstrating superior amplification kinetics. This platform also demonstrated compatibility with common thermal maintenance tools. Thus, Isothermal Ago-PCR has the potential to replace qPCR with broad applicability. Details refer to our patent (China Patent CN116479095A).