Introducing An Argonaute-facilitated Isothermal Amplification Technology

Abstract 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°C, 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°C. 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 low-copy 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).