Characterization and application of a thermophilic Argonaute from archaeon Thermococcus thioreducens

Abstract Prokaryotic Argonaute proteins (pAgos) play an important role in host defense against invading genetic elements. The functional diversities make pAgos very promising in development of novel nucleic acid manipulation tools and attract increasing attentions. Here, we reported the in vitro characterization of an Argonaute protein from archaeon Thermococcus thioreducens ( Ttr Ago) and its example of application in hepatitis B virus DNA detection. The results showed that Ttr Ago functions as a programmable DNA endonuclease by utilizing both short 5′‐phosphorylated and 5′‐hydroxylated single‐stranded DNA guides, and presents high efficiency and accuracy at optimal temperatures ranging from 75°C to 95°C. In addition, Ttr Ago also possesses stepwise cleavage activity like Pf Ago ( Pyrococcus furiosus ) and chopping activity toward double‐stranded DNA similar to Mj Ago ( Methanocaldococcus jannaschii ). This study increases our understanding of pAgos and expands the Ago‐based DNA detection toolbox.