Characterization and application of a thermophilic Argonaute from archaeon <i>Thermococcus thioreducens</i>
Mengjun Fang, Zhipeng Xu, Di Huang, Muhammad Naeem, Xiangcheng Zhu, Zhinan Xu, Zhinan Xu, Zhinan Xu
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 (TtrAgo) and its example of application in hepatitis B virus DNA detection. The results showed that TtrAgo 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, TtrAgo also possesses stepwise cleavage activity like PfAgo (Pyrococcus furiosus) and chopping activity toward double-stranded DNA similar to MjAgo (Methanocaldococcus jannaschii). This study increases our understanding of pAgos and expands the Ago-based DNA detection toolbox.