The stepwise endonuclease activity of a thermophilic Argonaute protein

Thermophilic Argonaute proteins (Agos) can function as endonucleases via specific guide-target base-pairing cleavage for host defense. The ability to cleave target DNA sequences at any arbitrary sites endows them with reprogramed DNA capacity. Here, we identify that an Ago from the hyperthermophilic archaeon Pyrococcus furiosus (PfAgo) shows a stepwise endonuclease activity, which is demonstrated by the double strand DNA cleavage directed by a single guide DNA rather than canonical one pair of guide DNAs. We reveal that the cleavage products with 5-phosphorylated ends can used as the renewed guide which is capable to induce next round of cleavage to complementary sequences of target DNA. By combining the PfAgo stepwise endonuclease activity followed by target DNA amplification, we establish a rapid and specific platform for the unambiguously multiplex gene detection, termed RADAR (Renewed-gDNA Assisted DNA-cleavage by Argonaute). In the end, RADAR was applied to distinguish of human papillomavirus of serotypes in patient samples in a single reaction, suggesting that our technique would be adopted for diagnosing application.