Argonaute with stepwise endonuclease activity promotes specific and multiplex nucleic acid detection.

Argonaute proteins (Agos) from thermophiles function as endonucleases via guide-target base-pairing cleavage for host defense. Since guides play a key role in regulating the catalytic specificity of Agos, elucidating its underlying molecular mechanisms would promote the application of Agos in the medical sciences. Here, we reveal that an Ago from Pyrococcus furiosus (PfAgo) showed a stepwise endonuclease activity, which was demonstrated through a double-stranded DNA cleavage directed by a single guide DNA (gDNA) rather than a canonical pair of gDNAs.

Argonaute integrated single-tube PCR system enables supersensitive detection of rare mutations.

Technological advances in rare DNA mutations detection have revolutionized the diagnosis and monitoring of tumors, but they are still limited by the lack of supersensitive and high-coverage procedures for identifying low-abundance mutations. Here, we describe a single-tube, multiplex PCR-based system, A-Star, that involves a hyperthermophilic Argonaute from Pyrococcus furiosus (PfAgo) for highly efficient detection of rare mutations beneficial from its compatibility with DNA polymerase. This novel technique uses a specific guide design strategy to allow PfAgo selective cleavage with single-nucleotide resolution at 94°C, thus mostly eliminating wild-type DNA in the denaturation step and efficiently amplifying rare mutant DNA during the PCR process.