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Thermophilic Argonaute proteins (Agos) have been shown to utilize small DNA guides for cleaving complementary DNA in vitro, which shows great potential for nucleic acid detection. In this study, we explored mesophilic Agos for the detection of small molecule by cooperating with allosteric transcription factors (aTFs). Two Agos from mesophilic bacteria,  Paenibacillus borealis  ( Pb Ago) and  Brevibacillus laterosporus  ( Bl Ago), showed nuclease activity for single-stranded DNA at moderate temperatures (37 °C) by using 5′-phosphorylated and 5′-hydroxylated DNA guides. Both Agos perform programmable cleavage of double-stranded DNA, especially in AT-rich regions of plasmid. Furthermore, we developed a simple and low-cost p-hydroxybenzoic acid detection method based on DNA-guided DNA cleavage of Agos and the allosteric effect of HosA, which expands the potential application of small molecule detection by Agos.

Characterization of Argonaute nucleases from mesophilic bacteria Paenibacillus borealis and Brevibacillus laterosporus