Novel nucleic acid detection based on the guide-independent nuclease activity of TbAgo

In addition to the well-characterized programmable nucleic acid cleavage activity, prokaryotic Argonaute proteins (pAgos) possess a guide-independent nuclease activity capable of degrading nucleic acids into small fragments in the absence of exogenous guide strands, a function commonly referred to as "chopping", which remains poorly understood. Here, we report the robust chopping activity of Thermus brockianus Argonaute (TbAgo) and leverage this activity to develop chop-NAD (chopping-based Nucleic Acid Detection), a minimal guide-free platform for sequence-specific nucleic acid sensing. When integrated with loop-mediated isothermal amplification (LAMP), chop-NAD enables sensitive, specific, and multiplexed detection of Bordetella pertussis, Bordetella parapertussis, and Mycoplasma pneumoniae in a single tube, with detection limits of 2.38 genome equivalents (gEq)/μL, 2.04 gEq/μL, and 59.7 copies/μL, respectively. Furthermore, a paraffin-encapsulated, lyophilized one-pot format facilitates contamination-resistant, sample-to-answer testing, showing 100 % concordance with clinical PCR results. This one-pot chop-NAD platform offers reduced reagent costs, simplified assay adaptation, improved biosafety, and a streamlined workflow, collectively establishing it as a versatile and scalable diagnostic platform with broad potential for point-of-care testing.