Thermus thermophilus Argonaute-based signal amplifier for highly sensitive and specific microRNA detection

The prokaryote-derived gene defense system as a new generation of nucleic acid detection tool exhibits impressive performance in the field of molecular diagnosis. Prokaryotic Argonaute (Ago) is a CRISPR-associated protein that is guided by a short DNA (gDNA) and then efficiently cleaves gDNA-complementary nucleic acids and presents unique characteristics that are different from the CRISPR/Cas system. However, the application of Ago in biosensing is still relatively scarce, and many properties of Ago need to be further clarified. In this study, we aim to systematically explore the properties of Thermus thermophilus Argonaute (TtAgo), including the dependence of TtAgo activity on guide DNA (gDNA) length, substrates' length, and the position of gDNA complementary region on the substrate. Based on these properties, we constructed an exonuclease III-assisted target-recycled amplification system (exoAgo) for sensitive miRNA detection. The result showed that exoAgo can be used for miRNA profiling with a detection limit of 12.2 pM and single-base-resolution and keep good performance for the detection of complex samples, which indicates that Ago has great application potential in the detection of nucleic acids. In conclusion, this study will provide guidance for further development and utilization of Ago in the field of biosensing.