Thermus thermophilus Argonaute-Based Isothermal Amplification Assay for Ultrasensitive and Specific RNA Detection

Recent advances in prokaryotic Argonauteproteins (pAgos)as potentialgenome-editing tools have provided new insights into the developmentof pAgos-based nucleic acid detection platforms. However, pAgos-basedisothermal detection remains challenging. Here, we report a true isothermalamplification strategy, termed Thermus thermophilus Argonaute-based thermostable exponential amplification reaction(TtAgoEAR), to detect RNA with ultrasensitivity andsingle-nucleotide resolution at a constant temperature of 66 degrees C.We demonstrate the reliable detection of lncRNA, mRNA, and virus RNAwith attomolar sensitivity and that TtAgoEAR canbe applied to detect RNA targets in in cell lines, saliva, and tissues.We utilize this assay to distinguish pancreatic cancer cells carryingthe mutation from wild-type cells with as little as 2 ng of RNA material.We also show that TtAgoEAR is easily adaptable toa lateral-flow-based readout. These results demonstrate that TtAgoEAR has great potential to facilitate reliable andeasy RNA detection in point-of-care diagnosis and field analysis.

Automated summary

Recent advances in prokaryotic Argonaute proteins (pAgos) have provided new insights into the development of pAgos-based nucleic acid detection platforms. However, the current challenge lies in achieving isothermal detection using pAgos. This study introduces a true isothermal amplification strategy called Thermus thermophilus Argonaute-based thermostable exponential amplification reaction (TtAgoEAR), which allows for RNA detection with ultra-sensitivity and single-nucleotide resolution at a constant temperature of 66 degrees C. The results demonstrate the potential of TtAgoEAR for reliable and easy RNA detection in point-of-care diagnosis and field analysis.