Moderate-temperature DNA cleavage activity of TtAgo activated by dCTP/TthSSB for one-step isothermal microRNAs detection.

Thermus thermophilus Argonaute (TtAgo) is a DNA-guided endonuclease promising for gene editing and molecular diagnostics. However, its high-temperature dependence (65-85 °C) restricts its utility in moderate-temperature scenarios. Here we show that combining deoxycytidine triphosphate (dCTP) with the single-stranded DNA-binding protein from T. thermophilus (TthSSB) enables robust TtAgo activity within 37-60 °C. Mechanistic studies reveal that dCTP increases TtAgo's conformational flexibility, while TthSSB promotes substrate recruitment, synergistically driving efficient DNA cleavage. Leveraging this mechanism, we develop ERCB-TtAgo (exponential amplification reaction by dCTP/TthSSB-activated TtAgo), a one-step isothermal diagnostic platform for microRNA detection. It achieves femtomolar sensitivity within 40 min and accurately distinguishes hepatocellular carcinoma patients from controls in a 151-sample clinical cohort, matching RT‑qPCR performance. Our work not only surmounts a key limitation of TtAgo but also offers an approach for modulating nuclease activity, paving the way for intelligent biosensing platforms with broad applicability.