A toehold mediated feedback rolling circle amplification with exponential signal amplification enables label-free nucleic acid sensing with high sensitivity and specificity

Abstract

Rolling circle amplification (RCA) has been widely used for nucleic acid and other biomarkers analysis, however, the limited amplification efficiency and low specificity in traditional RCA hinder its further application. Here, a toehold mediated feedback rolling circle amplification (TMFRCA) strategy with exponential signal amplification was proposed for label-free nucleic acids sensing. The rational engineered TH and CT ensured exponential signal amplification by utilizing the feedback of amplicons to the reactant TH for circularly initiating new RCA. Introduction of toehold strand displacement and G-quadruplex (G4) dimers in RCA reaction allowed enhanced specific recognition and further improved fluorescence signal due to higher quantum yield of G4 dimers/ThT. Taking classic swine fever virus (CSFV) as model target, this TMFRCA method exhibited great sensitivity and high specificity with a limit of detection down to 0.29 fM and enabled to discriminate target with single base differences. In addition, the proposed TMFRCA could be used for detecting CSFV RNA in actual samples with great accuracy comparable to RT-qPCR, and possessed great universality for detecting nucleic acid without length limitation. We believe this method can promote the development of nucleic acid signal amplification and be an effective tool for nucleic acid sensing-based fundamental research and clinical diagnosis.