Short-probe-based duplex-specific nuclease signal amplification strategy enables imaging of endogenous microRNAs in living cells with ultrahigh specificity.

Specific nucleic acids amplification at a constant and mild temperature is important for imaging assay of endogenous microRNAs (miRNAs) in living cells. Duplex-specific nuclease (DSN) is attractive in one-step isothermal assay of miRNA; however, its intrinsic limitations of low amplification specificity and high reaction temperature greatly restrict the application scope. Herein, we present a short-probe-based DSN signal amplification (spDSNSA) strategy enabling analysis of miRNAs at body temperature with significantly high specificity. From systematic investigation of amplification reaction on different types of DNA probes, we revealed that the annealing rate between probe and target miRNA greatly affects the dynamics of amplification process. By simply shortening the length of DNA probe, the spDSNSA remarkably improved specificity without loss of amplification efficiency at 37 °C. As a proof-of-concept, let-7a was sensitively detected by spDSNSA with a limit of detection down to 30 p.M., and a specificity 102 ‒ 104 folds higher than those of traditional DSNSA methods. The analysis of the let-7a in the lysates of A549 human lung cancer cells and BEAS-2B human lung normal bronchial epithelial cells exhibited well agreement with rt-qPCR method. Furthermore, the endogenous let-7a in A549 and BEAS-2B living cells was clearly imaged without damaging the original morphology of cells. The method provide a facile idea for extension of DNS related signal amplification strategies in the application in living cells and POCTs, and would pose a great impact on the development of simple and rapid molecular diagnostic applications for short oligonucleotides.