Duplex-specific nuclease-resistant triple-helix DNA nanoswitch for single-base differentiation of miRNA in lung cancer cells.

In this work, a duplex-specific nuclease (DSN)-resistant triplex-helix DNA nanoswitch was designed for assays of single-base differentiation of the let-7a family in lung cancer cells. Initially, although a 10-bp duplex stem in the nanoswitch was cleaved to pieces, a 10-bp triplex stem was resistant to DSN. Consequently, a triple-stranded DNA structure resistant to DSN was obtained. The pH-dependent formation of the triplex structure then produced the pH-related nanoswitch/miRNA hybrid, and the metastable nanoswitch generated an obvious signal increase at pH 6.8. Surprisingly, the pH condition at 6.8 for the best nanoswitch/miRNA hybrid is consistent with the optimal DSN catalysis, which paves the way for a first-rank DSN signal amplification (DSNSA) strategy for the single-base selective capacity of the homologous let-7a family with a limit of detection of 0.26 pM. The cyclic strategy based on the DSN-mediated triplex-helix DNA nanoswitch was verified in lung cancer cell samples and exhibited better discriminatory ability without user-unfriendly nucleotide modification or extra probe-mediated assistance, showing excellent potential for application in biomedical sensing and clinical diagnosis. Graphical abstract Based on the discovery that a triple-helix DNA nanoswitch is resistant to DSN and that the nanoswitch/miRNA hybridization was pH-related, pH at 6.8, which is suitable for the optimal nanoswitch/miRNA hybrid and DSN catalysis, reinforced the DSNSA strategy for the single-base selective capacity of the homologous let-7a family with a limit of detection of 0.26 pM.