Target-initiated labeling for the dual-amplified detection of multiple microRNAs

Herein we exploited a novel target-initiated labeling strategy for the multiplex detection of microRNAs (miRNAs) by coupling duplex-specific nuclease (DSN) with terminal deoxynucleotidyl transferase (TdT). In the presence of target miRNA, the immobilized and 3'-blocked capture probes hybridized with target and thus the formed DNA-RNA hybrid was recognized by DSN. DSN mediated the digestion of 3'-phosphated capture probes (CPs) in the hybrids and synchronously target was released and recycled for another round of hybridization and cleavage. The cleaved CP fragments with a free 3'-OH were then elongated and labeled with multiple biotin-dUTP nucleotides by TdT. Fluorescence reporter streptavidin-phycoerythin was finally added to react with the immobilized biotins and render fluorescence signals. This dual-amplification labeling strategy was successfully demonstrated to sensitively detect multiple miRNAs, taking advantage of DSN-mediated target recycling and TdT-catalyzed multiple signal modification with analysis by a commercial Luminex xMAP array platform. Our experimental results showed the simultaneous quantitative measurement of three sequence-specific miRNAs at concentrations from 1 pM to 2.5 nM. Attempts were also made to directly detect miRNAs in total RNA extracted from cancer cells. The dual-amplification labeling strategy reported here shows a great potential for the development of a method for the multiplexed, sensitive, selective, and simple analysis of multiple miRNAs in tissues or cells for biomedical research and clinical early diagnosis.