DSN/TdT recycling digestion based cyclic amplification strategy for microRNA assay

Sensitive and specific detection of microRNAs (miRNAs) is of great significance for early cancer diagnosis. Here we report a simple and sensitive fluorescence signal amplification strategy that based on DSN/TdT recycling digestion for miRNA detection. DSN initiates DNA digestion on 3'-phosphate-primer/miRNA heteroduplex which causes miRNA recycle. The digested DNA strands with 3'-OH ends enable TdT to synthesize a polydeoxyguanylic tails on the 3'-end. The DNAs with polydeoxyguanylic tails are converted to double-stranded-DNA prior to initiation of DSN/TdT recycling digestion. With the cooperation of TdT and DSN, a new round of digestion and extension is triggered, leading to massive fluorophores separating and signal amplification. The amplification strategy produces large amounts of 3'-OH probes that can be used directly for dsDNA enrichment and DSN digestion. Moreover, both DSN digestion and TdT extension are sequence-independent reaction without the need of complex sequences design. In addition, this strategy is utilized to analyze miRNA samples from MCF-7 cell lysates and Cu (II) ion samples, indicating its potential application in actual sample analysis. The method shows a promising analytical platform for DNA nicking-related studies and tumor biomarkers measuring in clinical diagnostics.