Sensitive detection of MiRNA and CircRNA through DSN enzyme cooperating NEase assisted dual signal amplification

MicroRNA (miRNA) and circular RNA (circRNA) are promising biomarkers for early-diagnosis of a variety of diseases, such as myocardial infarction and cancers. However, few methods reported simultaneous and sensitive detection of multiple miRNAs. Herein, we design a novel approach with an improved miRNA and circRNA detection sensitivity. There are only two probes designed with hairpin probe, namely catch probe and report probe, in the system. When target miRNA or circRNA existed, it can unfold catch probe through hybridizing with toehold section and form a rigid DNA-RNA duplex. The DNA sequence in the formed duplex is identified and digested by duplex-specific nuclease (DSN enzyme), and consequently, target miRNA is released to attend a next signal cycle. The rested DNA sequence in catch probe (initiator probe) recognizes report probe and separates its hairpin structure to generate fluorescence signals, forming a double-strand DNA (dsDNA) product. Unfold report probe sequence in the dsDNA product is then digested by DNA nicking endonuclease (NEase) and initiator is released to trigger amplified signals. Based on the DSN enzyme cooperating NEase assisted dual signal amplification, the method exhibits a greatly improved detection sensitivity.