Extension of duplex specific nuclease sensing application with RNA aptamer

Duplex specific nuclease (DSN) that can precisely cleave DNA portion in double-stranded DNA or DNA-RNA hybrid has engrossed immense attention owing to its great potential in emerging bioanalytical applications. Here, we present a novel approach to extend DSN sensing application by coupling RNA aptamer. Specially designed RNA ligand sequences are used to capture the target and simultaneously provide complementary sequences of DNA for DSN aided fluorescent signal enhancement. A clotting enzyme, thrombin, has been used as a model analyte. One RNA aptamer combined with the target molecule can generate fluorescent signals through cleavage of hybridized TaqMan DNA probe (P2) by DSN. The proposed assay has achieved the lowest detection limit of 0.039 pM. The assay has been applied for real-time detection of thrombin release from live cells and other biotic media for early disease diagnosis. The developed method is versatile and can detect various other targets by choosing the relevant aptamer and probe sequences. This method is promising to be applied to medical diagnosis, biosensing, food safety, environmental monitoring, and other fields.