Target‐Dependent Protection of DNA Aptamers against Nucleolytic Digestion Enables Signal‐On Biosensing with Toehold‐Mediated Rolling Circle Amplification

We report a generalizable strategy for biosensing that takes advantage of the resistance of DNA aptamers against nuclease digestion when bound with their targets, coupled with toehold mediated strand displacement (TMSD) and rolling circle amplification (RCA). A DNA aptamer containing a toehold extension at its 5′‐end protects it from 3′‐exonuclease digestion by phi29 DNA polymerase (phi29 DP) in a concentration‐dependent manner. The protected aptamer can participate in RCA in the presence of a circular template that is designed to free the aptamer from its target via TMSD. The absence of the target leads to aptamer digestion, and thus no RCA product is produced, resulting in a turn‐on sensor. Using two different DNA aptamers, we demonstrate rapid and quantitative real‐time fluorescence detection of two human proteins: platelet‐derived growth factor (PDGF) and thrombin. Sensitive detection of PDGF was also achieved in human serum and human plasma, demonstrating the selectivity of the assay. Digestion protection: a fluorescence assay is demonstrated based on target‐mediated protection of aptamers from 3′‐exonuclease digestion by phi29 DNA polymerase followed by toehold mediated strand displacement of the target by a circular template to initiate rolling circle amplification, providing sub‐nanomolar detection limits for protein targets, including in serum and plasma.