Universal Aptameric System for Highly Sensitive Detection of Protein Based on Structure-Switching-Triggered Rolling Circle Amplification

摘要

A universal approach is proposed in this study for thendevelopment of an aptameric assay system for proteinsnbased on aptamer structure-switching-triggered ligationrollingncircle amplification (L-RCA) upon target binding.nThe strategy chiefly depends on the competition fornbinding the aptamer probe between target protein and ancomplementary single-stranded DNA (CDNA) that canninduce the circularization of the padlock probe. Introductionnof target protein into the assay system inhibits thenhybridization of the CDNA with the aptamer probe becausenof the formation of the target/aptamer duplex. Thenfree CDNA can only hybridize with the padlock probe.nWith the assistance of DNA ligase, the padlock probe isncircularized, and the subsequent RCA process can benaccomplished by Phi 29 DNA polymerase. Each RCAnproduct containing thousands of repeated sequencesnmight hybridize with a large number of molecular beaconsn(detection probes), resulting in an enhanced fluorescencensignal. In contrast, in the absence of target protein, nonobvious change in the fluorescence intensity of the detectionnprobe is observed. This signaling mode for targetnrecognition and transduction events is based on thencombination of aptamer recognition elements and L-RCAntechnology with high specificity and sensitivity. The proposednassay system not only exhibits excellent analyticalncharacteristics (e.g., the detection limit on attomolar scalenand a linear dynamic range of more than 3 orders ofnmagnitude) but also possesses significant advantages overnexisting aptameric assays. The proposed strategy is universalnsince the sequences of aptamer probe, CDNA, andnpadlock probe could be easily designed to be compatiblenwith the L-RCA based detection of other proteins withoutnother conditions.