Assembly of Multiple DNA Components through Target Binding toward Homogeneous, Isothermally Amplified, and Specific Detection of Proteins

We describe a strategy of utilizing specific target binding to trigger assembly of three DNA components that are otherwise unable to spontaneously assemble with one another. This binding-induced DNA assembly forms a three-arm DNA junction, subsequently initiating nicking endonuclease-assisted isothermal fluorescence signal amplification. Real-time monitoring of fluorescence enables amplified detection of specific protein targets. The implementation of the strategy necessitates the simultaneous binding of a single target molecule with two affinity ligands each conjugated to a DNA motif. Simple alternation of affinity ligands enables different protein targets to induce the formation of the DNA junction and subsequent isothermal amplification. The use of the strategy allowed us to develop a sensitive assay for proteins with three appealing features: homogeneous analysis without the need for separation, isothermal amplification, and high specificity. Streptavidin was chosen as an initial target to establish and optimize the assay. Sensitivity of protein detection was improved by 1000-fold upon the application of isothermal amplification. A limit of detection of 10 pM was achieved for detection of prostate-specific antigen in buffer and diluted serum. The combination of its three appealing features makes the assay attractive for potential applications in molecular diagnosis, point-of-care testing, and on-site analysis.