Enzyme-free amplified detection of circulating microRNA by making use of DNA circuits, a DNAzyme, and a catalytic hairpin assembly

A homogeneous and enzyme-free fluorometric assay is described for the determination of microRNA-182. It is based on the use of DNA circuits and DNAzyme. The DNA circuits warrant strong signal amplification by virtue of catalytic hairpin assembly, a system that consists of two hairpin substrates. A part of the DNAzyme sequence is programmed to sequester into one of the two hairpin substrates. In the presence of target microRNA-182, the two hairpin substrates undergo catalytic assembling. This results in the formation of a DNA duplex and the release of the DNAzyme from the hairpin structure. Upon cyclic amplification, one target catalyzes the formation of Mg (II)-dependent DNAzymes. These bind to, and hydrolyze, the fluorescently labeled substrates for signal amplification and transduction. Based on nucleic acid programmability, this engineered assay has a limit of detection as low as 6.8 f. and a dynamic range that covers the 10 f. to10 nM microRNA-182 concentration range. Detection can be performed within 60 min. The assay is simple, rapid, homogenous, cost-effective, and enzyme-free. These features make the method an attractive tool in routine microRNA diagnosis and, conceivably, in point of care uses.