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

Abstract

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. Graphical abstract Schematic of a homogeneous and enzyme-free fluorometric assay for the determination of microRNA-182. It is based on the use of DNA circuits and DNAzymes. The DNA circuits warrant strong signal amplification by virtue of catalytic hairpin assembly that uses two hairpin substrates. The method represents an attractive tool for routine microRNA diagnosis and, conceivably, point of care uses.