DNA nanotechnology for nucleic acid analysis: detection of RNA and dsDNA amplicons using a multifunctional DNAzyme nanomachine (DNM)

Abstract

DNA has long been recognized as a promising material for biotechnological applications. Here, we demonstrate how DNA nanotechnology can enhance the sensing of practically important forms of nucleic acid analytes, including folded single-stranded RNA (ssRNA) and double-stranded DNA (dsDNA) amplicons. We designed and optimized a multifunctional DNA-based sensor incorporating the catalytic 10–23 DNAzyme, herein named a DNAzyme-based nanomachine (DNM), and compared its performance with that of the classical binary 10–23 DNAzyme (BiDz) probe in detecting ssDNA, ssRNA, and dsDNA targets. The newly developed sensor exhibited up to 100- and 20-fold lower limits of detection (LODs) for ssDNA and ssRNA, respectively. Unlike traditional BiDz, DNM was capable of detecting as little as 1 µL of dsDNA amplicon, corresponding to ∼2 nM PCR product in the assay, within 30 min, achieving excellent target specificity. We suggested an explanation for the behaviour of nucleic acid complexes with respect to Gibbs free energy.