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

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.