LAUSR

An enzyme-free dual catalytic DNA circuit for amplified detection of nucleic acids has been developed. The system functions based on a cyclic self-assembly of two auxiliary hairpins (H1 and H2) and three biotinylated hairpin oligonucleotides (H3, H4 and H5), in the format of two molecular circuits. In the upstream circuit, a target initiator (I) besides H1 and H2 hairpins constructs H1-H2 duplexes that trigger the operation of a subsequent circuit. In the downstream circuit, the H1-H2 duplex initiates cascaded self-assembly reactions, produces triplex H3-H4-H5, as sensing system, and releases the H1-H2 duplex as the catalyst for the self-assembly of additional hairpins. The H3-H4-H5 triplex acts as the scaffolds for assembling and orienting the streptavidin-functionalized gold nanoparticles (SA-AuNPs) into a lattice-like arrangement that generates a DNA-SA-AuNP cross-linked network, resulting in a dramatic pale red-to-blue color change. By ingeniously engaging two catalytic circuits with feedback amplification capabilities, the system can detect the target nucleic acid with an LOD value of 5 femtomolar and unambiguously discriminate spurious targets (i.e. targets containing substitution, insertion, and deletion nucleotides) without instrumentation. Simple and convenient operation of the assay makes the DNA circuit appropriate for point-of-care monitoring in resource-constrained settings.