Target DNA-Activating Proximity-Localized Catalytic Hairpin Assembly Enables Forming Split-DNA Ag Nanoclusters for Robust and Sensitive Fluorescence Biosensing

Abstract

Proximity-localized catalytic hairpin assembly (<i>pl</i>CHA) is intriguing for rapid and sensitive assay of an HIV-specific DNA segment (<i><b>T</b></i>*). Using template-integrated green Ag nanoclusters (<i>ig</i>AgNCs) as emitters, herein, we report the first design of a <i><b>T</b></i>*-activated <i>pl</i>CHA circuit that is confined in a three-way-junction architecture (3WJA) for the fluorescence sensing of <i><b>T</b></i>*. To this end, the <i><b>T</b></i>*-recognizable complement is programmed in a stem-loop hairpin (H1), and two split template sequences of <i>ig</i>AgNCs are separately overhung contiguous to the paired stems of H1 and another hairpin (H2). The hybridization among H1, H2, and two single-stranded linkers (L1 and L2) allows the stable construction of 3WJA. Upon presenting the input <i><b>T</b></i>*, the 3WJA-localized <i>pl</i>CHA is operated through toehold-mediated strand displacements of H1 and H2 reactants, and <i><b>T</b></i>* is rationally displaced and repeatably recycled, analogous to a specific catalyst, inducing more hairpin assembly events. Resultantly, the hybridized products enable the collective combination of two splits in the parent scaffold for hosting <i>ig</i>AgNCs, outputting <i><b>T</b></i>*-dependent fluorescence response. Because of 3WJA structural confinement, the spatial proximity of two reactive hairpins yielded high local concentrations to manipulate the <i>pl</i>CHA operation, achieving rapider reaction kinetics via <i><b>T</b></i>*-catalyzed recycling than typical catalytic hairpin assembly (CHA). This simple assay strategy would open the arena to develop various <i>pl</i>CHA-based circuits capable of modulating the fluorescence emission of <i>ig</i>AgNCs for applicable biosensing and bioanalysis.