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

Proximity-localized catalytic hairpin assembly (CHA) is intriguing for rapid and sensitive assay of an HIV-specific DNA segment (*). Using template-integrated green Ag nanoclusters (AgNCs) as emitters, herein, we report the first design of a *-activated CHA circuit that is confined in a three-way-junction architecture (3WJA) for the fluorescence sensing of *. To this end, the *-recognizable complement is programmed in a stem-loop hairpin (H1), and two split template sequences of 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 *, the 3WJA-localized CHA is operated through toehold-mediated strand displacements of H1 and H2 reactants, and * 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 AgNCs, outputting *-dependent fluorescence response. Because of 3WJA structural confinement, the spatial proximity of two reactive hairpins yielded high local concentrations to manipulate the CHA operation, achieving rapider reaction kinetics via *-catalyzed recycling than typical catalytic hairpin assembly (CHA). This simple assay strategy would open the arena to develop various CHA-based circuits capable of modulating the fluorescence emission of AgNCs for applicable biosensing and bioanalysis.