Invasive RNA Programs Optical Switching in DNA Nanoclusters for CRISPR Sensing

Abstract DNA-templated silver nanoclusters have regained attention due to recent advances in stability and tunable optical properties. Here, we report a highly regulated DNA-templated silver nanocluster (DFN2) that exhibits strong fluorescence at 561 nm and enables reversible, label-free fluorescence switching governed by nucleic acid hybridization. The fluorescence of DFN2 is efficiently quenched with complementary DNA or RNA and fully restored through invading RNA strand mediated displacement, enabling robust and programmable ON-OFF-ON optical control. We integrated this hybridization-responsive platform with CRISPR-Cas12a to construct a target-activated biosensing system. Upon recognition of a conserved genomic fragment from a foodborne pathogen Listeria monocytogenes, the activated Cas12a cleaves a regulator DNA, initiating a cascade of hybridization and strand-displacement reactions that restore nanocluster fluorescence through an invasive RNA. This strategy exhibits high specificity against conserved genomic regions from other foodborne pathogens. These findings establish DFN2 as programmable, enzyme-responsive optical reporters for rapid, sensitive, and modular point-of-care biosensing.