Inverted Molecular Beacons as Reaction-Based Hybridization Probes for Small-Molecule Activation by Nucleic Acid Inputs.

Nucleic acid-based hybridization probes that produce a fluorescent signal in the presence of DNA or RNA target molecules are essential components of nucleic acid computation and detection strategies. Commonly, the fluorescence activation of reporter gates is triggered by separation of a fluorophore-quencher pair upon target hybridization or strand displacement. In order to expand the utility of DNA computing by providing a chemical reaction as the ultimate output, reporter systems have been designed that carry reactive groups, which undergo a proximity-induced reaction upon oligonucleotide hybridization. The downside of published reporter gate designs is that they are composed of two separate, chemically modified oligonucleotides, which need to be taken into consideration when designing upstream circuits. Here, we report a novel hairpin-forming nucleic acid reporter probe that utilizes template-induced proximal reactivity to activate a small molecule in the presence of an unmodified nucleic acid input molecule. This DNA hairpin reporter gate consists of a duplex between a blocking strand and a hairpin-forming reporter strand. In the presence of input, the blocking strand is displaced, triggering hairpin formation allowing the proximity-driven templated activation of a vinyl ether-caged fluorophore by a tetrazine via an inverse electron demand Diels-Alder reaction. This new approach demonstrates robust small-molecule activation in vitro and in cells through logic operations in the presence of input DNA molecules.