Shared‐Output Aptamer‐Gated DNA Nanodevices Program NbaSPARDA Trans‐Cleavage for Metabolite and Drug Sensing

ABSTRACT Routing chemically distinct small‐molecule aptamers into a common nuclease trans‐cleavage pathway is constrained by the target‐specific output handles of conventional structure‐switching aptamers (SSAs). Shared‐output aptamer‐gated DNA nanodevices are designed so that recognition cores control release of the same short trigger DNA (tDNA). The released tDNA hybridizes with a common guide RNA and activates NbaSPARDA, a short prokaryotic Argonaute‐associated nuclease complex, for reporter trans‐cleavage. Analyses across two sequence scaffolds reveal sequence‐dependent activation and identify a practical 14–16 nt range for the primary scaffold. Native PAGE supports target‐induced tDNA release. Under matched conditions, NbaSPARDA produces lower target‐free signals than LbCas12a for the compact SSA configurations tested. Adenosine‐gated devices achieve detection limits of 0.28 and 0.40 µM in buffer, while Ade‐SSA14 supports matrix‐spiked relative readout of pentostatin‐induced adenosine‐related changes in processed HeLa lysate filtrates. A mefloquine aptamer reconfigured with the same 16 nt output achieves detection limits of 0.018 µM in buffer and 0.061 µM by matrix‐matched calibration in an acetonitrile‐processed blank‐serum extract. Serum samples spiked before processing give recoveries of 107 ± 19% and 101 ± 7.5%. An exploratory estradiol construct accesses the output channel. Together, these results establish a shared‐output interface for programming NbaSPARDA trans‐cleavage in metabolite and drug analysis.