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

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.