PANDA: AND Logic-Gated RNA Sensing Enabled by Photo-Activated RCA-Argonaute Cascade

Abstract

Achieving stable, multiplexed, and affordable nucleic acid detection in a true one-pot format remains a long-standing challenge for molecular diagnostics. Here, we report PANDA, a photo-activated rolling circle amplification (RCA)-Argonaute (Ago) cascade that encodes a stringent molecular AND logic gate between optical activation and target RNA recognition to enable highly sensitive, programmable RNA detection. In this system, ultraviolet (UV) irradiation triggers photolysis of precursor probe, simultaneously generating a 5′-phosphorylated DNA guide and circularizable padlock templates, while target RNA selectively initiates ligation and RCA. Signal generation occurs only upon convergence of these two orthogonal inputs, enforcing strict dual-input gating prior to Ago activation. Once engaged, Ago catalyzes continuous guide regeneration and sequence-directed reporter cleavage, producing signal boosting outputs within a single reaction vessel. The modular architecture further enables parallel assembly of multiple AND logic gates synchronized by a common optical trigger yet paired with distinct RNA targets, allowing controllable multichannel detection without physical compartmentalization. By integrating optochemical control with RCA-Ago-mediated catalytic turnover, PANDA establishes a streamlined, stable, and cost-efficient framework for logic-gated nucleic acid diagnostics and multiplexed RNA sensing.