Topology-Gated λ Exonuclease Enables Amplification-Free Signal Boosting.

Amplification-free detection remains a fundamental challenge in CRISPR-based RNA diagnostics. Here, we identify a previously unrecognized topological property of λ exonuclease, whereby duplex substrates bearing 5' phosphates at both termini undergo a self-sustained cyclic cleavage-reforming process. This topology-gated behavior enables signal renewal without external amplification. Through systematic biochemical and structural analyses, we elucidate the underlying mechanism and establish λ exonuclease as a topology-driven signal amplifier. Then, we design a topology-gated dumbbell probe that sequesters 5' phosphates within dual RNA hairpin loops. Upon target recognition, CRISPR/Cas13 specifically cleaves the loops, exposing the hidden phosphates and thereby activating the λ exonuclease-mediated cyclic reaction. The resulting cascade, termed Topo-CRISPR (Topology-gated λ exonuclease enables CRISPR amplification-free), achieves attomolar sensitivity within 25 min without preamplification. Applied to clinical samples, Topo-CRISPR enables robust and specific detection of enterovirus RNA, miR-21, and ciR1445, demonstrating performance comparable to RT-qPCR. We further extend the Topo-CRISPR to non-nucleic-acid targets via aptamer-mediated conformational gating. This work uncovers a previously overlooked enzymatic topology, positioning λ exonuclease as a cyclic signal transducer and offering a general framework for ultrasensitive molecular sensing.