Phosphorothioate-Enabled Cas12a Autocatalytic Network for Amplification-Free Ultrasensitive Nucleic Acid Detection

Abstract Autocatalytic CRISPR/Cas systems offer a promising route toward amplification-free nucleic acid detection, yet their performance is limited by indiscriminate transcleavage of ssDNA activators and the reliance on structurally complex, thermodynamically unstable DNA architectures. A phosphorothioate (PS)-enabled Cas12a autocatalytic network (SCAN) is presented to address these challenges by leveraging the unique compatibility of F. novicida Cas12a (FnCas12a) with fully PS-modified ssDNA activators. An ssDNA mediator is engineered with fully PS-modified activator domains flanking a cleavable, unmodified spacer. The intact mediator remains sterically inactive toward Cas12a ribonucleoproteins, whereas trace-target-induced cleavage liberates split activators that trigger a self-propagating amplification network. The SCAN platform achieves ultrasensitive detection with limits of detection of 889 aM and 341 aM for ssDNA and dsDNA targets, respectively. The platform further enables amplification-free detection of Salmonella enterica and Escherichia coli in real samples, with detection limits of 30 and 20 CFU/mL, respectively. This work establishes fully PS-modified ssDNA as a robust and generalizable design element for CRISPR-based autocatalytic systems, providing a simple, flexible, and efficient strategy to enhance stability, suppress background, and improve analytical performance.