Abstract

A dual-locking cascade integrating dCas13a/crRNA and DSN enables orthogonal recognition of circRNA BSJ and a second distinct region. • Circular probe design and bead-based separation effectively suppress non-specific background for reliable circRNA detection. • The platform achieves ultrasensitive detection with a limit of 0.16 fM and a broad linear range from 0.5 fM to 200 pM. • Excellent discrimination against single-base mismatches and homologous circRNAs is demonstrated via the dual-recognition mechanism. • Successful validation in cancer cells and spiked serum samples supports its potential for ovarian cancer liquid biopsy. Circular RNAs (circRNAs) have emerged as promising non-invasive biomarkers for ovarian cancer, yet their accurate detection remains challenging due to their low abundance and the difficulty in discriminating true circRNAs from linear RNA homologs. Herein, we report a dual-locking fluorescent cascade platform that integrates dCas13a/crRNA and duplex-specific nuclease (DSN) for sensitive and specific detection of ovarian cancer-related circRNAs. Unlike conventional single-target strategies, our design employs an orthogonal dual-recognition mechanism: dCas13a/crRNA captures the back-splice junction region, while a circular probe hybridizes to a second distinct region; subsequent DSN cleavage occurs only upon perfect DNA–RNA heteroduplex formation, converting the circular probe into a padlock that triggers rolling circle amplification (RCA) and Cas12a trans-cleavage for fluorescence readout. The circular probe effectively suppresses non-specific background, and the cascade amplification provides ultrasensitivity with a detection limit of 0.16 fM and a broad linear range from 0.5 fM to 200 pM. The platform exhibits excellent specificity, discriminating single-base mismatches and homologous circRNAs, and demonstrates robust performance in complex biological matrices. Using the clinically relevant circRNA hsa_circ_0007534, we confirmed its signif