Dual-loop-probe cooperating CRISPR/switch-crRNA for triple check-based highly specific cancer-related microRNA analysis

Abstract Strategies for detecting microRNA (miRNA) via rolling circle amplification (RCA), combined with additional signal amplification techniques, have shown promising potential. However, achieving rapid, sensitive, and precise detection of target miRNA remains a major challenge in clinical diagnostics. The inherent limitations of insufficient selectivity have impeded the advancement of RCA-based methods. To address these challenges, we developed an innovative RCA-based approach that integrates the unique target recognition capability of a dual-loop probe with multiple signal amplification strategies, enabling highly sensitive detection of miRNA-21. In this method, miRNA-21 specifically unfolds the hairpin probe within the dual-loop structure, triggering target recycling, RCA, and Cas12a/crRNA-mediated signal generation. The proposed technique exhibits exceptional selectivity for miRNA-21, showing only a 17.8% response to single-base mismatch sequences, thereby demonstrating significantly enhanced specificity. The high specificity of this method arises from a triple-check mechanism, which includes (1) the dual-loop structure of the padlock probe, (2) the crRNA complex switching upon RCA product recognition, and (3) the trans-cleavage activity of Cas12a/crRNA. Furthermore, the assay achieves an ultra-low detection limit of 412 aM, meeting the stringent requirements for trace miRNA analysis. In summary, we demonstrate that this methodology offers a novel and effective pathway for miRNA detection, with potential applications in tumor monitoring and post-therapeutic management.