Deoxyribonucleic Acid Looped-Ag Nanocluster-Based Fluorescent Sensor for Accurate and Sensitive Circular Ribonucleic Acid Analysis via Dual Catalytically Deactivated Cas13a/crRNA Recognition-Mediated Proximity Ligation

Accurate and specific detection of circular RNAs (circRNAs) is critical for vascular biology research and the clinical diagnosis of diabetes, particularly diabetic angiopathies. A major challenge in circRNA detection stems from the presence of abundant linear RNA isoforms that share identical sequences with circRNAs except for the back-splice junction. To overcome this limitation, we developed a novel detection strategy based on dual catalytically deactivated Cas13a/crRNA (dCas13a/crRNA) complexes that simultaneously recognize both ends of the circRNA back-splice junction. This system initiates a proximity ligation-triggered rolling circle amplification (RCA) reaction, producing long single-stranded DNA with tandemly repeated functional sequences. By combining dual dCas13a-guided recognition with proximity-mediated RCA, our method achieves exceptional specificity, enabling direct circRNA detection in complex RNA backgrounds, including linear isoforms, without requiring RNase R pretreatment. Coupled with triple catalytic hairpin assembly amplification, the assay detects circRNA with a detection limit of 0.083 fM within 150 min. The high specificity and sensitivity of this dCas13a/crRNA complex recognition-induced exponential amplification platform were validated in complex biological samples, demonstrating its broad potential as a versatile tool for sequence-specific RNA analysis and biomarker development in both basic research and clinical diagnostics of diabetic vascular complications.