Deoxyribonucleic Acid Looped-Ag Nanocluster-Based Fluorescent Sensor for Accurate and Sensitive Circular Ribonucleic...

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.