Development of a dCas13a/crRNA-Linked Immunosorbent Mimic Assay (dCLIMA) for Sensitive and Accurate Circular RNA...

Circular RNAs (circRNAs) are emerging as promising biomarkers in liquid biopsy, yet their accurate detection in circulating tumor cells remains challenging due to low abundance and interference from linear RNA counterparts. Here, we develop a dCas13a/crRNA-Linked Immunosorbent Mimic Assay (dCLIMA) for sensitive and specific colorimetric detection of circRNAs. The dCas13a/crRNA complex immobilized on a microplate specifically captures target circRNAs via their back-splicing junction (BSJ) region, eliminating linear RNA interference. Upon capture, the circRNA unlocks a hairpin probe and facilitates padlock circularization, triggering rolling circle amplification (RCA) to produce tandem G-rich repeats. Notably, this design enables RCA to recognize and probe dual functional domains on the same circRNA, allowing more comprehensive functional characterization. The G-rich repeats fold into G-quadruplex/hemin DNAzyme that catalyzes TMB oxidation to generate a blue product for label-free final readout. The dCLIMA assay achieves a detection limit of 0.63 fM for circRNA_100876, discriminates single-base mismatches, and exhibits excellent anti-interference capability. We successfully quantify circRNA_100876 in lung cancer-derived cells, cell lysates, and clinical serum samples, with results correlating well with qRT-PCR. This method holds great promise for advancing circRNA-based liquid biopsy and precision oncology.