A Programmable Y-Shaped Catalytic Hairpin Assembly Amplification Enabling Ultrasensitive Fluorescent Analysis of Circular RNAs in Living Cells

Circular RNAs (circRNAs) have become a research hotspot due to their high stability, tissue specificity, and disease-regulating effects. Among them, circRNA mitochondrial tRNA translation optimization 1 (circMTO1) is abnormally expressed in cancer. However, the low abundance of circRNAs in biological samples and the complex matrix render traditional detection techniques inadequate for practical requirements. This study designed a programmable Y-shaped catalytic hairpin assembly (CHA) strategy with the back-splice junction (BSJ) sequence of circMTO1 as the trigger to hybridize with H1, initiating the ordered assembly of H2, H3, and H4 into a stable complex for cyclic target release and fluorescence signal amplification. This strategy relies on the BSJ recognition and the Y-shaped structure to avoid linear RNA interference, reduce nonspecific amplification, and enhance the target cycling efficiency for multilevel amplification by leveraging the unique spatial structure. Moreover, the assembly module (H1-H2-H3) and reporting module (H4) separation could be flexibly replaced with different signal types. To verify the programmability of the designed system, a CHA2 amplification was further integrated, which effectively realized a higher signal amplification efficiency. These results showed that the constructed CHA fluorescence sensing platform could detect circMTO1 with a limit of detection (LOD) of 10.63 nM in a linear range of 20-200 nM as well as excellent anti-interference capability and programmability, providing a multifunctional tool for early cancer diagnosis and mechanism research and laying a technical foundation for the clinical transformation of circRNAs as a diagnostic marker.