Simultaneous and sensitive detection of two tumor-related circRNAs by enzyme-free isothermal amplification assisted microchip electrophoresis

Covalently closed single-stranded circular RNAs (circRNAs) are a class of naturally occurring special single-stranded non-coding RNAs with a covalently closed circular structure. CircRNAs have a profound relationship with human cancers and are identified as reliable tumor markers. This study developed an enzyme-free strategy based on catalytic hairpin assembly (CHA) combined with microchip electrophoresis (MCE) to achieve simultaneous detection of circMTO1 and circCDYL. During the CHA process, the well-designed hairpin probes (Hp₁ and Hp₂) could specifically recognize the back-splicing junction (BSJ) sequence of circRNAs and generate Hp₁-Hp₂ double-stranded products. Owing to their different numbers of bases, the Hp₁-Hp₂ double-stranded products corresponding to the two target circRNAs after CHA could be separated and sensitively detected by MCE. Under the optimized conditions, the limits of detection (LOD) of circMTO1 and circCDYL were as low as 2.13 pM and 1.68 pM (S/N = 3), respectively. This approach was also applied to detect circRNAs in HeLa cells, demonstrating excellent applicability and accuracy in biological samples.