A fluorescence strategy for circRNA quantification in tumor cells based on T7 nuclease-assisted cycling enzymatic amplification

Circular Ribonucleic Acid (CircRNA) plays regulatory roles in many biological processes, such as tumors and metabolic diseases. Due to the fact that circRNA is more stable and conservative than linear RNA, circRNA has become a potential biomarker in early clinical diagnosis and biomedical research. Therefore, the quantification of circRNA expression level is of importance for understanding their functions and their applications for disease diagnosis and treatment. Nevertheless, due to the low abundance of circRNA, it is still a challenge for the analysis of circRNA in cells. Herein, we proposed a sensitive detection method for circRNA based on the T7 exonuclease-assisted cycling enzymatic amplification. The fluorescent sensor was constructed by a hairpin molecular beacon and T7 exonuclease. With the cycling enzymatic amplification process, this sensor achieved the limit of detection of 1 pM with a good linear correlation in the range of 0-100 pM (R2 = 0.9891) using circBART2.2 as a model. Furthermore, we applied the proposed method in the determination of circBART2.2 in cell lysates. The results demonstrated that this method has promising applications in early diagnosis of Epstein-Barr virus (EBV) infection-related diseases using circRNA as the biomarker.