Label-free fluorometric detection of microRNA using isothermal rolling circle amplification generating tandem G-quadruplex

MicroRNAs (miRNAs) play an important role in various biological processes and have been regarded as promising diagnostic biomarkers for solid tumors in the field of clinical diagnostics. In this study, we developed a simple label/quencher-free fluorometric system for sensitive and selective miRNA detection using isothermal gene amplification such as rolling circle amplification generating tandem G-quadruplex DNA structures (GQ-RCA). The closed-circular dumbbell-shaped padlock DNA was designed to be complementary to its corresponding target miRNA. In the presence of the target miRNA, a long stretch of ssDNA with tandem G-quadruplex sequence repeats was readily generated by RCA, initiated by phi29 DNA polymerase through DNA synthesis priming at the 3'-OH of the target miRNA annealed to the padlock DNA. The RCA product harboring tandem G-quadruplex was monitored with fluorophore Thioflavin T (ThT) that emits strong fluorescence only when it intercalates into the G-quadruplex. The GQ-RCA assay enabled us to detect miRNA as low as 4.9 fM with a linear range from 25.6 fM to 80 pM within 0.5 h. In addition, our system was applied to the miRNA samples present in human plasma, showing its potential use in the clinical diagnosis of cancer.