G-quadruplex-deficient precursor hairpin probes for ultra-low background dual-mode detection of miRNAs

Design of oligonucleotide probe-based isothermal amplification with the ability to identify miRNA biomarkers is crucial for molecular diagnostics. Herein, we engineered a miRNA-21 responsive G-quadruplex-deficient precursor hairpin probe (PHP) to achieve dual-mode detection of fluorescent signal and colorimetric signal. Due to lack of complete G-quadruplex sequence, PHP becomes shorter in length, lower background signal and less interference. Based on the polymerase-driven amplification mechanism, in the presence of miRNAs, two simultaneous amplification reaction processes will occur in PHP: miRNA-based amplification process and endogenous amplification process along the 3′ end. Due to the positional difference between the starting points of the two amplification processes, the orderly and efficient occurrence of the two amplification processes can be achieved. Based on an interesting concept, PHP can achieve high detection performance with only simple amplification cycles. In such a way, the detection limits for fluorescence and colorimetry were 2.93 fM and 8.81 fM, which would cover most of clinical qualitative and quantitative needs. Thus, the accurate quantitative and visual miRNA detection technology based on PHP is beneficial to carry out extensive disease screening and treatment monitoring in various complex occasions.