Programmable DNA barcode-encoded exponential amplification reaction for the multiplex detection of miRNAs

Abstract

Multiple analysis of miRNAs is essential for the early diagnosis and monitoring of diseases. Here, a programmable, multiplex, and sensitive approach was developed for one-pot detection of miRNAs by melting temperature encoded sequences and exponential isothermal amplification (E-EXPAR). In the presence of target miRNAs, the corresponding templates initiate the cycles of nicking and polymerization/displacement, generating numerous barcode strands with unique encoding sequences. Subsequently, generated barcode strands hybridize with fluorescent probes and quench the fluorophore by a triplet of G base through a photo-induced electron transfer mechanism. Finally, a melting curve analysis is performed to quantify miRNAs by calculating the rate of fluorescence change at the corresponding melting temperature. Based on this, miRNA-21, miRNA-9, and miRNA-122 were detected with the detection limits of 3.3 fM, 2.9 fM, and 1.7 fM, respectively. This E-EXPAR was also employed to simultaneously detect three miRNAs in biological samples, showing consistent results with RT-qPCR. Overall, this study provides a programmable and universal platform for multiplex analysis of miRNAs, and holds great promise as an alternative to the multiplex analysis in clinical diagnostics and prognostics for nucleic acid detection.