Sensitive and selective detection of microRNA using hyperbranched rolling circle amplification based on symmetric...

Examining the changes in microRNA expression in vivo enhances the understanding of their tumor-associated functions and facilitates clinical assays based on their roles as disease markers. However, due to the low abundance and high homology, their detection remains challenging. Herein, we developed an exponential amplification method by combining a symmetric dumbbell-type probe (SDTP) and toehold-initiated hyperbranched rolling circle amplification (HRCA) to obtain high sensitivity and selectivity. The toehold region of the dumbbell-type probe specifically binds to the target, triggering a toehold-mediated strand displacement reaction and thus activating SDTP into a circular structure. In the presence of phi29 DNA polymerase, primer P2, and dNTPs, the HRCA reaction proceeds continuously using the circular structure as a template, enabling exponential amplification of let-7a. When the concentration of let-7a ranges from 1.00 fmol·L^-1 to 1.00 nmol·L^-1, the fluorescence intensity obtained by this method exhibits a linear correlation with the logarithm of let-7a concentration (unit: pmol·L^-1), with a detection limit of 214 amol·L^-1. This method can distinguish homologous miRNAs with single-base mismatches. Serum spiking recovery experiments verified the accuracy of the method in actual serum sample detection; meanwhile, the simplicity was demonstrated by the detection of let-7a in different cell lysates without RNA extraction. Therefore, our work exhibited a simple, sensitive, and specific detection of miRNAs in biological samples, which holds promise as a potential tool for miRNA analysis in clinical testing.