Isothermal Digital Detection Of MicroRNA Using Background-Free Molecular Circuit

MicroRNA, a class of transcripts involved in the regulation of gene expression, are emerging as promising disease-specific biomarkers accessible from tissues or bodily fluids. However, their accurate quantification from biological samples remains challenging. We report a sensitive and quantitative microRNA method using an isothermal amplification chemistry adapted to a droplet digital readout. Building on molecular programming concepts, we design DNA circuit that converts, threshold, amplifies and report the presence of a specific microRNA, down to the femtomolar concentration. Using a leak-absorption mechanism, we were able to suppress non-specific amplification, classically encountered in other exponential amplification reactions. As a result, we demonstrate that this isothermal amplification scheme is adapted to digital counting of microRNA: by partitioning the reaction mixture into water-in-oil droplets, resulting in single microRNA encapsulation and amplification, the method provides absolute target quantification. The modularity of our approach enables to repurpose the assay for various microRNA sequences.