Convenient and multiplexed detection of microRNAs based on an exonucleation reaction by conformational switch of hairpin probes

Abstract

The ability to detect and quantify microRNA (miRNA) could greatly facilitate the comprehension of miRNA-mediated biological process and related disease in more detail. We developed, for the first time, a convenient and efficient strategy for miRNA detection based on direct molecular recognition between initially quenched DNA probes and miRNA targets. The loop region of dual labeled DNA probe with hairpin shape was complementary to target miRNA, which could induce a corresponding conformational switch, a DNA:RNA hybrid duplex was formed with a single-strand DNA overhang, which could be recognized and degraded by various exonucleases, including Exonuclease I (Exo I) and T4 DNA polymerase. Fluorescence enhancement was generated by the degradation, which occurred in the 3′–5′ direction. This method represented a reliable way to distinguish different miRNAs with high homology, such as different members of the miR-200 family. Furthermore, multiplexed miRNAs detection could be achieved with our developed approach. More importantly, this proposed method can distinguish the expression level of miR-21 in human liver tissues between liver cancer tissues and normal tissues. This exonucleation-based fluorescence enhancement strategy holds great promise for further applications in early miRNA-related disease diagnosis and prognosis.