Sensitively distinguishing intracellular precursor and mature microRNA abundance

Mature microRNAs (miRNAs) produced from precursor microRNAs (pre-miRNAs) by the RNase Dicer have showed significant potential for cancer diagnosis and prognosis due to their key regulatory roles in various pathological processes. However, discriminatory detection of low-abundance miRNAs and pre-miRNAs remains a key challenge since the mature sequence is also present in the pre-miRNA forms. Herein, we report a novel cascade reaction to sensitively distinguish miRNAs versus pre-miRNAs in living cells based on two pairs of programmable hairpin oligonucleotide probes with a simple sequence design. The programmable hairpin probes can metastably coexist until the introduction of miRNAs or pre-miRNAs, which can trigger a specific hybridization chain reaction (HCR), respectively, leading to the self-assembly of nicked DNA duplex structures and a remarkable specific fluorescence intensity increase. The system can readily and sensitively assess the miRNA or pre-miRNA abundance in a homogeneous solution. The intracellular miRNA and pre-miRNA expression level assessment in different living cells is realized. Thus, we provide a novel investigation tool for discriminatorily and accurately assessing miRNA and pre-miRNA abundance, which could be useful for the biomedical application of miRNAs.