Intracellular MicroRNA Imaging with MoS₂-Supported Nonenzymatic Catassembly of DNA Hairpins

Abstract

Amplification strategies for low-level microRNA detection in living cells are pivotal for gene diagnosis and many cellular bioprocesses. In this work, we develop an amplification strategy for microRNA-21 (miRNA-21) imaging in living cells with MoS₂-supported catassembly of DNA hairpins. The MoS₂ nanosheet with low cytotoxicity serves as the nanocarrier and excellent fluorescence quencher, which can transfer fluorescent metastable hairpin DNA into the cells easily in a nondestructive manner and significantly reduce background signals. The three-branched catalyzed hairpin assembly (TB-CHA) probes contain three types of designed DNA molecular beacons with the modification of Cy3 in the terminal. In the presence of miRNA-21, the catalyzed hairpin assembly (CHA) reaction would be triggered and a "Y"-shaped three-branched duplex nanostructure would be formed, which would release from the surface of the MoS₂ nanosheet due to the reduced affinity between the DNA duplex and MoS₂ nanosheet. The multisite fluorescence modification and the circular reaction of TB-CHA probes allowed a significant fluorescence recovery in a live-cell microenvironment. The ultrasensitive detection of miRNA-21 is achieved with a detection limit of 75.6 aM, which is ∼5 orders of magnitude lower than that of a simple strand displacement-based strategy (detection limit: 8.5 pM). This method offers great opportunities for the ultrasensitive live-cell detection of miRNAs and helps in gaining a deeper understanding of the physiological functions of miRNAs in cancer research and life processes.