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.