Live‐Cell MicroRNA Imaging through MnO₂ Nanosheet‐Mediated DD‐A Hybridization Chain Reaction

Abstract

Innovative techniques to visualize native microRNAs (miRNAs) in live cells can dramatically impact current research on the roles of miRNA in biology and medicine. Here, we report a novel approach for live-cell miRNA imaging using a biodegradable MnO nanosheet-mediated DD-A FRET hybridization chain reaction (HCR). The MnO nanosheets can adsorb DNA hairpin probes and deliver them into live cells. After entering cells, the MnO nanosheets are degraded by cellular GSH. Then, the target miR-21 triggers cascaded assembly of the liberated hairpin probes into long dsDNA polymers, which brings each two FAMs (donor) and one TAMRA (acceptor) into close proximity to generate significantly enhanced DD-A FRET signals, which was discovered and proven by our previous report. We think the developed approach can serve as an excellent intracellular miRNAs detection tool, which promises the potential for biological and disease studies.

<p>Manganese dioxide nanosheets: from preparation to biomedical applications</p>

“…It is also remarkable that the effort of Zhao et al to fabricate MnO 2 NS-aptamer nanoprobes early in 2014 verified that 79% of CCRF-CEM and Ramos human B lymphoma cells remained alive following by exposure to their nanoprobes at a concentration of 1 mM for 24 hrs 150Table 3Cytotoxicity results of various nanomaterials based on MnO 2 NSsNanomaterialsCell linesResponse, maximum exposure concentration, and durationTesting assaysReferenceCQDs-MnO 2 NSHeLaNo apparent loss of cell viability, 30 µg/mL, 24 hrsMTT170GQDs-MnO 2 nanoprobeMCF-7Low cytotoxicity, 40 µg/mL, 24 hrsMTT87g-C 3 N 4 -MnO 2 nanosandwichHeLaNo apparent loss of cell viability, 50 µg/mL, 24 hrsCCK-8151MnO 2 NS-FAM + TMR hairpinsHeLaInsignificant viability loss, 86% alive, 60 µg/mL, 24 hrsMTT111MnO 2 NS-FAM + TMR hairpinsMCF-7 & HepG2Low cytotoxicity, 90 µg/mL, 24 hrsMTT171MnO 2 NS-Janus DNA machineMCF-7Good biocompatibilty, 100 µg/mL, 24 hrsMTT69MnO 2 NS-aptamer nanoprobeCCRF-CEM and Ramos79% of cells remained alive, 1 mM, 24 hrsMTS150Ru(BPY) 3 @MnO 2 nanoprobeHeLaThe viabilities remained higher than 87%, 160 µM, 24 hrsMTT157MnO 2 NS-“DD-A” binary probeHepG2Low cytotxicity, 90 µg/mL, 24 hrsMTT172 Abbreviations: CCK-8, cell counting kit-8; NS, nanosheet; GQD, graphene quantum dot. …”

Simple, Amplified, and Multiplexed Detection of MicroRNAs Using Time-Gated FRET and Hybridization Chain Reaction

“…The simplicity of HCR can be improved by attaching FRET (Förster resonance energy transfer) donor–acceptor pairs to the HCR probes. ,,,− With this approach, HCR amplification leads to double-stranded DNA concatemers with many donors and acceptors in close proximity. Only HCR amplification can lead to FRET, whereas free HCR FRET probes are separated and cannot engage in energy transfer due to the spatial separation of donor and acceptor.…”

Nucleic‐Acid Structures as Intracellular Probes for Live Cells

“…This strategy proved viable for detecting low abundance miR‐21 in HeLa cells. Ou et al recently used the same strategy, but incorporated two donor fluorophores into one of the DNA strands and an acceptor fluorophore in the other DNA strand . In particular, the two donor/one acceptor (DD‐A) system had higher FRET efficiency than the one donor/one acceptor (D‐A) system.…”