3D DNA Scaffold-Assisted Dual Intramolecular Amplifications for Multiplexed and Sensitive MicroRNA Imaging in Living Cells

Abstract

The simultaneous live-cell imaging of multiple intracellular and disease-related microRNAs (miRNAs) with low abundances is highly important to enhance specificity and accuracy for disease diagnosis. On the basis of the improved cell internalization and accelerated reaction kinetics, we develop a three-dimensional (3D) DNA nanoprobe that integrates intramolecular DNAzyme (intra-Dz) and catalytic hairpin assembly (intra-CHA) amplifications to simultaneously monitor multiple miRNAs in living cells. The sensing components are loaded on a DNA scaffold via the sticky-end hybridization of the DNA sequences to increase the local concentrations of the signal probes. The miRNA-21 and miRNA-155 target sequences can trigger intra-Dz and -CHA amplifications on the nanoprobes to show significantly amplified and distinct fluorescence at different wavelengths for simultaneously monitoring low levels of miRNAs. Real-time fluorescence microscopy reveals that such a 3D DNA nanoprobe design with the intra-Dz and -CHA amplifications can accelerate the reaction rate compared to that of the conventional free Dz and CHA because of the increased local concentrations of the sensing components. Importantly, the 3D DNA nanoprobe has desirable stability and biocompatibility and can be readily delivered into living cells to achieve multiplexed and highly sensitive sensing of intracellular miRNA-155 and miRNA-21 sequences. With the demonstration of its intracellular application, the developed 3D DNA nanoprobe thus holds promising potential for biological studies and accurate disease diagnosis.