Live-Cell Imaging of MicroRNA Expression via Photoinduced Electron Transfer Controlled by Catalytic Hairpin Assembly.

MicroRNAs (miRNAs) serve as emerging biomarkers for a range of diseases, and their quantitative analysis draws increasing attention. Yet, current invasive methods limit continuous tracking within living cells. To overcome this, we developed a non-enzymatic DNA-based nanoprobe for dynamic, non-invasive miRNA tracking via live-cell imaging. This probe features a unique hairpin DNA structure with five guanines that act as internal quenchers, suppressing fluorescence from an attached fluorophore via photoinduced electron transfer. Target miRNA initiates toehold-mediated strand displacement, restoring and amplifying the fluorescence signal. Additionally, by introducing a single mismatch to the hairpin DNA, the nanoprobe's sensitivity was significantly enhanced, lowering the detection limit to about 60 pM without compromising specificity. To optimize intracellular delivery for prolonged monitoring, the nanoprobe was encapsulated within multilamellar lipid nanovesicles, fluorescently labeled for dual-wavelength ratiometric analysis. The proposed nanoprobe demonstrates a significant advance in live-cell miRNA detection, promising enhanced in situ analysis for a better understanding of miRNAs' pathophysiological function. This article is protected by copyright. All rights reserved.