Live-cell imaging of microRNA expression via photoinduced electron transfer controlled by catalytic hairpin assembly

Abstract

MicroRNA has been recognized as an important biomarker for various diseases, including cancer, and has been applied in early-stage diagnosis, therapeutic, and biomedicine. However, current methods for microRNA profiling and imaging are limited for temporal analysis, due to essential processes such as microRNA extraction or cell fixation. Therefore, a molecular probe capable of detection in living cells is necessary. In this thesis, I proposed an imaging platform designed for quantification of dynamic intracellular levels of microRNA. I investigated effectiveness of strategies, encompassing microRNA assays, transfection carriers, and ratiometric analysis techniques. The single-labeled probe was employed to minimize cytotoxicity arising from organic dyes and to reduce batch-to-batch variations in labeling. This approach is based on photoinduced electron transfer between guanine and FAM. The innovative design, where five guanines are placed adjacent to the fluorescent dye regardless of the target, ensures efficient and consistent quenching. The targeted microRNA specifically binds to hairpin DNA, and the subsequent cascade of toehold-mediated strand displacement releases the target, allowing for recycling and signal amplification. A single base-pair mismatch in the stem of the hairpin DNA boots the target-triggered strand displacement, resulting in an over 20-fold increase in sensitivity and achieving a pM level of limit of detection. The designed hairpin DNAs, encapsulated within the interlayers of multilamellar liposome vesicles, was delivered into cells with a high transfection yield, maintaining high stability. The microRNA imaging platform successfully monitored changes in microRNA levels in living cells over several hours of drug treatment. This long-term microRNA imaging system have great potential in biomedical field.