Programmed Fluorescence-Encoding DNA Nanoflowers for Cell-Specific-Target Multiplexed MicroRNA Imaging

The precise identification and differentiation of multiple microRNAs (miRNAs) with high spatial resolution in specific cells remain a significant challenge, primarily due to the limited availability of spectrally distinguishable fluorophores and the absence of cell-specific recognition capabilities. In this study, we introduce a programmed fluorescence-encoding DNA nanoflower (CNFs) system based on the self-assembly of rolling circle amplification (RCA), enabling multiplexed miRNA imaging in living cells. The CNFs system is rationally designed to consist of three key components: a CD63 aptamer region, dual fluorophore encoding regions, and an miRNA recognition region. The polyvalent tandem CD63 aptamer enhances the cellular targeting specificity and endocytic uptake efficiency. By controlling dual fluorophores and three levels of intensity within encoding regions, it generates 9 distinct barcodes for labeling multiple targets. Additionally, when conjugated with molecular beacons (MBs), CNFs facilitate the simultaneous detection of multiplexed intracellular miRNAs. Using this CNFs system, we successfully evaluated the expression profiles of nine miRNAs in breast cancer. Overall, we expect that this CNFs system will be a valuable tool for disease-related multiplex miRNAs biomarker imaging in specific cells and the exploration of miRNAs’ molecular regulation mechanisms.