Multiplexed profiling of microRNAs' subcellular expression and distribution is essential to understand their spatiotemporal function information, but it remains a crucial challenge. Herein, we report an encoding approach that leverages combinational fluorescent dye barcodes, organelle targeting elements, and an independent quantification signal, termed Multiplexed Organelles Portrait Barcodes (MOPB), for high-throughput profiling of miRNAs from organelles. The MOPB barcodes consist of heterochromatic fluorescent dye-loaded shell-core mesoporous silica nanoparticles modified with organelle targeting peptides and molecular beacon detection probes. Using mitochondria and endoplasmic reticulum as models, we encoded four Cy3/AMCA ER-MOPB and four Cy5/AMCA Mito-MOPB by varying the Cy3 and Cy5 intensity for distinguishing eight organelles' miRNAs. Significantly, the MOPB strategy successfully and accurately profiled eight subcellular organelle miRNAs' alterations in the drug-induced Ca2+ homeostasis breakdown. The approach should allow more widespread application of subcellular miRNAs and multiplexed subcellular protein biomarkers' monitoring for drug discovery, cellular metabolism, signaling transduction, and gene expression regulation readout.
Keywords: Ca2+ Homeostasis; Living Cell Imaging; MicroRNAs Detection; Multiplexed Barcode; Organelles Targeting; Subcellular Heterogeneity.