Developing genetically encodable probes for multimodal tracking of exosomal RNA cargo

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Project Summary Exosomes, also referred to as small extracellular vesicles, play important roles in cellular communication under physiological and pathophysiological conditions. Exosomes contain a wide range of both short and long non- coding RNAs that regulate many aspects of gene expression including epigenetic processes that modulate cellular fate, phenotype, polarization, and morphogenesis. Despite the important functional roles played by exosomal RNAs, there are currently no methods that allow live exosomal RNA tracking. This is because RNA is by nature non-fluorescent and difficult to label while maintaining its intended biological function. Access to exosomal RNA is further complicated by the fact that each RNA species is present at extremely low copy numbers in exosomes. This emphasizes both the need for a novel marker capable of tracking the intercellular movement of exosomal RNA, and the need to enhance loading of RNAs into exosomes. Given the central importance of exosomal RNAs in dictating cellular behavior, there is a need and demand for exosomal RNA imaging methods to determine how (a) cells use exosomes and their cargoes to communicate with each other and (b) how exosomes modulate their microenvironment and travel to distant organs and tissues. Existing methods focus on tracking exosomes by labeling the lipid membrane via a lipid-based fluorophore or exosomal protein labeling. None of these methods allow the tracking of exosomal RNA via genetic encoding or barcoding without exogenously modifying the exosomes after extensive collection and alteration steps. The overall goal of this proposal is to develop genetically encodable RNA EXO-Code probes that allow multimodal tracking and imaging of exosomal RNAs. The EXO-Code probe will allow multimodal tracking of exosomal RNA via (1) genetic encoding, (2) non-destructive labeling with fluorescent dyes, and (3) unique identification and quantification based on barcoding. This combination is powerful as it allows tracking of exosomal RNA via multiple modes for high content biodistribution mapping. Because EXO-Code barcodes are composed of unique nucleotide sequences, they can be accurately decoded using sequencing with sensitivity in the attomolar range. The combination with a fluorogenic RNA aptamer allows for complementary tracking of exosomal RNA via simple incubation with dyes. The fluorescent exosome toolkit will be developed for investigators to detect disruptions in membrane stability, exosomal fusion events, and endocytic processes as a result of exosome biogenesis, distribution, and uptake. This will enable researchers to track exosomal RNAs through organisms, cells, and their ultimate destinations within subcellular compartments.