Developing genetically encodable probes for multimodal tracking of exosomal RNA cargo

项目摘要 外体，也被称为细胞外小泡，在细胞通讯中发挥着重要的作用。 生理和病理生理条件。Exosome包含广泛的短和长非- 编码RNA调节基因表达的许多方面，包括调节表观遗传过程 细胞命运、表型、极化和形态发生。尽管它扮演着重要的职能角色 对于外体RNA，目前还没有允许实时追踪外体RNA的方法。这是因为RNA是 本质上是非荧光的，在保持其预期的生物功能的同时很难标记。进入 外体rna由于每个rna物种都以极低的拷贝存在这一事实而进一步复杂化。 外显体中的数字。这强调了对能够跟踪细胞间信号的新型标记的需求 外体核糖核酸的运动，以及增加核糖核酸到外体的负载的必要性。 鉴于外体RNA在决定细胞行为方面的核心重要性，有必要和需求 确定(A)细胞如何利用外切体及其货物进行通讯的外体RNA成像方法 以及(B)外切体如何调节其微环境并向远处的器官和组织移动。 现有的方法集中在通过基于脂质的荧光团标记脂膜或 胞外体蛋白标记。这些方法都不允许通过遗传编码或基因编码来跟踪外体RNA 条形码，在广泛的收集和改变步骤之后，不需要外源修改外体。 这项提案的总体目标是开发可遗传编码的RNA EXO-Code探针，以允许 胞外体RNA的多模式跟踪和成像。EXO代码探测器将允许多模式跟踪 外体RNA通过(1)遗传编码，(2)荧光染料非破坏性标记和(3)独特 基于条形码的识别和量化。这种组合是强大的，因为它允许跟踪 通过多种模式的外体RNA进行高含量的生物分布图谱。因为EXO-Code条形码 它们由独特的核苷酸序列组成，可以使用具有敏感性的测序来准确解码 阿托莫尔范围。与荧光RNA适配子的结合允许互补追踪 通过简单地与染料孵育来获得外体RNA。荧光外体工具包将被开发用于 研究人员检测膜稳定性、胞外体融合事件和内吞过程的破坏情况 外切体生物发生、分布和摄取的结果。这将使研究人员能够追踪外体RNA 通过生物体、细胞和它们在亚细胞隔间中的最终目的地。

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.