Developing genetically encodable probes for multimodal tracking of exosomal RNA cargo

开发用于外泌体 RNA 货物多模式追踪的基因可编码探针

• 批准号: 10681827
• 负责人: Juliane Nguyen
• 金额: $ 38.88万
• 依托单位: UNIV OF NORTH CAROLINA CHAPEL HILL
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-09-15 至 2027-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10681827
• 关键词: Area; Bar Codes; Base Sequence; Biodistribution; Biogenesis; Biological Process; Cancer Biology; Cells; Code; Collection; Communication; Destinations; Discipline; Disease; Distant; Dyes; Effectiveness; Embryo; Environment; Epigenetic Process; Event; Fluorescent Dyes; Gene Expression; Generations; Genetic; Genetic Transcription; Goals; Health; Human; Image; Immunology; Incubated; Label; Lipids; MDA MB 231; Macrophage; Maps; Membrane; Membrane Lipids; Mesenchymal Stem Cells; Methods; Modeling; Morphogenesis; Movement; Multimodal Imaging; Mus; Nature; Neurobiology; Organ; Organism; Phenotype; Physiological; Play; Process; Proteins; Publishing; RNA; Reagent; Research; Research Personnel; Role; Sorting; Suspensions; Technology; Time; Tissues; Travel; Tumor-Derived; Untranslated RNA; Vesicle; Work; aptamer; cancer cell; cell behavior; cell type; design; exosome; extracellular vesicles; fluorophore; imaging modality; imaging probe; in vivo; in vivo imaging system; intercellular communication; kidney cell; multimodality; new technology; novel; novel marker; single cell analysis; tool; trafficking; tumor; uptake

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.

项目摘要 外泌体，也被称为小的细胞外囊泡，在细胞通讯中起重要作用。 生理和病理生理条件。外泌体含有广泛的短和长的非- 调节基因表达的许多方面的编码RNA，包括调节 细胞命运、表型、极化和形态发生。尽管这些机构发挥着重要的职能作用， 尽管存在活的外泌体RNA，但目前没有允许活的外泌体RNA追踪的方法。这是因为RNA 本质上是非荧光的，并且难以标记，同时保持其预期的生物学功能。获得 外泌体RNA由于每个RNA种类以极低拷贝存在而进一步复杂化 exosomes的数量。这强调了对一种能够跟踪细胞间 外泌体RNA的移动，以及增强RNA加载到外泌体中的需要。 鉴于外泌体RNA在决定细胞行为方面的核心重要性，需要和要求 外泌体RNA成像方法，以确定（a）细胞如何使用外泌体及其货物进行通信 以及（B）外泌体如何调节它们的微环境并行进到远处的器官和组织。 现有方法的重点是通过基于脂质的荧光团标记脂膜来追踪外泌体，或 外泌体蛋白标记。这些方法都不允许通过遗传编码或基因测序来追踪外泌体RNA。 在广泛的收集和改变步骤后，无需外源修饰外泌体的条形码化。 该提案的总体目标是开发可遗传编码的RNA EXO-Code探针， 外泌体RNA的多模式追踪和成像。EXO-Code探测器将允许多模式跟踪 外泌体RNA通过（1）遗传编码，（2）荧光染料非破坏性标记，和（3）独特的 基于条形码的鉴定和定量。这种组合是强大的，因为它允许跟踪 外泌体RNA通过多种模式用于高含量生物分布作图。因为EXO-Code条形码是 它们由独特的核苷酸序列组成，可以使用测序准确解码， 阿托摩尔范围。与荧光RNA适体的组合允许互补追踪 外泌体RNA通过简单的染料孵育。荧光外泌体工具包将被开发用于 研究人员检测膜稳定性、外泌体融合事件和内吞过程的破坏，作为一种 外泌体生物发生、分布和摄取的结果。这将使研究人员能够追踪外泌体RNA 通过有机体、细胞和它们在亚细胞区室中的最终目的地。