Aptamers for Analysis of Extracellular Vesicles

在研究项目资助中，我提出了细胞外囊泡（EV）亲和分离、EV检测和计数以及EV生物标志物综合分析的新技术。EV是由许多细胞类型分泌到细胞外环境中的小的膜封闭结构。这些颗粒包括小（外来体，30-150 nm）和大（微泡，100 - 1000 nm;凋亡小体，50 - 5000 nm）囊泡。这些囊泡从细胞中分泌并在身体的不同液体中释放。EV含有保护货物的脂质双层，所述货物可以由核酸、蛋白质、脂质和代谢物组成，免于降解。电动汽车现在是生物科学的核心，因为它们似乎构成了一个新的细胞通讯系统。 由于EV的货物反映了细胞的精确状态，因此在生物标志物分析中利用EV具有重大意义。EV是创伤性脑损伤（TBI）和创伤后应激障碍（PTSD）等疾病的生物标志物的丰富来源。最近，人们认识到，来自脑细胞的EV携带来自其母细胞的蛋白质和核酸并穿过血脑屏障，可以在外周血和唾液中检测到，这为评估PTSD期间和TBI后神经元和神经胶质的分子变化提供了新的机会。 在接下来的5年里，我和我的研究团队计划提供两种新技术，用于生产针对EV的特异性亲和探针，称为核酸适体。所得适体将对源自脑细胞的外来体和微泡具有可切换的结合和高选择性。两个电动汽车隔离技术（磁珠和柱为基础）也将开发和出版，为电动汽车研究界的利益。我们期望从人类样品中获得高回收率的分离EV，用于下游应用，如miRNA和蛋白质测定。我们将开发和分享至少两种基于毛细管电泳和质谱的技术，用于对电动汽车的蛋白质和microRNA进行综合分析。通过这些方法获得的数据将被纳入公共数据库。特别是，我们将开发一种基于适配体辅助分离脑EV和分析TBI/PTSD生物标志物以区分损伤状态的突破性诊断方法。 从长远来看，我计划在适体的选择，它们的结构，功能和相关的结合伴侣方面进行大规模的研究。我想在miRNA数据和生物信息学分析的支持下，使用蛋白质组学和代谢组学中的高通量方法研究EV。总的来说，我将解决EV起源和最终目的地，分子多样性和生物功能的基本问题。脑细胞衍生EV的生物测定的发展将为研究TBI，PTSD和退行性脑疾病的基本过程的研究人员带来新的分析工具。

In the research program grant, I propose new technologies for the affinity isolation of extracellular vesicles (EVs), the detection and counting of EVs, and comprehensive analysis of EV biomarkers. EVs are small membrane-enclosed structures that are secreted into the extracellular milieu by many cell types. These particles include small (exosomes, 30-150 nm), and large (microvesicles, 100 1000 nm; apoptotic bodies, 50 5000 nm) vesicles. These vesicles are secreted from cells and released in different fluids of the body. EVs contain a lipid bilayer that protects the cargo, which can consist of nucleic acids, proteins, lipids and metabolites, from degradation. EVs are now central to biological sciences because they seem to constitute a new system of cellcell communication. Since the cargo of EVs reflects the precise state of a cell, there has been significant interest in utilizing EVs in biomarker analysis. EVs are a plentiful source of biomarkers of traumatic brain injury (TBI) and posttraumatic stress disorder (PTSD) among other conditions. Recently, the recognition that EVs derived from brain cells that carry proteins and nucleic acid from their mother cells and cross the blood brain barrier can be detected in peripheral blood and saliva, offers a new opportunity to evaluate molecular changes in neurons and glia during PTSD and after TBI. Over the next 5 years, my research team and I plan to provide two novel technologies for the production of specific affinity probes for EVs, named nucleic acid aptamers. The resulting aptamers will have switchable binding and high selectivity to exosomes and microvesicles originating from brain cells. Two EV isolation technologies (magnetic bead and column - based) will be also developed and published, for the benefit of the EV research community. We expect to achieve high recovery of isolated EVs from human samples for downstream applications such as miRNA and protein assays. We will develop and share at least two technologies based on capillary electrophoresis and mass spectrometry for comprehensive analysis of proteins and microRNAs of EVs. The data obtained from these approaches will be integrated into public databases. In particular, we will develop a breakthrough diagnostic based on aptamer-assisted isolation of brain EVs and analysis of TBI/PTSD biomarkers to discriminate injury states. In the long-term, I plan to perform large-scale research on selection of aptamers, their structures, functions and related binding partners. I want to study EVs using high-throughput approaches in proteomics and metabolomics, supported by miRNA data and bioinformatic analysis. In general, I will address fundamental issues of EV origins and final destinations, molecular diversity and biological functions. The development of bioassays for brain cell-derived EVs will bring new analytical tools for researchers investigating fundamental processes of TBI, PTSD and degenerative brain disorders.