Aptamers for Analysis of Extracellular Vesicles

在研究计划资助中，我提出了细胞外小泡(EVS)的亲和分离、EVS的检测和计数以及EV生物标志物的综合分析的新技术。EVS是一种小的膜封闭结构，由多种细胞分泌到细胞外环境中。这些颗粒包括小的(外切体，30-150 nm)和大的(微泡，100,1000 nm；凋亡小体，50,000 nm)小泡。这些囊泡由细胞分泌，并在体内不同的液体中释放。电动汽车包含一个脂质双层，可以保护货物不被降解，这些货物可能由核酸、蛋白质、脂类和代谢物组成。电动汽车现在是生物科学的核心，因为它们似乎构成了一种新的细胞通讯系统。 由于EVS的货物反映了细胞的精确状态，因此将EVS用于生物标志物分析一直是人们非常感兴趣的事情。EVS是创伤性脑损伤(TBI)和创伤后应激障碍(PTSD)等疾病的丰富生物标志物来源。最近，从携带母细胞蛋白质和核酸的脑细胞衍生的EV可以在外周血和唾液中检测到跨越血脑屏障的EV，这为评估PTSD和脑创伤后神经元和胶质细胞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项新技术，用于生产电动汽车的特定亲和力探针，称为核酸适配子。由此产生的适配子将具有可切换的结合和对来自脑细胞的外切体和微囊的高选择性。还将开发和出版两种电动汽车隔离技术(磁珠和柱基)，以造福电动汽车研究界。我们希望从人体样本中实现分离的EV的高回收率，用于下游应用，如miRNA和蛋白质分析。我们将开发和分享至少两项基于毛细管电泳法和质谱仪的技术，用于全面分析电动汽车的蛋白质和microRNAs。通过这些方法获得的数据将纳入公共数据库。特别是，我们将开发一种突破性的诊断方法，基于适体辅助分离脑EV和分析TBI/PTSD生物标记物来区分损伤状态。 从长远来看，我计划对适配子的选择、结构、功能以及相关的结合伙伴进行大规模的研究。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来研究电动汽车。总的来说，我将讨论电动汽车的起源和最终目的地、分子多样性和生物功能等基本问题。脑细胞衍生电动汽车的生物检测的发展将为研究脑外伤、创伤后应激障碍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.