Novel Separation Methods for exRNA Carriers: Extracellular Vesicles, Lipoprotein Particles, and Protein Aggregates

摘要 细胞外RNA（exRNA）是液体活检的特别有吸引力的分子组分，因为RNA 物种可以特异性扩增。在三种主要的exRNA载体-细胞外囊泡中， (EVs)脂蛋白颗粒（LPP）和游离核糖核蛋白（RNP）-EV迄今为止收到的最多 关注在每一个类，也有巨大的多样性的物理特征的大小，密度， 表面电荷事实上，据我们所知，迄今为止还没有研究在多个成员中描述exRNA。 三种携带者类别已经从相同的样本中严格分离出来。迫切需要 开发新的策略和控制措施，以确保exRNA载体的比较不受共 分离（在同一级分中存在不同种类的载体）或污染（检测未复合的 和/或在样品收集和处理过程中引入的外源RNA）。为此，我们组建了一个 电动汽车，LPP和RNP的专家，沿着尖端分离和表征方法的专家。 在最初的UG 3阶段，我们将首先（目标1）使用最先进的物理和生物化学的组合， 分离方法，以从相同的生物样品中分离EV、LPP和RNP的八种亚型的文库， 样品和最佳可实现的纯度。“黄金标准”蛋白质组学、脂质组学、糖组学和RNA组学 将生成数据集。精心设计的“过程”控制将首次建立一个全面的 污染物和其他可能使解释复杂化的人为因素的板基线。在目标2中，我们将测试 不对称场流分级分离（AF 4）和亲和捕获平台，包括ExoView平台和 灵敏的电化学传感器作为最常用的传统方法的上级替代物， 差速离心法与传统技术相比，我们将寻求在速度、分辨率和纯度方面的收益。 如果在第二年（UG 3）结束时符合通过/不通过标准，我们将进入UH 3阶段。这个阶段 将包括Aim 3，在多个位置验证结果，使用大约6倍的原始样本 数字来解释性别和年龄的影响。AF 4方法将进一步发展， 基于我们的工程和分析化学专业知识进行修改，而ExoView技术将 用于筛选抗体和其他亲和材料，以快速分离并检测大量RNA 物种直接在固定的exRNA载体中。最后，目标4将评估饮食的生物因素， 从干预研究中获得有价值的样本，沿着可能需要在RNase中收集样本 抑制剂来保护更脆弱的RNA种类。总的来说，我们假设1）AF 4，单独或与 方法学上的改进，以及2）新的亲和分离方法将大大改善 基于超微粒子的方法在简便和纯度和当前最先进的，但繁琐和冗长的 exRNA载体分离方法。

ABSTRACT Extracellular RNA (exRNA) is a particularly attractive molecular component of liquid biopsy because RNA species can be specifically amplified. Of the three major classes of exRNA vehicle—extracellular vesicles (EVs), lipoprotein particles (LPPs), and free ribonucleoproteins (RNPs)—EVs have so far received the most attention. Within each class, there is also tremendous diversity by physical characteristics of size, density, and surface charge. Indeed, to our knowledge, no study to date has profiled exRNA in multiple members of the three carrier classes that have been isolated rigorously from the same samples. There is a strong need to develop new strategies and controls to ensure that comparisons of exRNA carriers are not confounded by co- isolation (different classes of carriers present in the same fraction) or contamination (detection of uncomplexed and/or foreign RNAs introduced during sample collection and processing). To this end, we assemble a team of experts on EVs, LPPs, and RNPs, along with experts in cutting-edge separation and characterization methods. In an initial UG3 phase, we will first (Aim 1) use a combination of state-of-the-field physical and biochemical separation methods to separate a library of eight subtypes of EVs, LPPs, and RNPs from the same biological samples and with the best achievable purity. “Gold standard” proteomic, lipidomic, glycomic, and RNomic datasets will be generated. Carefully designed “process” controls will for the first time establish an across-the- board baseline of contaminants and other artifacts that may complicate interpretation. In Aim 2, we will test asymmetric field-flow fractionation (AF4) and affinity capture platforms including the ExoView platform and sensitive electrochemical sensors as superior alternatives to the most commonly used legacy method, differential centrifugation. We will seek gains in speed, resolution, and purity compared with legacy techniques. If go/no-go criteria are met by the end of the second year (UG3), we will proceed to a UH3 phase. This phase will include an Aim 3, validating results in multiple locations and with approximately 6 times the original sample numbers to account for influence of sex and age. The AF4 method will be further developed with additional modifications based on our engineering and analytical chemistry expertise, while ExoView technology will be harnessed to screen antibodies and other affinity materials for rapid isolations and to detect abundant RNA species directly in immobilized exRNA carriers. Finally, an Aim 4 will assess the biological factor of diet with valuable samples from intervention studies, along with the possible desirability of collecting samples in RNase inhibitors to preserve more fragile RNA species. Overall, we hypothesize that 1) AF4, on its own or with methodologic modifications, as well as 2) novel affinity separation approaches will improve substantially on ultracentrifuge-based methods in ease and purity and on current state-of-the-art but tedious and lengthy exRNA carrier separation methods.