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

exRNA 载体的新型分离方法：细胞外囊泡、脂蛋白颗粒和蛋白质聚集体

基本信息

批准号：
10483185
负责人：
Kenneth W Witwer
金额：
$ 54.9万
依托单位：
JOHNS HOPKINS UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2019
资助国家：
美国
起止时间：
2019-08-01 至 2024-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10483185
关键词：
Affinity Age Analytical Chemistry Antibodies Attention Binding Biochemical Biological Biological Factors Biology Catalogs Cells Centrifugation Characteristics Charge Data Set Detection Development Diet Disease Engineering Ensure Field Flow Fractionation Gold Health High Density Lipoproteins Higher Order Chromatin Structure Immobilization Intervention Studies Libraries Lipid Bilayers Lipoproteins Liquid substance Location Low-Density Lipoproteins MLLT2 gene Measures Methodology Methods MicroRNAs Modification Molecular Morphologic artifacts Phase Process Proteomics RNA Resolution Ribonucleases Ribonucleoproteins Sampling Speed Surface Techniques Technology Testing Therapeutic Time Tissues Validation base biological sex density design extracellular extracellular vesicles improved inhibitor lipidomics liquid biopsy member molecular carrier novel particle preservation protein aggregation sample collection screening sensor sex validation studies

项目摘要

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.

抽象的细胞外RNA（EXRNA）是液体的特别有吸引力的分子成分，因为RNA 物种可以特别放大。（EVS），脂蛋白颗粒（LPP）和游离核糖核蛋白（RNP） - Eve到目前为止收到的最多注意每个班级，这也是大小，密度的物理特征的巨大多样性据我们所知，表面充电。与同一样品严格隔离的三个载体类别。制定新的策略和控制，以确保Exrna载体的比较不会被共同混淆隔离（相同分数中存在的不同类别的载体）或污染和/或在样本收集和处理过程中引入的外国RNA。电动汽车，LPP和RNP的专家，以及前沿分离和表征方法的专家。在最初的UG3阶段，我们将首先（AIM 1）使用现场物理和生化的组合分离方法将八个亚型的电动汽车，LPP和RNP的库与同一生物学分开。 “黄金标准”蛋白质组学，脂质组，糖和rNomic，rnomic，rnomic，rnomic和rnomic和rnomic，rnomic 数据集将被生成精心设计的“过程”控件污染物的基线和其他可以在AIM 2中进行的工件。不对称的场流分馏（AF4）和亲和力捕获平台，包括Exoview平台和敏感的电化学传感器是最常用的遗产方法的优越选择，与传统技术相比，我们将寻求速度，分辨率和纯度的提高。如果在第二年结束时满足GO/No-Go标准（UG3）将包括一个目标3，验证多个位置的结果，大约是原始样本的6倍为了进一步开发的性别和年龄的影响。基于我们的工程分析化学专业知识的修改，而Exoview技术将是利用筛选抗体和其他材料进行快速隔离并检测丰富的RNA 直接在固定的埃克纳携带者中，目标4将评估饮食的生物学因素干预研究中的宝贵样本，以及在RNase中收集样品的可能性抑制剂保留更脆弱的RNA物种，我们假设1）AF4或方法论修改以及2）新型亲和力分离方法将在很大程度上改善基于纯度和纯度的超级离心方法以及当前最先进的乏味和冗长的方法 EXRNA载体分离方法。

项目成果

期刊论文数量（11）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Circulating SNORD57 rather than piR-54265 is a promising biomarker for colorectal cancer: common pitfalls in the study of somatic piRNAs in cancer.

DOI：
10.1261/rna.078444.120
发表时间：
2021-04
期刊：
RNA (New York, N.Y.)
影响因子：
0
作者：
Tosar JP;García-Silva MR;Cayota A
通讯作者：
Cayota A

Letter by Halushka and Witwer Regarding Article, "Circulating MicroRNA-122-5p Is Associated With a Lack of Improvement in Left Ventricular Function After Transcatheter Aortic Valve Replacement and Regulates Viability of Cardiomyocytes Through Extracellula

Halushka 和 Witwer 关于文章“循环 MicroRNA-122-5p 与经导管主动脉瓣置换术后左心室功能缺乏改善有关，并通过细胞外调节心肌细胞的活力”的信函

DOI：
10.1161/circulationaha.122.061834
发表时间：
2023
期刊：
Circulation
影响因子：
37.8
作者：
Halushka,MarcK;Witwer,KennethW
通讯作者：
Witwer,KennethW

RI-SEC-seq: Comprehensive Profiling of Nonvesicular Extracellular RNAs with Different Stabilities.

RI-SEC-seq：具有不同稳定性的非囊泡细胞外 RNA 的综合分析。

DOI：
10.21769/bioprotoc.3918
发表时间：
2021
期刊：
Bio-protocol
影响因子：
0.8
作者：
Tosar,JuanPablo;Gámbaro,Fabiana;Castellano,Mauricio;Cayota,Alfonso
通讯作者：
Cayota,Alfonso

Lipid-Based Nutrient Supplementation Increases High-Density Lipoprotein (HDL) Cholesterol Efflux Capacity and Is Associated with Changes in the HDL Glycoproteome in Children.

DOI：
10.1021/acsomega.1c04811
发表时间：
2021-11-30
期刊：
ACS omega
影响因子：
4.1
作者：
Hong BV;Zhu C;Wong M;Sacchi R;Rhodes CH;Kang JW;Arnold CD;Adu-Afarwuah S;Lartey A;Oaks BM;Lebrilla CB;Dewey KG;Zivkovic AM
通讯作者：
Zivkovic AM

Urinary extracellular vesicles contain mature transcriptome enriched in circular and long noncoding RNAs with functional significance in prostate cancer.