Enabling exosome biomarker development via digitized single vesicle analysis

通过数字化单囊泡分析实现外泌体生物标志物的开发

基本信息

批准号：
10359052
负责人：
Don L DeVoe
金额：
$ 42.83万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
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：
2019-02-01 至 2024-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10359052
关键词：
Alkanes Antibodies Biological Biological Assay Biological Markers Cholesterol Clinical DNA DNA Sequence DNA-Directed DNA Polymerase DNA-Directed RNA Polymerase Deposition Detection Devices Disease Exonuclease Fluorescence Resonance Energy Transfer Generations Genetic Transcription Goals Heterogeneity Individual Investigation Label Liquid substance Measurement Membrane Proteins Methods MicroRNAs Microfluidic Microchips Modeling Molecular Analysis Nucleic Acids Oils Oligonucleotides Periodicity Poisson Distribution Polymers Population Population Analysis Population Heterogeneity Proteins RNA RNA Sequences RNA amplification Reaction Reagent Sampling Sensitivity and Specificity Specificity Stroke System Techniques Technology Time Vesicle amplification detection base biomarker development biomarker discovery circulating biomarkers circulating microRNA controlled release cost design diagnostic biomarker digital exosome frontier high throughput analysis improved isothermal amplification microsystems molecular marker novel operation potential biomarker protein biomarkers scale up seal statistics tool

项目摘要

PROJECT SUMMARY/ABSTRACT Exosomes represent the next “omic” frontier in diagnostic biomarkers. As such, numerous technologies have been developed to exploit detection of exosomal proteins or microRNA (miRNA) cargo for potential biomarker development. In general, these approaches are based on the assumption that detection of a particular exosomal protein or miRNA profile will correlate with a specific disease. Yet, this assumption is undermined by the inherent variability resulting from heterogeneity of exosome populations collected from biological fluids. Further, previous attempts to develop protein and, especially, miRNA-based biomarkers have revealed that a single miRNA sequence can be correlated with numerous diseases even without the confounding heterogeneity of exosomes. Thus, we hypothesize that to achieve appropriate specificity for the discovery of exosomal biomarkers, correlation of exosomal proteins and miRNAs – simultaneously detected and resolvable at the single-exosome level – is necessary. Here we propose a highly dense multiplexed microsystem implementing a new isothermal nucleic acid amplification method for digital exosome analysis, enabling the specific correlation of membrane protein markers and miRNA sequences at the single exosome level. A low- cost and easy-to-use thermoplastic chip with integrated amplification reagents enables self-discretization of exosomes for spatially multiplexed analysis, scalable up to one million reactions. For specific detection of miRNA sequences and membrane proteins, we have designed an isothermal reaction optimized for the amplification of short DNA and RNA sequences that identifies miRNA sequences as well as DNA oligonucleotides conjugated to antibodies that label the exosome surface proteins. The reaction, referred to as transcription cycling amplification (TCA), utilizes DNA polymerase with exonuclease activity to degrade donor/quencher-labeled probes (i.e., FRET probes) for specific and real-time quantitative detection. In tandem, RNA polymerase acts on the polymerized oligo for the cyclical generation of RNA that leads to exponential amplification, providing highly sensitive detection. By integrating these two tools, we will develop a biomarker discovery platform that first digitizes the exosomes across a dense reaction array and correlates miRNA sequences with membrane proteins in single exosomes.

项目概要/摘要外泌体代表了诊断生物标志物的下一个“组学”前沿，因此，许多技术已经出现。已开发用于利用外泌体蛋白或 microRNA (miRNA) 货物的检测来寻找潜在的生物标志物一般来说，这些方法基于检测特定开发的假设。外泌体蛋白或 miRNA 谱与特定疾病相关，然而，这一假设被破坏了。从生物体液中收集的外泌体群体的异质性导致的固有变异性。此外，之前开发蛋白质，特别是基于 miRNA 的生物标志物的尝试表明，即使没有混杂因素，单个 miRNA 序列也可以与多种疾病相关因此，我们追求外泌体的异质性，以实现适当的特异性以发现外泌体。外泌体生物标志物、外泌体蛋白和 miRNA 的相关性 – 同时检测和解析在单外泌体水平上 - 是必要的。实施一种用于数字外泌体分析的新等温核酸扩增方法，使膜蛋白标记物和 miRNA 序列在单个外泌体水平上的特异性相关性。具有集成放大试剂的成本和易于使用的热塑性芯片可以实现自我离散化用于空间多重分析的外泌体，可扩展至一百万个反应，用于特定检测。 miRNA 序列和膜蛋白，我们设计了针对扩增短 DNA 和 RNA 序列，识别 miRNA 序列以及 DNA 与标记外泌体表面蛋白的抗体缀合的反应，称为转录循环扩增（TCA），利用具有核酸外切酶活性的DNA聚合酶进行降解用于特异性和实时定量检测的供体/淬灭剂标记探针（即 FRET 探针）。 RNA 聚合酶作用于聚合寡核苷酸，循环生成 RNA，从而导致指数增长通过整合这两种工具，我们将开发一种生物标记物。发现平台，首先将密集反应阵列中的外泌体数字化并关联 miRNA 单个外泌体中膜蛋白的序列。

项目成果

期刊论文数量（13）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Assessment of anti-inflammatory bioactivity of extracellular vesicles is susceptible to error via media component contamination.

细胞外囊泡抗炎生物活性的评估很容易因培养基成分污染而出错。

DOI：
10.1016/j.jcyt.2022.12.002
发表时间：
2023
期刊：
Cytotherapy
影响因子：
4.5
作者：
Kronstadt,StephanieM;VanHeyningen,LaurenHoorens;Aranda,Amaya;Jay,StevenM
通讯作者：
Jay,StevenM

Chitosan Particles Complexed with CA5-HIF-1α Plasmids Increase Angiogenesis and Improve Wound Healing.

DOI：
10.3390/ijms241814095
发表时间：
2023-09-14
期刊：
International journal of molecular sciences
影响因子：
5.6
作者：
Born LJ;Bengali S;Hsu ATW;Abadchi SN;Chang KH;Lay F;Matsangos A;Johnson C;Jay SM;Harmon JW
通讯作者：
Harmon JW

Enhanced Loading of Functional miRNA Cargo via pH Gradient Modification of Extracellular Vesicles