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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.

项目摘要/摘要外切体代表了诊断生物标记物的下一个“组学”前沿。因此，许多技术都有已开发用于利用外体蛋白或microRNA(MiRNA)货物的检测作为潜在的生物标记物发展。一般而言，这些方法基于这样的假设：检测到特定的外体蛋白或miRNA图谱将与特定疾病相关。然而，这一假设被以下因素破坏了从生物体液中收集的外体种群的异质性所产生的内在变异性。此外，以前开发蛋白质，特别是基于miRNA的生物标记物的尝试表明，即使没有混淆，单个miRNA序列也可以与多种疾病相关外切体的异质性。因此，我们假设，为了获得适当的特异性，发现外体生物标记物，外体蛋白和miRNAs的相关性--同时检测和可分辨在单一外显体水平上是必要的。在这里，我们提出了一种高密度的多路传输微系统实施一种新的用于数字外切体分析的等温核酸放大方法，使膜蛋白标记物和miRNA序列在单个外切体水平上的特异性相关性。一个低点- 具有集成扩增试剂的成本低、使用方便的热塑性芯片使用于空间多重分析的外切体，可扩展到一百万个反应。对于特定的检测 MiRNA序列和膜蛋白，我们设计了一种优化的等温反应扩增短DNA和RNA序列，以识别miRNA序列和DNA 寡核苷酸连接到标记外体表面蛋白的抗体上。这种反应，被称为转录循环扩增(TCA)，利用具有核酸外切酶活性的DNA聚合酶来降解供体/猝灭剂标记的探针(即FRET探针)，用于特异性和实时定量检测。同时， RNA聚合酶作用于聚合的寡核苷酸，循环产生RNA，从而导致指数放大，提供高灵敏度的检测。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，首先对密集反应阵列中的外切体进行数字化，并将miRNA 在单个外切体中含有膜蛋白的序列。

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.

与CA5-HIF-1α质粒复合的壳聚糖颗粒会增加血管生成并改善伤口愈合。

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