Water Soluble Nanoarrays for Single Cell Proteomics

用于单细胞蛋白质组学的水溶性纳米阵列

• 批准号: 8473226
• 负责人: Hao Yan
• 金额: $ 28.77万
• 依托单位: ARIZONA STATE UNIVERSITY-TEMPE CAMPUS
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-08-01 至 2015-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8473226
• 关键词: Address; Adsorption; Affinity; Apoptosis; Atomic Force Microscopy; Base Sequence; Behavior; Binding; Biochemical; Biodegradation; Cell Cycle Arrest; Cells; Cellular Structures; Cytolysis; DNA; DNA Damage; DNA Repair; Data; Deposition; Detection; Detergents; Development; Disease; Elements; Engineering; Environment; Gene Expression; Generations; Genome; Genomics; Goals; Health; Histone H4; Image; Incubated; Individual; Kinetics; Knowledge; Language; Lead; Life; Ligands; Location; Lysine; Malignant Neoplasms; Methodology; Microfluidic Microchips; Microfluidics; Modeling; Modification; Nanoarray Analytical Device; Nanotechnology; Nucleic Acid Hybridization; Nucleic Acids; Nucleic acid sequencing; Organism; Pathology; Pattern; Polymerase Chain Reaction; Positioning Attribute; Post-Translational Protein Processing; Preparation; Printing; Protein Analysis; Protein Binding; Protein p53; Proteins; Proteomics; Protocols documentation; Reading; Reagent; Resolution; Rest; Role; Sampling; Scanning; Single Parent; Site; Sodium Chloride; Solutions; Specificity; Surface; System; Systems Biology; Technology; Testing; Time; Titrations; Tumor Suppressor Proteins; Variant; Water; Work; aptamer; base; density; human CCDC6 protein; improved; interest; lithography; molecular array; molecular recognition; molecular scale; nanolitre; nanometer; nanoscale; new technology; protein distribution; response; scaffold; self assembly; single cell analysis; single molecule; success; thrombin aptamer; tool; Address; Adsorption; Affinity; Apoptosis; Atomic Force Microscopy; Base Sequence; Behavior; Binding; Biochemical; Biodegradation; Cell Cycle Arrest; Cells; Cellular Structures; Cytolysis; DNA; DNA Damage; DNA Repair; Data; Deposition; Detection; Detergents; Development; Disease; Elements; Engineering; Environment; Gene Expression; Generations; Genome; Genomics; Goals; Health; Histone H4; Image; Incubated; Individual; Kinetics; Knowledge; Language; Lead; Life; Ligands; Location; Lysine; Malignant Neoplasms; Methodology; Microfluidic Microchips; Microfluidics; Modeling; Modification; Nanoarray Analytical Device; Nanotechnology; Nucleic Acid Hybridization; Nucleic Acids; Nucleic acid sequencing; Organism; Pathology; Pattern; Polymerase Chain Reaction; Positioning Attribute; Post-Translational Protein Processing; Preparation; Printing; Protein Analysis; Protein Binding; Protein p53; Proteins; Proteomics; Protocols documentation; Reading; Reagent; Resolution; Rest; Role; Sampling; Scanning; Single Parent; Site; Sodium Chloride; Solutions; Specificity; Surface; System; Systems Biology; Technology; Testing; Time; Titrations; Tumor Suppressor Proteins; Variant; Water; Work; aptamer; base; density; human CCDC6 protein; improved; interest; lithography; molecular array; molecular recognition; molecular scale; nanolitre; nanometer; nanoscale; new technology; protein distribution; response; scaffold; self assembly; single cell analysis; single molecule; success; thrombin aptamer; tool

DESCRIPTION (provided by applicant): Remarkable progress in single cell genomics rests on the polymerase chain reaction. There is no analogous tool to probe the cell-to-cell distribution of protein diversity and posttranslational modification (PTM) patterns at the few- or single-cell level. If protein-capture arrays can be synthesized on a molecular scale, then the arrays themselves become reagents suitable for incubation with the contents of even a single cell. Molecular arrays could be used in titrations, giving a potentially enormous dynamic detection range and facilitating quantification of the protein content and PTMs for each generation of the progeny of a single parent cell. Nanotechnology already provides the components for such a system, and we propose to integrate four new technologies to develop self-assembled nanoarrays for protein analysis. The first is construction of an array of single molecule probes arranged at nanometer density using nucleic acid self-assembly. The arrays are themselves giant molecules and are used like a reagent in a solution analysis, only being deposited onto a surface for a final readout. The second is a nanoscale readout using atomic force microscopy (AFM).The AFM is capable of reading out >30,000 array sites per minute. The third is the development of new molecular recognition elements based on aptamers and multivalent aptamers for proteins and their PTM variants. Because aptamers are synthetic and also nucleic acid sequences, individual aptamers can bind to a unique position on the array through nucleic acid hybridization and require no printing or lithography. The fourth is microfluidic technology to allow the nanoarrays to interact with lysed or intact cells, and to deliver the reacted arrays to predefined locations for readout. Our goal is to fill a unique niche in proteomics: parallel analysis of minute amounts of protein from small numbers of (potentially individual) cells. Can we make suitable ligands and will they work on arrays? Can we read the arrays accurately with AFM? Can we synthesize ligands that are highly selective for posttranslational modifications? What factors cause biodegradation of the arrays, and how can we control them while maintaining sensitivity and selectivity? What conditions are required to exploit the arrays for proteomics in a microfluidic system? What methodologies can we employ to deposit nanoliter solutions of reacted arrays at precise locations for AFM readout? These questions are the focus of this proposal. If successful, this work will make it possible to correlate nucleic acid diversity with the corresponding protein PTM diversity on a cell-by- cell basis, yielding, for the first time, data on the interplay between genome, gene expression, the environment and the spectrum of protein posttranslational modifications: the 'protein language'.

描述（由申请人提供）：单细胞基因组学的显着进展取决于聚合酶链反应。没有类似工具可以探测蛋白质多样性和翻译后修饰（PTM）模式在几个或单细胞水平上的细胞分布。如果可以以分子尺度合成蛋白质捕获阵列，则阵列本身成为适合与单个细胞含量孵育的试剂。分子阵列可用于滴定，从而为每一生的单亲细胞的后代提供了潜在的巨大动态检测范围，并促进了蛋白质含量和PTM的定量。纳米技术已经为这种系统提供了组件，我们建议整合四种新技术，以开发自组装的纳米阵列进行蛋白质分析。首先是使用核酸自组装以纳米密度排列的一系列单分子探针阵列。阵列本身是巨型分子，在溶液分析中像试剂一样使用，仅沉积在表面上以进行最终读数。第二个是使用原子力显微镜（AFM）的纳米级读数。AFM能够每分钟读取> 30,000个阵列位点。第三是基于适体和蛋白质及其PTM变体的多价适体的新分子识别元件的发展。由于适体是合成的，也是核酸序列，因此单个适体可以通过核酸杂交与阵列上的独特位置结合，不需要打印或光刻。第四是微流体技术，允许纳米阵列与裂解或完整的细胞相互作用，并将反应阵列传递到预定义的位置进行读数。我们的目标是填补蛋白质组学的独特利基：对少量（潜在个体）细胞的微小蛋白质的平行分析。我们可以制作合适的配体，它们可以在阵列上工作吗？我们可以使用AFM准确阅读阵列吗？我们可以合成对翻译后修饰具有高度选择性的配体吗？哪些因素导致阵列的生物降解，以及在保持灵敏度和选择性的同时如何控制它们？利用微流体系统中的蛋白质组学阵列需要什么条件？我们可以采用哪些方法将反应阵列的纳米材料解决方案存放在AFM读数的精确位置？这些问题是该提议的重点。如果成功，这项工作将使核酸的多样性与相应的蛋白质PTM多样性在细胞基础上相关联，从而首次屈服于基因组，基因表达，环境，环境和蛋白质后蛋白质后的相互作用数据的数据：“蛋白质语言”。