Next Generation Glycan Microarray using DNA-coded glycans and Next Generation Sequencing (NGS)

使用 DNA 编码聚糖和下一代测序 (NGS) 的下一代聚糖微阵列

基本信息

批准号：
10455643
负责人：
Xuezheng Song
金额：
$ 33.54万
依托单位：
EMORY UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-09-15 至 2024-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10455643
关键词：
Address Affinity Antibodies Avidity Bacteria Bar Codes Binding Binding Proteins Biological Biological Assay Biological Process Biology Biomedical Research Carbohydrates Cell Communication Cells Chemicals Chemistry Clinical Research Code Collaborations Communities Complex Consumption Coupling Custom DNA DNA Binding DNA Library DNA Sequence DNA sequencing DNA-Binding Proteins Data Analyses Development Disease Funding Glass Glycoconjugates Glycoproteins Glycoside Hydrolases Goals Human Immobilization Immunize Immunoprecipitation Incubated Individual Investigation Investments Laboratories Lampreys Lectin Libraries Location Manuals Mediating Methods Microarray Analysis Microscope Oligonucleotides Pathway interactions Patients Polysaccharides Positioning Attribute Printing Procedures Process Proteins Protocols documentation Quality Control Reading Reagent Research Personnel Role Saliva Salivary Sampling Scanning Serum Shotguns Slide Solid Specificity Spottings Structure Substrate Specificity Surface System Techniques Technology Time United States National Institutes of Health Vial device Virus Yeasts base clinical investigation clinical translation commercialization cost experimental study fluorescence imaging glycosyltransferase image processing instrumentation interest microorganism next generation next generation sequencing novel oral bacteria prevent research and development screening success tool

项目摘要

Project Summary/Abstract The development and application of glycan microarray technology and the availability of a large glycan microarray to investigators by the NIH-funded Consortium for Functional Glycomics (CFG) in the last decade revolutionized functional studies in glycomics. Hundreds of investigator-driven projects were carried out during this time in collaboration with the CFG, and there is continued interest in glycan microarray based experiments for studies in Functional Glycomics. However, these studies continue to be associated primarily with laboratories specialized in manufacturing and processing the glycan microarrays due to the high cost of instrumentation and special expertise required, including our Emory Comprehensive Glycomics Core. One of the major goals of the NIH Common Fund investment in Glycoscience is to develop tools that will make glycomic studies more available to non-specialized laboratories. In order to address this issue, we used a Common Fund supported project to demonstrate the feasibility for developing the Next Generation Glycan Microarray (NGGM) that eliminates the high cost of arrayers and scanners by introducing DNA sequences as codes for glycans and Next Generation Sequencing (NGS) for decoding to amplify and analyze protein-glycan interactions. A microarray is essentially the presentation of a library of coded-molecules where the code for each individual structure is its physical location on the glass slide. By switching the physical location code to a DNA sequence, we eliminate the complexity of manufacturing and reading a physical microarray. For each glycan structure, we install a unique oligonucleotide sequence (code). The coded glycans are mixed together in a single vial and incubated with a potential glycan binding protein (GBP). The GBP-Glycan-DNA complexes are then separated from the mixture using a common immunoprecipitation procedure, and the oligonucleotide codes are amplified and quantitatively sequenced by NGS. Each unique code corresponds to a unique glycan structure, and the copy number of the sequence represents the amount of glycan bound, which will be directly proportional to relative affinity of the GBP to different glycans. Eliminating the cost of instrumentation and developing a technology that is familiar to most laboratories will make glycan microarray studies available to the biomedical R&D community by simply providing the appropriate library of DNA-coded glycans. The optimization of the NGGM and the development of a large library of DNA-coded glycans will address the limitations of the current glycan microarray format including limited numbers of available glycans, instrumentation costs and the labor intensive process that prevents screening large numbers of sample required for clinical investigations. We will also study the quantitative feature of this new platform. Furthermore, we will also develop glycan microarray analysis of intact cells including bacteria and yeast cells, which have been technically challenging using current glycan microarray format.

项目概要/摘要聚糖微阵列技术的开发和应用以及大聚糖的可用性 NIH 资助的功能糖组学联盟 (CFG) 在过去十年中向研究人员提供的微阵列彻底改变了糖组学的功能研究。在此期间开展了数百个由研究者驱动的项目这次与 CFG 合作，人们对基于聚糖微阵列的实验持续感兴趣用于功能糖组学的研究。然而，这些研究仍然主要与实验室相关由于仪器和成本高昂，专门从事聚糖微阵列的制造和加工所需的特殊专业知识，包括我们的埃默里综合糖组学核心。该组织的主要目标之一 NIH 共同基金对糖科学的投资旨在开发工具，使糖组学研究更容易实现到非专业实验室。为了解决这个问题，我们使用了共同基金支持的项目展示了开发下一代聚糖微阵列 (NGGM) 的可行性，该微阵列消除了通过引入DNA序列作为聚糖和下一代的代码，阵列仪和扫描仪的成本很高用于解码的测序 (NGS) 以放大和分析蛋白质-聚糖相互作用。微阵列本质上是编码分子库的呈现，其中每个单独结构的代码是其物理结构载玻片上的位置。通过将物理位置代码转换为 DNA 序列，我们消除了制造和读取物理微阵列的复杂性。对于每个聚糖结构，我们安装了一个独特的寡核苷酸序列（代码）。将编码的聚糖混合在一个小瓶中并与潜在的聚糖结合蛋白（GBP）。然后将 GBP-聚糖-DNA 复合物从混合物中分离出来使用常见的免疫沉淀程序，对寡核苷酸代码进行扩增和定量通过NGS测序。每个唯一的代码对应一个唯一的聚糖结构，以及该聚糖的拷贝数序列代表聚糖结合的量，其与聚糖的相对亲和力成正比 GBP 到不同的聚糖。消除仪器成本并开发熟悉的技术大多数实验室将通过简单的方式向生物医学研发界提供聚糖微阵列研究提供适当的 DNA 编码聚糖文库。 NGGM的优化和发展大型 DNA 编码聚糖文库将解决当前聚糖微阵列格式的局限性，包括可用聚糖数量有限、仪器成本和劳动密集型过程阻碍了筛选临床研究所需的大量样本。我们还将研究定量特征这个新平台的。此外，我们还将开发完整细胞的聚糖微阵列分析，包括细菌和酵母细胞，使用当前的聚糖微阵列格式在技术上具有挑战性。