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

• 批准号: 10671639
• 负责人: Xuezheng Song
• 金额: $ 33.54万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 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/10671639
• 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; clinical investigation; clinical translation; commercialization; cost; experimental study; fluorescence imaging; glycosyltransferase; image processing; instrumentation; interest; large print; manufacture; meter; microorganism; next generation; next generation sequencing; novel; oral bacteria; prevent; research and development; screening; success; tool

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.

项目摘要/摘要 聚糖微阵列技术的开发和应用以及大型聚糖的可用性 NIH资助的功能性糖基质（CFG）在过去十年 彻底改变了糖基质的功能研究。在 这次与CFG合作，对基于聚糖微阵列实验的持续感兴趣 用于功能性糖化的研究。但是，这些研究继续主要与实验室有关 由于仪器的高成本和 需要的特殊专业知识，包括我们的Emory综合糖基质核心。主要目标之一 NIH共同基金对糖科学的投资是开发可以使糖研究更多可用的工具 致非专业实验室。为了解决这个问题，我们使用了一个普通基金的项目来 证明开发下一代聚糖微阵列（NGGM）的可行性 通过引入DNA序列作为聚糖和下一代的代码，阵列和扫描仪的高成本很高 测序（NGS）用于解码和分析蛋白质 - 聚糖相互作用。微阵列本质上是 呈现一个编码 - 分子的库，其中每个单独的结构的代码是其物理的 位于玻璃滑梯上。通过将物理位置代码切换到DNA序列，我们消除了 制造和阅读物理微阵列的复杂性。对于每个聚糖结构，我们安装一个唯一的 寡核苷酸序列（代码）。编码的聚糖在单个小瓶中混合在一起，并与 潜在的聚糖结合蛋白（GBP）。然后将GBP-GlyCan-DNA复合物与混合物分离 使用常见的免疫沉淀程序，并放大寡核苷酸代码并进行定量 由NGS测序。每个唯一的代码对应于唯一的聚糖结构，以及 序列代表聚糖结合的量，该量将与该结合的相对亲和力成正比 英镑到不同的聚糖。消除仪器的成本并开发一项熟悉的技术 大多数实验室将简单地通过生物医学研发社区获得聚糖微阵列研究 提供适当的DNA编码聚糖库。 NGGM的优化和 大型DNA编码的聚糖库将解决当前聚糖微阵列格式的局限性 有限数量的可用聚糖，仪器成本和劳动密集型过程阻止 筛选临床研究所需的大量样本。我们还将研究定量功能 这个新平台。此外，我们还将对完整细胞的聚糖微阵列分析，包括 细菌和酵母菌细胞，在技术上使用当前的聚糖微阵列格式在技术上具有挑战性。