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

项目摘要/摘要 多聚糖微阵列技术的发展应用及大分子多聚糖的可获得性 由美国国立卫生研究院资助的功能糖链联盟(CFG)在过去十年向研究人员提供的微阵列 彻底改变了糖组学中的功能研究。在此期间，开展了数百个由调查员推动的项目 这一次是与CFG合作，并继续对基于多糖微阵列的实验感兴趣 用于功能糖喜剧的研究。然而，这些研究仍然主要与实验室有关。 由于仪器和设备的高成本，专门制造和加工多糖微阵列 需要特殊的专业知识，包括我们的埃默里综合血糖核心。该委员会的主要目标之一是 NIH共同基金对血糖科学的投资是为了开发工具，使血糖研究更容易获得 转移到非专业实验室。为了解决这个问题，我们使用了一个共同基金支持的项目 论证开发下一代葡聚糖微阵列(NGGM)的可行性，以消除 通过引入DNA序列作为糖链和下一代的代码，阵列和扫描仪的成本很高 测序(NGS)，用于解码，以扩增和分析蛋白质-多糖相互作用。微阵列基本上是 编码分子库的呈现，其中每个单独结构的编码是其物理结构 玻璃幻灯片上的位置。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。对于每个多聚糖结构，我们安装了一个独特的 寡核苷酸序列(代码)。将编码的多糖混合在一个小瓶中，并与 潜在的多糖结合蛋白(GBP)。然后从混合物中分离GBP-葡聚糖-DNA复合体 使用普通的免疫沉淀程序，寡核苷酸密码被扩增并定量 经NGS测序。每个唯一的编码对应一个唯一的糖链结构， 序列表示结合的糖链的量，它将直接与 GBP到不同的糖链。消除仪器成本并开发一种熟悉的技术 大多数实验室将通过以下方式向生物医学研发社区提供葡聚糖微阵列研究 提供适当的DNA编码的糖链文库。NGGM的优化与发展 一个DNA编码的大型糖链文库将解决当前糖链微阵列格式的局限性，包括 可利用的糖链数量有限、仪器成本和劳动密集型工艺 筛选临床调查所需的大量样本。我们还将研究数量特征 这个新的平台。此外，我们还将开发完整细胞的葡聚糖微阵列分析，包括 细菌和酵母细胞，使用目前的糖链微阵列形式在技术上一直具有挑战性。

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.