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

项目概要/摘要 聚糖微阵列技术的发展、应用及大聚糖的获得 在过去的十年里，NIH资助的功能性糖组学联盟（CFG）向研究人员提供了基因芯片 彻底改变了糖组学的功能研究。在此期间，开展了数百个由发电机驱动的项目， 这一次是与CFG合作，并且对基于聚糖微阵列的实验持续感兴趣 功能性糖组学的研究然而，这些研究仍然主要与实验室有关， 由于仪器成本高， 需要特殊的专业知识，包括我们的埃默里综合糖组学核心。的主要目标之一， 美国国立卫生研究院共同基金在糖科学方面的投资是开发工具，使糖组学研究更容易获得 非专业实验室。为了解决这个问题，我们利用共同基金支持的项目， 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编码，阵列和扫描仪的成本较高 用于解码以扩增和分析蛋白质-聚糖相互作用的测序（NGS）。微阵列基本上是 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， 在载玻片上的位置。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，我们安装一个独特的 寡核苷酸序列（代码）。将编码的聚糖在单个小瓶中混合在一起，并与一个或多个微球一起孵育。 潜在聚糖结合蛋白（GBP）。然后从混合物中分离GBP-Glycan-DNA复合物 使用普通的免疫沉淀程序，扩增寡核苷酸代码并定量 通过NGS测序。每个独特的代码对应于独特的聚糖结构，并且聚糖结构的拷贝数是相同的。 序列表示结合的聚糖的量，其将与聚糖的相对亲和力成正比。 GBP到不同的聚糖。消除仪器的成本，并开发一种熟悉的技术， 大多数实验室将使聚糖微阵列研究可用于生物医学研究与发展社区， 提供适当的DNA编码聚糖文库。NGGM的优化与NGGM的发展 DNA编码聚糖的大型文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工艺， 筛选临床研究所需的大量样本。我们还将研究数量特征 这个新平台。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，这在技术上具有挑战性，使用目前的聚糖微阵列格式。

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.