Next generation glycan microarray (NGGM) enabled by next generation sequencing (NGS) and DNA-coded glycan library

由下一代测序 (NGS) 和 DNA 编码聚糖文库支持的下一代聚糖微阵列 (NGGM)

• 批准号: 9336953
• 负责人: Xuezheng Song
• 金额: $ 30.26万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-01 至 2018-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9336953
• 关键词: Address; Alkynes; Antibodies; Azides; Binding; Binding Proteins; Biological Assay; Biological Process; Chemicals; Chemistry; Code; Communities; Computer software; DNA; DNA Library; DNA Sequence; DNA-Protein Interaction; Data Analyses; Development; Disease; Ensure; Glass; Glycoconjugates; Grant; Immobilization; Immunoprecipitation; Incubated; Individual; Label; Laboratories; Lectin; Libraries; Ligands; Magnetism; Manuals; Methods; Microarray Analysis; Oligonucleotides; Patients; Play; Polysaccharides; Precipitation; Preparation; Problem Solving; Process; Proteins; Reaction; Role; Route; Sampling; Serum; Slide; Solid; Specificity; Spottings; Streptavidin; Structure; Surface; Technology; base; biological systems; cost; experimental study; fluorescence imaging; genome-wide; improved; instrumentation; microorganism; next generation; next generation sequencing; novel; novel strategies; sequencing platform; success; tool; transcriptome sequencing

Project Summary/Abstract Glycans play important roles in biological systems and many diseases, often through their specific interactions with other biomolecules such as glycan-binding proteins (GBPs). In the past decades, the systematic study of protein-glycan interactions has been greatly improved by the development of glycan microarray technology, in which a library of glycan structures is immobilized by a microarray printer onto solid surfaces such as glass slides and interrogated with fluorescently labeled GBPs. The binding specificities of GBPs can be quickly deduced from the fluorescent image acquired by a microarray scanner. Despite its success, the current glycan microarray technology is seriously limited in several aspects. First, the number of glycans included in current glycan microarrays is limited by chemical/enzymatic synthesis. Even in the most popular glycan microarray provided by the Consortium for Functional Glycomics, only a small fraction (~600 glycans) of glycome is represented. This number is growing quickly owing to novel chemoenzymatic synthesis and novel methods to utilize natural glycans; however, using current glycan microarray platforms, it would be difficult to accommodate more than 1,000 glycans. Second, while glycan microarray is considered a high throughput platform due to the large number of glycans that can be analyzed simultaneously, it actually suffers from a bottleneck in processing that requires a manual alignment of a grid in the fluorescent image to quantify the fluorescent intensity at each individual spot. Thus, processing of many samples such as patient serum samples is a very labor intensive and slow process. Third, despite the simple concept, glycan microarray technology is limited to a number of very specialized laboratories due to the high cost of instrumentation including microarray printer and scanner. To address these challenges, we propose a novel approach termed Next Generation Glycan Microarray (NGGM) enabled by Next Generation Sequencing (NGS). In the new approach, we will present glycan microarray as a mixture of glycans and/or glycoconjugates that are coded with oligonucleotide sequences. The immunoprecipitation of glycans with GBPs will select specifically bound glycans with DNA codes. The codes can be decoded using the powerful quantitative NGS technology to elucidate the relative binding specificities of glycan structures to GBPs. The sequence reads will be converted to binding specificities of GBPs. We expect that this new approach will solve the problems of capacities, throughput, easy accessibility and affordability. It will greatly lower the threshold for the general scientific community to study protein-glycan interactions.

项目摘要/摘要 通常通过其特定互动，在生物系统和许多疾病中扮演重要角色 与其他生物分子（例如聚糖结合蛋白（GBP））。在过去的几十年中，对 通过开发聚糖微阵列技术，蛋白质 - 聚糖相互作用得到了极大的改善 一个聚糖结构库被微阵列打印机固定在玻璃等实体表面上 幻灯片并用荧光标记的Gbps询问。 Gbps的结合特异性可以很快 从微阵列扫描仪获得的荧光图像中推导。尽管取得了成功，但目前的聚糖 微阵列技术在几个方面受到严重限制。首先，当前包含的聚糖数量 聚糖微阵列受化学/酶促合成的限制。即使在最受欢迎的聚糖微阵列中 由功能性糖果财团提供 代表。由于新型的化学酶合成和新方法，该数字正在迅速增长 利用天然聚糖；但是，使用当前的聚糖微阵列平台，很难 容纳1000多个聚糖。第二，而聚糖微阵列被认为是高吞吐量 平台由于可以同时分析的大量聚糖，它实际上遭受了 处理中的瓶颈需要在荧光图像中手动对齐网格以量化 每个位置的荧光强度。因此，处理许多样品，例如患者血清样品 是一个非常劳动密集型和缓慢的过程。第三，尽管有一个简单的概念，但聚糖微阵列技术是 由于仪器的高成本，包括微阵列，仅限于许多非常专业的实验室 打印机和扫描仪。为了应对这些挑战，我们提出了一种称为下一代的新方法 下一代测序（NGS）启用了Glycan微阵列（NGGM）。在新方法中，我们将 当前的聚糖微阵列是用寡核苷酸编码的聚糖和/或糖缀合物的混合物 序列。用Gbps对聚糖的免疫沉淀将选择具有DNA的特异性结合聚糖 代码。这些代码可以使用强大的定量NGS技术来解码，以阐明相对 聚糖结构与GBP的结合特异性。序列读取将转换为结合特异性 Gbps。我们预计这种新方法将解决能力，吞吐量，轻松的问题 可访问性和负担能力。这将大大降低一般科学界学习的阈值 蛋白质 - 聚糖相互作用。