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

项目摘要/摘要 葡聚糖在生物系统和许多疾病中发挥着重要作用，通常是通过它们特定的相互作用 与其他生物分子，如糖结合蛋白(GBP)。在过去的几十年里，系统的研究 随着糖链微阵列技术的发展，蛋白质与多糖的相互作用得到了极大的改善 通过微阵列打印机将糖链结构库固定在诸如玻璃的固体表面上 幻灯片，并用荧光标记的GBPS进行讯问。Gbps的绑定特性可以快速地 从微阵列扫描仪获取的荧光图像中推导出。尽管它取得了成功，但目前的多糖 微阵列技术在几个方面受到严重限制。首先，当前包含的糖链数量 糖链微阵列受到化学/酶合成的限制。即使在最流行的葡聚糖微阵列中 由功能性糖链联盟提供，只有一小部分(~600个糖罐)的糖胺是 有代表性的。这一数字正在迅速增长，这要归功于新的化学酶合成和新的方法来 利用天然多聚糖；然而，使用目前的多聚糖微阵列平台，很难 可容纳1000多个糖罐。第二，虽然多聚糖微阵列被认为是高通量的 平台由于可以同时分析大量的多糖，它实际上遭受了 处理中的瓶颈，需要手动对齐荧光图像中的网格以量化 每个单独点的荧光强度。因此，许多样本的处理，如患者血清样本 这是一个非常劳动密集型和缓慢的过程。第三，尽管概念简单，但葡聚糖微阵列技术 限于一些非常专业的实验室，因为包括微阵列在内的仪器成本很高 打印机和扫描仪。为了应对这些挑战，我们提出了一种名为下一代的新方法 由下一代测序(NGS)实现的糖链微阵列(NGGM)。在新的方法中，我们将 目前的葡聚糖微阵列是用寡核苷酸编码的葡聚糖和/或糖结合物的混合物 序列。葡聚糖的免疫沉淀将选择与DNA特异结合的葡聚糖 密码。可以使用强大的定量NGS技术来解码这些代码，以阐明相关的 将多聚糖结构的特异性结合到Gbps。序列读数将被转换为结合特异性 千兆位数。我们预计，这种新的方法将解决容量、吞吐量、 可获得性和可负担性。它将大大降低普通科学界研究的门槛 蛋白质-葡聚糖相互作用。

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.