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

项目总结/摘要 聚糖在生物系统和许多疾病中发挥着重要作用，通常通过它们的特定相互作用 与其他生物分子，如聚糖结合蛋白（GBP）。在过去的几十年里， 蛋白质-聚糖相互作用已经通过聚糖微阵列技术的发展而得到了极大的改善， 其中通过微阵列打印机将聚糖结构文库固定在固体表面上 载玻片并用荧光标记的GBP询问。GBP的结合特异性可以快速地被识别。 从微阵列扫描仪获得的荧光图像推断。尽管它的成功，目前的聚糖 微阵列技术在几个方面受到严重限制。首先，包括在当前的聚糖中的聚糖的数量是已知的。 聚糖微阵列受到化学/酶合成的限制。即使在最流行的聚糖微阵列中 由功能性糖组学协会提供，只有一小部分（约600个聚糖）的糖组是 代表。由于新的化学酶促合成和新的方法， 利用天然聚糖;然而，使用当前的聚糖微阵列平台， 容纳超过1,000个聚糖。其次，虽然聚糖微阵列被认为是高通量的， 由于可以同时分析大量的聚糖，该平台实际上遭受了 需要手动对准荧光图像中的网格以量化 每个单独斑点的荧光强度。因此，许多样品如患者血清样品的处理 是一个非常劳动密集和缓慢的过程。第三，尽管概念简单，但聚糖微阵列技术 由于包括微阵列在内的仪器的高成本， 打印机和扫描仪。为了应对这些挑战，我们提出了一种新的方法，称为下一代 聚糖微阵列（NGGM）由下一代测序（NGS）实现。在新方法中，我们将 将聚糖微阵列呈现为用寡核苷酸编码的聚糖和/或糖缀合物的混合物 序列的聚糖与GBP的免疫沉淀将选择与DNA特异性结合的聚糖 代码.这些代码可以使用强大的定量NGS技术解码，以阐明相关的 聚糖结构与GBP的结合特异性。序列读数将被转换为结合特异性 的GBP。我们期望这种新的方法将解决容量、吞吐量、易操作性等问题。 可获得性和可负担性。它将大大降低一般科学界研究的门槛 蛋白质-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.