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.

Project Narrative Although glycan microarray technology has revolutionized studies of protein-glycan interactions mediating cell communication, the limitations of the current microarray platform are becoming obvious as glycome-wide studies are increasing in size and numbers. We address these limitations by fundamentally changing the current microarray platform by introducing DNA sequences as codes for glycans and next generation sequencing (NGS) for decoding or detecting protein-glycan interactions. This “next generation glycan microarray (NGGM)” technology will increase the capacity, throughput, sensitivity, dynamic range and versatility of assays to study protein-glycan interactions and, more importantly, make such investigations accessible to the general scientific community by using techniques familiar to biologists and by eliminating the technical complexities of current microarray technology.