Development of Genetically Encoded Multivalent Liquid Glycan Array (LiGA) Displayed on M13 Phages
Creators: Sojitra, Mirat
Publisher: name: University of Alberta Library
Year: 2023

<p>The template-based flow of information, DNA→RNA→protein, allows the study of these biomolecules using the next-generation sequencing. By contrast, studying the role of post-translational modifications (PTMs) such as glycans cannot be done readily using DNA sequencing. Within a cell, the synthesis of glycan occurs via a set of competing enzymes in a stochastic manner, thus the structural information is not encoded by the cell’s DNA. In this thesis, I describe a technology that establishes a one-to-one correspondence between a DNA sequence and a carbohydrate structure that enables DNA sequencing approaches to study the glycan binding preferences of receptors on cells in vitro and in vivo.In Chapter 1, I briefly review the current state of chemically synthesized glycan probes and specifically focus on examples where glycosylated nanocarriers have been applied to study glycan binding protein (GBPs) on the surface of live cells in vivo. The summary of examples in Chapter 1 highlighted that there remains an unmet need for a multiplexed glycan array to profile glycan binding proteins in a target-agnostic manner; for example, the ability to profile glycan-binding properties of not only purified GBPs but also for GBPs displayed on the surface of live cells and inside live organisms is currently limited. This thesis addresses the challenge by performing bioorthogonal chemistry on prospectively DNA barcoded M13 phage to create a genetically encoded liquid glycan array (LiGA). Specifically, synthesis of LiGA starts with a covalent modification of prospectively barcoded M13 phages bearing bi-cyclooctene moieties by pre-synthesized glycans bearing an azido linker. From a mixture of such DNA barcoded multivalent glycan probes displaying 30–1500 copies of a glycan, we were able to uncover avidity and affinity-based binding to cell surface receptors such as CD22, in vitro and in vivo (Chapter 2).By fine-tuning the cell-binding, cell-elution, and PCR conditions, I developed a robust method to investigate five receptors of the Siglec family of immunomodulators (Chapter 3). Elution of phages using a combination of RNase A and Proteinase K treatment followed by a two-step PCR, resulted in a robust method for deep sequencing of the barcodes in the bound phages. Among the five tested members of Siglecs family, the cells expressing Siglec 2, -7 and -9 pulled down sialylated glycans from a LiGA containing ~100 glycophage conjugates. Interestingly, the nature of the interacting glycans changed significantly when the Siglec-7 was displayed on different cell types (Jurkat vs. CHO cells). Pull down of LiGA by CHO cells expressing Siglec-6 yielded a seemingly random collection of sialo and asialoglycans. Pull down of LiGA by CHO cells expressing Siglec-1 resulted in weak, noisy enrichment profile. To explore the avidity-based gangliosides binding to Siglec-1 further, I used a focused LiGA with gangliosides GM1, GM2, GM3, and GD1a displayed at 160–1300 copies. The Siglec 1 bound to glycophage carrying 680 or higher copies of GM2 and GD1a per bacteriophage. Interestingly, ganglioside binding preferences of Siglec-1(+) cells were not identical between LiGA and display of GM gangliosides on liposomes indicating that either linker chemistry, presentation in the bilayer or mobility might be important factors in ganglioside–Siglec-1 interactions.  Chapter 4 focuses on a new chemical conjugation method to synthesize LiGA by forming an amide bond between the carboxylic acid group on the anomeric linker and the N-terminal amine of the major coat protein pVIII. The motivations for this development were to explore whether the hydrophobic DBCO-triazole moiety present in the linker of previously developed LiGA modulates interaction with target proteins. I adopted a published synthesis of glycan-NHS esters and their ligation to proteins to modify the pVIII major coat protein and introduce a specific copy number of glycans on phage. This strategy was applied to build “amide linked LiGA” using a collection of 10 glycans with a carboxylic acid linker, many of which belonged to A-type of B-type blood group. We came across a remarkable interplay of linker structure and it displayed the density of glycan in their recognition by the lectins, SBA and ECL. We also observed that the HepG2 cells significantly bound to phage modified with DBCO and capped with azidoethanol, thus indicating a potential for non-specific binding of DBCO moiety. Future work is required for evaluations of triazole-DBCO vs. amide linker to uncovered liabilities of linkers.</p>