On October 16, 2024, Ratmir Derda's team at the University of Alberta published their innovative technology, Liquid Glycan Array (LiGA), in Nature Protocols. This technology, based on DNA-barcoded M13 bacteriophage, enables the first high-throughput determination of the recognition specificity of glycan-binding proteins (GBPs) in living cells. Compared to traditional Glycan Arrays, LiGA eliminates the need for complex immobilization platforms and can be applied directly to living cells, providing a revolutionary tool for dissecting cell surface glycan-protein interaction networks and opening a new chapter in glycobiology research.

Overview

Glycans are ubiquitous on the cell surfaces of various organisms and constitute a significant component of the diversity of biological macromolecules. Because glycan structures are not directly encoded in DNA, high-throughput DNA-based techniques have been unable to investigate the role of cellular glycosylation in biological processes, hindering the understanding of how cell-surface glycoconjugates (glycocalyx) are recognized by GBPs. To address this issue, this study developed a novel platform, LiGA, that enables the analysis of glycan-protein interactions on the surface of living cells using high-throughput sequencing, both in vitro and in vivo. The core of the LiGA platform is a library of DNA-barcoded M13 phages. Each clonal phage displays 5 to 1500 glycans on its surface, and the internal DNA barcodes encode the types and densities of the displayed glycans. Deep sequencing of these "glycophages" bound to living cells allows for the generation of binding preferences for GBPs on the Cell Surface. This study details the LiGA technology workflow, including glycophage preparation, MALDI-TOF mass spectrometry analysis, LiGA library construction, and high-throughput sequencing. This technology was used to analyze the carbohydrate binding properties of five immunomodulatory sialic acid-binding immunoglobulin-like lectins (Siglec-1, -2, -6, -7, and -9) on the surfaces of different cells. Compared to traditional methods that rely on complex instrumentation, LiGA requires only basic molecular biology skills and can measure the carbohydrate binding profile of any exogenously expressed GBP cell surface within 2–3 days.

Main Content

Research Background and Technical Challenges

The Key Role of Carbohydrate Molecules

Glycosylation is ubiquitous on the cell surface and participates in physiological processes such as cellular communication, immune response, and pathogen recognition. The interaction between cell-surface glycoconjugates (glycocalyx) and GBPs lies at the heart of these processes. However, because glycan structures are not directly encoded by DNA, traditional high-throughput technologies struggle to decipher their dynamic mechanisms.

Limitations of Existing Technologies

• Traditional "glass-slide glycanarrays" require immobilized glycan molecules and fail to mimic the natural environment of living cells.
• The "cis-action" of the cell's own glycans can occupy GBP binding sites, impairing the "trans-action" recognition of exogenous glycan
• Multivalent glycanprobes (such as glycopolymers) lack glycan identity tags and are therefore limited to monosaccharide experiments.

Technical Requirements

A high-throughput technology is needed that can simultaneously assess multiple glycan structures and their display density within a living cell environment.

Fig.1. Workflow for cell-surface binding assay using LiGA. (Sojitra, et al. 2025)

LiGA Technology Core and Innovation Highlights

Technical Design Principle

• DNA Barcoded Phage Library: Utilizes M13 phage as a carrier, displaying 5-1500 glycanmolecules on its surface. The internal DNA barcode encodes the glycan types and density.
• Multivalent GlycanDisplay Control: Precisely control the number of glycan molecules per phage (20-1500) through chemical/enzymatic methods.
• High-Throughput Sequencing Analysis: Combined with deep phage sequencing, obtains a map of GBP binding preferences on the surface of living cells.

Core Advantages

• Physiological Relevance: Closely matches natural multivalent glycan-protein interactions, suitable for living cells and in vivo
• Interference Resistance: The DNA barcode is located within the phage, resisting degradation in complex environments.
• Ease of Use:Requires only basic molecular biology skills, and assays can be completed within 2-3 days.
• Scalability:A single experiment can evaluate hundreds of glycan structures and display densities.

Fig. 2. High-throughput sequencing and data processing. (Sojitra, et al. 2025)

Experimental Workflow and Data Analysis

LiGA Library Construction

• Glycans were attached to the phage capsid protein pVIII using a bioorthogonal reaction (e.g., DBCO-NHS).
• The SB1/SB2 barcode system was used to insert the barcodes into the M13 genome, establishing a traceable glycan-barcode mapping relationship.
• Glycan modification density was controlled, and the degree of modification was confirmed by MALDI-TOF mass spectrometry.

Live Cell Binding Assay

• Day 1: The LiGA library was incubated with target cells, and bound phages were recovered.
• Day 2: Phages were eluted, DNA was extracted, and amplified using a two-step PCR (without overprinting primers in the first step and with Illumina adapters in the second step), followed by high-throughput sequencing.

Data Analysis Strategy

• EdgeR differential expression analysis was used to calculate the fold change and significance (q-value) of glycan enrichment in GBP-positive/negative cells.
• The TMM normalization algorithm was used to correct for differences in experimental groups, and binding specificity was displayed using a "volcano plot."
• Repeated experiments on the same day showed higher correlation (Pearson R = 0.97). Simultaneous control experiments are recommended to reduce bias.

LiGA Technology Application Examples

Analyzing Siglec Receptor Binding Spectra

• Analyzing the carbohydrate recognition properties of immunomodulatory Lectins such as Siglec-1, -2, -6, -7, and -9.
• It was found that differences in recognition of the same Siglec in different cell types (e.g., CHO vs. Jurkat) were influenced by expression level and cellular background.

Validation of Binding Specificity

• By mutating key arginine residues in Siglecs, it was confirmed that their recognition is dependent on sialic acid.
• The binding of Siglec-1 to gangliosides (GM2, GD1a) was found to be density-dependent: little binding at low densities and significant binding at high densities.

Technical Scalability

• Applicable to a variety of receptors (e.g., viral envelope proteins, tumor-associated GBPs) and cell types.
• It can be used to study immune regulation mechanisms, drug target discovery, and pathogen invasion mechanisms.

Summary

LiGA technology, by integrating phage display, DNA barcoding, bioorthogonal chemistry, and high-throughput sequencing, has achieved a paradigm shift in glycobiology research. It not only provides unprecedented tools for analyzing cell surface Gycan-Protein Interactions, but also lays the foundation for uncovering life processes such as immune evasion, tumorigenesis, and pathogen recognition. With the advancement of glycan synthesis technology and bioinformatics, LiGA is expected to become a core platform for the development of carbohydrate-targeted therapies, promoting the deep integration of glycobiology and translational medicine.

Related Services & Products

• Traditional Glycan Display Array
• Cell-based Glycan Display Array
• Neoglycolipid (NGL) Display Array
• Liquid Glycan Display Array (LiGA)
• Glycophage Display
• De Novo Glycan Display
• Glycome Arrays
• Lectin Arrays
• Glycan Arrays
• Special Glycan Array
• Glycan Modification Array

Reference

1. Sojitra, M., et al. Measuring carbohydrate recognition profile of lectins on live cells using liquid glycan array (LiGA). Nat Protoc, 20, 989–1019 (2025). DOI: 1038/s41596-024-01070-3.