Single Cell Glycan Profiling via Chemoenzymatic Barcoding
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Solution Overview
Problem
Current methods for analyzing glycans are complex, lack specificity and sensitivity, and cannot be used in functional biological assay systems, and existing single-cell sequencing technologies do not provide a comprehensive snapshot of the cell glycosylation state or glycome.
Innovation Solution
A method involving chemoenzymatic labeling and bioorthogonal chemical reactions, combined with single-cell profiling, to detect and analyze glycans using barcoding and proximity ligation assays, enabling precise identification of glycans and glycan motifs in single cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional glycan analysis methods are used, then glycan detection can be performed, but the methods lack specificity and sensitivity and cannot achieve single-cell resolution
Solution Approach 1:
The method segments the glycan analysis process into distinct molecular components: flag molecules (nucleotide sugars with reactive groups) that specifically label glycans, reporter molecules with oligonucleotide barcodes that detect the flags, and partition-specific barcodes that enable single-cell resolution. This segmentation allows each component to be optimized independently for specificity and sensitivity while maintaining overall system manageability.
Solution Approach 2:
The invention introduces intermediary molecules to bridge the gap between glycan structures and detectable signals. Flag molecules act as intermediaries that first bind to glycans via glycosyltransferases, then serve as targets for reporter molecules. This intermediary approach enables indirect detection that amplifies sensitivity while maintaining specificity through multiple selective binding steps.
2Quantity of substance
If existing single-cell sequencing technologies are used, then single-cell analysis is achieved, but comprehensive glycosylation state information is not provided
Solution Approach 1:
The method changes the detection parameter from direct glycan observation to detection of oligonucleotide barcodes associated with glycans. By converting glycan information into a nucleic acid-based readout, the system leverages the sensitivity and versatility of sequencing technologies while providing comprehensive glycosylation state information including presence, abundance, and diversity of glycan structures.
Solution Approach 2:
The invention replaces direct physical/chemical detection of glycans with a biochemical conversion system. Instead of attempting to directly detect and measure glycans (which is difficult due to their structural complexity and low abundance), the system substitutes a biochemical pathway that converts glycan information into detectable oligonucleotide barcodes that can be analyzed using established sequencing methods.
3Adaptability or versatility
If current glycan analysis methods are used, then glycan presence can be detected, but the methods are too complex for functional biological assay systems
Solution Approach 1:
The method employs universal components that can be applied across different functional biological assay systems. The flag-reporter-barcode architecture is modular and can be integrated with various cell types, cultivation conditions, and downstream analyses. The use of standardized oligonucleotide barcodes and common biochemical reagents enables broad adaptability while keeping individual assay steps relatively simple.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
Provides a comprehensive snapshot of glycosylation states and identifies unique glycosylation profiles at a single-cell level, offering insights into health and disease, and enabling diagnosis and treatment of conditions related to abnormal glycosylation patterns.
Implementation Method 1
incubating the sample with (i) a first flag molecule comprising a nucleotide sugar and a first reactive molecule of a reaction pair and (ii) a first glycan specific transferase, wherein the first flag molecule is a substrate for the first glycan specific transferase; wherein the first flag molecule is incorporated onto a glycan-modified glycoprotein in the sample
Implementation Method 2
admixing the sample with a first reporter molecule comprising (i) a second reactive molecule of the reaction pair and (ii) a first reporter oligonucleotide... wherein the second reactive molecule of the reaction pair couples to the first reactive molecule of the reaction pair, whereby the first reporter molecule is conjugated to the glycan-modified glycoprotein via the first flag molecule
Data Source
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AI summary
The present disclosure relates to methods, compositions, systems, and kits for detecting and analyzing the glycosylation of healthy and diseased cells and protein-specific glycosylation patterns using single-cell profiling methodologies.