Affinity-Oligonucleotide Conjugates for Single-Cell Sequencing
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Solution Overview
Problem
Current flow cytometry-based methods for immunophenotyping are limited by the number of fluorophores that can be used concurrently and suffer from spectral overlap, making them inadequate for biologically relevant assays and DNA sequencing.
Innovation Solution
The use of affinity-oligonucleotide conjugates, such as antibody-oligonucleotide or tetramer-oligonucleotide conjugates, which allow for the analysis of proteins in individual cells through sequence readouts without the need for fluorophores, enabling the identification of surface proteins, T cell receptors, and B cell receptors, and determining their binding affinities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If flow cytometry-based approaches are used for immunophenotyping, then cell surface proteins can be identified and categorized, but the number of fluorophores that can be used concurrently is limited and spectral overlap occurs
Solution Approach 1:
The patent replaces the optical detection system (flow cytometry with fluorophores) with a sequence-based detection system. Affinity molecules are conjugated to oligonucleotide barcodes that can be identified through DNA sequencing, eliminating the need for fluorophores and spectral resolution while enabling higher multiplexing capacity through the vast diversity of nucleotide sequences.
2Measurement precision
If flow cytometry is used for cell analysis, then surface-bound antibodies can be measured, but the method is not amenable to DNA sequencing and biologically-relevant assays
Solution Approach 1:
The patent merges affinity-based cell surface protein detection with sequence-based identification. By conjugating affinity molecules (antibodies, antigen-binding fragments) to oligonucleotide barcodes, the system combines the specificity of affinity binding with the versatility of nucleic acid technologies, enabling compatibility with DNA sequencing, PCR, and other biologically-relevant assays.
3Measurement precision
If cell sorting is performed to characterize single cells, then cell identity can be determined, but the process is slow and reduces target yield
Solution Approach 1:
The patent uses oligonucleotide barcodes as molecular copies or proxies for cell surface proteins. Instead of physically sorting cells based on surface markers, the method attaches sequence-based barcodes to cells via affinity binding, then sequences the barcodes to identify cell phenotypes. This copying approach eliminates slow physical sorting while preserving single-cell resolution and target yield.
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
This approach overcomes limitations of flow cytometry by allowing for the characterization of single cells without fluorophore constraints, facilitating simultaneous measurement of TCR pair sequences, clone abundance, and relative tetramer affinities, and is compatible with emulsion-based single-cell analysis and subsequent DNA sequencing.
Implementation Method 1
the affinity portion of the affinity-oligonucleotide conjugate specifically binds to a target antigen of a single cell
Data Source
Figure 1
Figure 2
Figure 3A~3B
AI summary
Provided herein are methods and compositions for single cell characterization using affinity-oligonucleotide conjugates. Provided herein are methods and compositions for single cell charaterization using tetramer-oligonucleotide conjugates.