Single-Cell Proteomic Sequencing with Oligonucleotide Barcode Tags
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Solution Overview
Problem
Existing methods for quantitative analysis of proteins at the single-cell level, such as FACS, ELISA, and mass cytometry, are limited by sensitivity, sample throughput, and the number of markers that can be analyzed simultaneously, and require expensive and specialized heavy atom labeling.
Innovation Solution
Encoding the level of biological components, such as proteins, into oligonucleotide barcode sequences using libraries of binding elements like antibodies or aptamers, which are tagged with identifiable oligonucleotide barcodes, allowing for high-throughput sequencing to detect binding-element ligand complexes in a highly parallel fashion.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If FACS, ELISA, or bead-based multiplexing methods are used for protein quantification, then quantitative analysis can be performed, but sensitivity, sample throughput, and the number of markers that can be analyzed simultaneously are limited
Solution Approach 1:
The patent introduces oligonucleotide barcodes as intermediary molecules that bridge protein detection and sequencing technology. Each antibody is conjugated to a unique oligonucleotide barcode, allowing protein-antibody interactions to be translated into detectable nucleic acid signals that can be amplified and sequenced with high throughput and sensitivity
Solution Approach 2:
The patent replaces traditional flow cytometry mechanical detection systems with a nucleic acid-based detection system. Instead of using fluorescent markers and flow cytometry instruments, the invention uses oligonucleotide barcodes that can be detected through PCR amplification and next-generation sequencing, enabling simultaneous analysis of thousands of markers
2Adaptability or versatility
If mass cytometry is used to increase the number of simultaneous markers, then more markers can be analyzed, but expensive and specialized heavy atom labeling is required
Solution Approach 1:
The patent replaces expensive heavy atom labels with inexpensive oligonucleotide barcodes. These nucleic acid tags are significantly cheaper to synthesize and conjugate than heavy atom labeling reagents, while enabling comparable or superior multiplexing capabilities through combinatorial barcode designs
Solution Approach 2:
The patent changes the detection parameter from mass-based detection (mass cytometry) to sequence-based detection (sequencing). This parameter change allows for higher multiplexing since oligonucleotide barcodes can be designed with combinatorial diversity, enabling thousands of unique identifiers rather than the tens limited by mass spectrometry resolution
3Adaptability or versatility
If traditional protein analysis methods are used, then analysis can be performed, but the number of markers that can be analyzed simultaneously is limited
Solution Approach 1:
The patent creates a universal platform where a single oligonucleotide barcode system can detect thousands of different protein markers simultaneously. The same sequencing infrastructure and analysis pipeline can be used for any combination of markers, providing universal applicability without requiring marker-specific detection systems
Solution Approach 2:
The patent segments the detection system into modular components: antibodies for specific binding, oligonucleotide barcodes for identification, and sequencing for detection. This segmentation allows for flexible assembly and scaling of the system to analyze different numbers of markers without increasing overall system complexity
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
Enables quantitative analysis of multiple proteins in single cells with high sensitivity and throughput, overcoming limitations of existing methods by using oligonucleotide barcodes to encode and detect protein levels through sequencing.
Implementation Method 1
a library of at least about 10 structurally distinct antibodies, wherein the structurally distinct antibodies have a specific binding affinity for, and are bound to, structurally distinct target epitopes of the fixed proteins
Implementation Method 2
The levels of the binding-element ligand complexes can be detected by recovering and sequencing the oligonucleotide barcodes bound to the binding elements
Data Source
AI summary
Methods and compositions are described for single cell resolution, quantitative proteomic analysis using high throughput sequencing.