Ligand-Polymer Constructs for Multiplexed Single-Cell Proteomics
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Solution Overview
Problem
Current methods for single-cell RNA sequencing lack the ability to simultaneously detect and measure transcripts and proteins effectively, particularly in complex cell populations, due to limitations in scale and the loss of phenotypic information beyond protein levels and cell-surface protein expression.
Innovation Solution
A composition comprising a ligand attached to a polymer construct with an Amplification Handle, Barcode, and Unique Molecular Identifier (UMI), allowing for the simultaneous detection of epitopes and transcriptomes by hybridizing to capture sequences and amplifying specific sequences for identification and quantification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If droplet-based microfluidic approaches are used for single-cell RNA sequencing, then throughput is dramatically increased, but phenotypic information (proteins and cell-surface markers) is lost
Solution Approach 1:
The patent combines protein detection and RNA sequencing in a single droplet-based assay. Antibodies conjugated to oligonucleotides with barcodes enable simultaneous measurement of protein expression and transcriptome, resolving the contradiction by merging two previously separate detection modalities into one integrated system that maintains both high throughput and phenotypic information
Solution Approach 2:
The patent introduces oligonucleotide barcodes as an intermediary that bridges protein detection and RNA sequencing. These barcodes are conjugated to antibodies, allowing protein signals to be captured and linked to cellular RNA content through a molecular intermediary, thereby enabling simultaneous measurement without loss of phenotypic data
2Reliability
If FACS/scRNA-seq approaches are used, then cell sorting and sequencing are achieved, but throughput is low and experimental bias exists
Solution Approach 1:
The patent replaces the mechanical FACS sorting process with a passive droplet-based encapsulation system. Instead of using flow cytometry to physically sort cells based on fluorescent markers, the system uses droplet microfluidics to randomly partition cells into droplets, eliminating mechanical sorting biases while maintaining high throughput
3Loss of information
If targeted methods to simultaneously measure transcripts and proteins are used, then multi-omics data is obtained, but scale is limited
Solution Approach 1:
The patent segments the measurement process into discrete droplets, each containing a single cell and its associated protein-RNA complexes. This segmentation enables parallel processing of thousands of cells simultaneously, achieving both comprehensive multi-omics data and large scale through the droplet-based approach
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 high-throughput, multiplexed analysis of cellular targets, providing comprehensive cellular characterization by combining protein and transcript data, enhancing the detection of multiple targets and reducing experimental bias.
Implementation Method 1
The ligand is designed to bind specifically to a target in a biological sample
Implementation Method 2
hybridizing to a capture sequence that comprises a sequence complementary to the Anchor
Data Source
AI summary
Compositions, kits and methods are described that comprise one or more constructs, each construct comprising a ligand attached or conjugated to a polymer construct, e.g., an oligonucleotide sequence, by a linker, each ligand binding specifically to a single target located in or on the surface of a cell. The polymer construct comprises a) an Amplification Handle; b) a Barcode that specifically identifies a single ligand; c) an optional Unique Molecular Identifier that is positioned adjacent to the Barcode on its 5′ or 3′ end; and d) an Anchor for hybridizing to a complementary sequence, e.g., for generation of a double-stranded oligonucleotide. These compositions are used in methods, including high throughput methods, for detecting one or more targets or epitopes in a biological sample. These compositions are also used in a high throughput method for characterizing a cell by simultaneous detection of one or more epitopes located in or on the cell and its transcriptome.