Oligonucleotide-Tagged Binding Reagents for Simultaneous Protein and Gene Analysis
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Solution Overview
Problem
Current methods lack the capability to quantitatively analyze protein expression in cells simultaneously with gene expression, especially in a high-throughput manner, and fail to efficiently determine the number of copies of cellular component targets.
Innovation Solution
The method involves contacting cells with cellular component-binding reagents associated with specific oligonucleotides that have a detectable moiety and an identifier sequence, allowing for the generation of labeled nucleic acids through barcode hybridization and extension, enabling the determination of the number of copies of cellular component targets by sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current technology is used to measure gene expression of single cells in a massively parallel manner, then high-throughput gene expression analysis is achieved, but the capability to quantitatively analyze protein expression simultaneously is lacking
Solution Approach 1:
The patent combines protein expression analysis and gene expression analysis into a single unified workflow. Cellular component-binding reagents (for protein detection) and oligonucleotide barcodes (for gene detection) are integrated in the same system, allowing simultaneous measurement of both protein and gene expression in the same cell population through a single high-throughput process.
Solution Approach 2:
The patent creates a universal platform that can perform multiple functions: detecting protein expression via cellular component-binding reagents, detecting gene expression via oligonucleotide barcodes, and quantitatively analyzing both simultaneously. This multi-functional system resolves the limitation of existing technologies that could only perform gene expression analysis.
2Measurement precision
If cellular component-binding reagents are associated with oligonucleotides containing detectable moieties and identifier sequences, then quantitative analysis of cellular component targets is enabled, but the complexity of the reagent system increases
Solution Approach 1:
The patent segments the quantitative analysis system into distinct functional modules: cellular component-binding reagents for specific target recognition, oligonucleotide sequences for identification and quantification, detectable moieties for signal generation, and barcode sequences for high-throughput processing. Each segment performs a specific function, making the overall complex system manageable and scalable.
Solution Approach 2:
The patent introduces oligonucleotide sequences as intermediaries that bridge the cellular component-binding reagents and the detection/quantification system. These oligonucleotides carry identifier sequences that link specific binding reagents to their target cellular components, enabling precise quantitative analysis without requiring direct complex integration of all detection components with each reagent.
3Measurement precision
If barcode hybridization and extension is performed to generate labeled nucleic acids, then the number of copies of cellular component targets can be determined, but the process time and procedural steps increase
Solution Approach 1:
The patent performs preliminary actions by pre-synthesizing and preparing the barcode sequences and oligonucleotide constructs before the actual analysis. The cellular component-binding reagents are pre-associated with oligonucleotides containing identifier sequences. This preliminary preparation allows the actual measurement process to proceed more quickly without sacrificing the accuracy of copy number determination.
Solution Approach 2:
The patent implements continuous useful action by designing a workflow where barcode hybridization and extension reactions proceed without interruption once initiated. The labeled nucleic acids are generated continuously through the extension process, maintaining reaction efficiency and reducing total process time while preserving the precision needed for accurate copy number determination.
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 allows for the quantitative analysis of protein and gene expression in cells, providing accurate counts of cellular component targets and enabling simultaneous high-throughput analysis.
Implementation Method 1
contacting a plurality of cellular component-binding reagents with a plurality of cells of a sample, each cell comprising cellular component targets of a plurality of cellular component targets, for specific binding with the plurality of cellular component targets
Implementation Method 2
contacting a plurality of barcodes with the cellular component-binding reagent specific oligonucleotides, or products thereof, associated with the cellular component-binding reagents associated with the cells of interest obtained, wherein each of the plurality of barcodes comprises a target binding region and a molecular label sequence to generate barcodes hybridized to the cellular component-binding reagent specific oligonucleotides
Implementation Method 3
extending the barcodes hybridized to the cellular component-binding reagent specific oligonucleotides to produce a plurality of labeled nucleic acids
Data Source
AI summary
Disclosed herein include systems, methods, compositions, and kits for quantitative analysis of a plurality of cellular component targets of cells of interest. In some embodiments, an oligonucleotide associated with a cellular component-binding reagent (e.g., an antibody) is associated with one or more detectable moieties (e.g., luminescent moieties, fluorescent moieties, a phosphorescent moieties). In some embodiments, the presence of the detectable moiety in the binding reagent oligonucleotide enables the cellular component-binding reagent to be employed for both fluorescence analysis (e.g., cell sorting) and sequence analysis (e.g., protein expression profiling).