RCA-Assisted ccs-FISH Protein Detection Multiplexing
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Solution Overview
Problem
Current protein detection methods in tissue samples are time-consuming, labor-intensive, and inefficient due to the need for repetitive antibody incubation and signal amplification, especially in high autofluorescence tissues, and they struggle with high multiplexity and data processing complexity, making them inaccessible to most laboratories.
Innovation Solution
The method employs antibody-conjugated oligonucleotides for protein labeling, using rolling circle amplification (RCA) and color-combinatorial and sequential fluorescence in situ hybridization (ccs-FISH) to achieve high multiplexity, all-in-one staining, and signal amplification compatible with various tissue types, enabling efficient protein detection with minimal cycles and low data storage requirements.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If cyclic immunofluorescence or multi-epitope-ligand cartography is used to achieve high multiplexity (>50 markers), then the number of detectable proteins is improved, but the time required and labor intensity increase significantly, lasting weeks to complete
Solution Approach 1:
The patent applies preliminary action by pre-conjugating multiple different oligonucleotides to a single antibody in advance. This allows the antibody to carry multiple oligonucleotide sequences that can be subsequently amplified, eliminating the need for repeated antibody incubation cycles. The pre-prepared multi-oligonucleotide conjugated antibody enables high multiplexity detection in a single staining step rather than requiring weeks of cyclic processing.
Solution Approach 2:
The patent uses copying by employing rolling circle amplification to generate multiple copies of oligonucleotide sequences attached to each antibody. A single antibody-oligonucleotide conjugate can produce thousands of copies through RCA, thereby amplifying the signal and enabling detection of multiple protein targets simultaneously without requiring multiple antibody incubation cycles.
2Measurement precision
If signal amplification is applied to overcome high autofluorescence in tissues, then detection sensitivity is improved, but the penetration efficiency into deep tissue decreases due to long DNA concatemers
Solution Approach 1:
The patent applies parameter changes by modifying the physical and chemical properties of the DNA structures used for signal amplification. Instead of using long linear DNA concatemers that penetrate tissue poorly, the invention uses short circular DNA templates for rolling circle amplification that generate branched DNA structures with improved tissue penetration while maintaining high signal amplification capability to overcome autofluorescence.
3Device complexity
If all-in-one staining with antibody cocktail is used to reduce experimental complexity, then the number of incubation cycles is reduced, but achieving high multiplexity with sufficient signal amplification becomes difficult
Solution Approach 1:
The patent uses copying by implementing rolling circle amplification that generates thousands of copies of oligonucleotide sequences from a single antibody-oligonucleotide conjugate. This amplification occurs in a single staining step, enabling all-in-one staining to achieve both reduced experimental complexity and high signal amplification capability simultaneously, as each antibody carries multiple amplified oligonucleotide copies that can be detected by complementary probes.
4Quantity of substance
If high multiplexity detection is achieved using single color per protein target, then all target proteins can be detected, but data processing becomes cumbersome and storage requirements increase massively
Solution Approach 1:
The patent applies universality by making each oligonucleotide sequence serve multiple functions: it acts as a unique identifier for a specific protein target, a template for rolling circle amplification to generate signal, and a binding site for complementary detection probes. This multi-functionality enables high multiplexity detection where each oligonucleotide carries multiple pieces of information, reducing the need for separate detection channels and thereby simplifying data processing while maintaining high multiplexity.
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 high-resolution, high-multiplex protein profiling with reduced experimental complexity and storage needs, overcoming the limitations of existing methods by enabling efficient detection of multiple proteins in a single image, even in auto-fluorescent tissues, and is more accessible to laboratories.
Implementation Method 1
using rolling circle amplification (RCA) and color-combinatorial and sequential fluorescence in situ hybridization (ccs-FISH) to achieve high multiplexity
Implementation Method 2
color-combinatorial and sequential fluorescence in situ hybridization (ccs-FISH)
Data Source
AI summary
Materials and methods for capturing target proteins and analyzing the proteins using an antibody conjugated to an oligonucleotide are provided. Methods of using rolling circle amplification (RCA) to amplify to the oligonucleotide are also provided. The oligonucleotide contains multiple sites for probe binding, which allows for multiplexity by means of color-combinatorial and sequential fluorescence in situ hybridization (ccs-FISH).