DNA Polymerization Signal Amplification for Protein Detection
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Solution Overview
Problem
Current methods for protein detection, especially low-abundance proteins, face challenges such as the need for cell lysis and protein separation, which are not suitable for maintaining cell integrity, and lack the ability to analyze multiple proteins with high sensitivity while preserving their original cellular location.
Innovation Solution
A method involving polymerization, detection, and depolymerization of DNA oligonucleotides, where initiator ssDNA oligonucleotides hybridize with hairpin DNA oligonucleotides to form detectable dsDNA polymers that can be conjugated to molecules like quantum dots or fluorophores, allowing for sensitive detection of proteins in their native location without cell lysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If cell lysis and protein separation methods are used, then protein detection sensitivity is improved, but cell integrity is lost and spatial information is destroyed
Solution Approach 1:
The patent uses DNA oligonucleotides as intermediaries to bridge the target protein and the detectable signal. The DNA probe binds to the protein of interest, and subsequent polymerization reactions amplify the signal without requiring cell lysis or protein separation, thus maintaining cell integrity while achieving sensitive detection.
Solution Approach 2:
The patent replaces mechanical cell lysis and protein separation procedures with a biochemical polymerization-based detection system. The DNA-based signal amplification mechanism substitutes for the mechanical disruption of cells, enabling sensitive protein detection while preserving cellular structure and spatial information.
2Adaptability or versatility
If conventional protein detection methods are used, then detection capability is achieved, but the ability to analyze multiple proteins with high sensitivity is limited
Solution Approach 1:
The patent designs a universal detection platform based on DNA polymerization that can detect multiple different proteins simultaneously. By using different DNA probe sequences that bind to different proteins, the same polymerization-based detection system can sensitively detect various proteins in their native cellular locations without requiring separate detection methods for each protein.
3Measurement precision
If signal amplification is implemented, then detection sensitivity is improved, but the complexity of the detection system increases
Solution Approach 1:
The patent employs self-service signal amplification where the DNA polymerization reaction automatically amplifies the signal based on the binding of the DNA probe to the target protein. The system uses the target protein itself and the bound DNA probe as the template for polymerization, eliminating the need for external amplification reagents or complex enzymatic systems, thus reducing overall system complexity while achieving high sensitivity.
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 the sensitive detection of multiple proteins in their original cellular location, overcoming the limitations of existing methods by providing high sensitivity and maintaining cell integrity, suitable for comprehensive analysis of protein pathways and networks.
Implementation Method 1
a polymerization step comprising hybridizing an initiator ssDNA oligonucleotide with one or more hairpin DNA oligonucleotides to form a double-stranded DNA (dsDNA) polymerization product
Implementation Method 2
The dsDNA polymerization product is conjugated to a DNA-conjugated fluorophore or a quantum dot
Data Source
AI summary
The present invention relates generally to compositions, methods and kits for detection of a molecule of interest. The present invention is based, at least in part, on the design of nucleic acid oligonucleotides such that they can be used as molecular building blocks which can be assembled (polymerized) to form a dsDNA polymerization product which can be detected and further, can be disassembled (depolymerized) such that the polymerization product is no longer detectable. The invention can be performed multiple times on the same cell population, and is therefore useful for highly sensitive in situ detection of multiple biomarkers.