Protein Identification via DNA Barcoding for Low-Abundance Samples
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Solution Overview
Problem
Traditional protein identification methods, such as mass spectrometry, require high purity and high abundance of proteins, limiting their applicability and efficiency in proteomics research.
Innovation Solution
A method that uses molecular instruments to transform protein sequence information into nucleic acid records by attaching barcoded DNA strands to proteins, enabling single-molecule level detection and analysis without the need for high purity or abundance, using polymerase-mediated nucleic acid polymerization and strand displacement.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If mass spectrometry is used for protein identification, then protein sequence information can be obtained, but high purity and high abundance of proteins are required
Solution Approach 1:
The patent introduces nucleic acid molecules as intermediary carriers that bind to proteins and transfer sequence information. These nucleic acid intermediaries enable the detection of low-abundance proteins by converting protein sequence information into nucleic acid sequences that can be amplified and detected with high sensitivity, thus resolving the contradiction between maintaining identification accuracy and reducing protein abundance requirements
Solution Approach 2:
The patent creates nucleic acid copies of protein sequence information. By synthesizing nucleic acid molecules that complement the protein sequence and then amplifying these copies through PCR or other nucleic acid amplification methods, the system can detect trace amounts of protein while maintaining high identification accuracy, effectively bypassing the limitation of requiring high protein abundance
2Measurement precision
If mass spectrometry is used for protein identification, then protein analysis can be performed, but the method is inherently ensemble-based requiring high purity
Solution Approach 1:
The patent segments the protein analysis process into individual molecular events. Instead of measuring ensemble averages, the system uses single-molecule nucleic acid detection to observe individual protein-nucleic acid binding events and sequence information transfer, enabling high-purity identification from complex mixtures by analyzing one molecule at a time
Solution Approach 2:
The patent replaces the mechanical/physical separation and detection approach of mass spectrometry with a biochemical information transfer system. Nucleic acid molecules specifically recognize and bind to target proteins through sequence complementarity, transferring sequence information in a highly specific manner that provides both high identification capability and tolerance for impurities
3Measurement precision
If traditional protein fingerprinting is used, then protein identification is achieved, but the acquisition speed is slow and resolution is limited
Solution Approach 1:
The patent establishes continuous nucleic acid polymerization reactions that proceed without interruption. Polymerases continuously synthesize nucleic acid sequences complementary to the protein target, and multiple polymerization reactions occur simultaneously in parallel, maintaining continuous useful action that both accelerates data acquisition and enhances resolution through sustained signal accumulation
Solution Approach 2:
The patent employs periodic cycling of nucleic acid amplification and detection steps. Through repeated cycles of denaturation, annealing, and extension, the system exponentially amplifies the nucleic acid signal while maintaining high fidelity, achieving both fast acquisition through exponential amplification and high resolution through iterative refinement
4Productivity
If ensemble assays are used for protein analysis, then analysis can be performed, but single-molecule level detection is not achieved
Solution Approach 1:
The patent employs self-assembling nucleic acid structures that automatically organize into detection-ready configurations. The nucleic acid molecules self-hybridize to form hairpin structures or other defined conformations that present the protein-binding interface in an optimal geometry, enabling single-molecule detection without complex external manipulation while maintaining high throughput through autonomous molecular organization
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 faster, high-resolution protein identification in complex mixtures with multiplexed and parallel detection, facilitating proteomics research and applications like drug screening and disease modeling.
Implementation Method 1
combining in reaction buffer comprising a polymerase having strand displacement activity (a) a substrate to which a protein chain comprising amino acids labeled with barcoded DNA strands is attached, and (b) barcoded molecular instruments that bind to the DNA strands and produce nucleic acid records of the barcoded DNA strands, and incubating the reaction mixture under conditions that result in nucleic acid polymerization
Implementation Method 2
barcoded molecular instruments that bind to the DNA strands and produce nucleic acid records of the barcoded DNA strands
Implementation Method 3
polymerase having strand displacement activity
Data Source
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AI summary
Provided herein, in some embodiments, are methods and compositions for protein identification.