Sequential Nucleic Acid Encoding for High-Throughput Protein Analysis
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Solution Overview
Problem
Highly-parallel characterization and recognition of macromolecules, such as proteins, remains a challenge due to limitations in existing assays like immunoassays and mass spectrometry, which face issues with sensitivity, dynamic range, cross-reactivity, and background signals, making high-throughput macromolecule analysis inefficient and inaccurate.
Innovation Solution
A method involving nucleic acid encoding and barcoding of molecular recognition events, utilizing a recording tag joined to a support, binding agents with coding tags, and enzymatic reactions like ligation, extension, and cleavage to transfer identifying information from coding tags to recording tags, enabling efficient and high-throughput macromolecule analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If immunoassays and mass spectrometry are used for macromolecule analysis, then protein identification and quantitation can be achieved, but sensitivity, dynamic range, and accuracy are limited due to cross-reactivity and background signals
Solution Approach 1:
The patent introduces nucleic acid encoding tags as intermediary molecules that bridge the binding event and the detection system. Instead of directly detecting protein-protein or protein-ligand interactions, the method encodes binding information into nucleic acid sequences that can be amplified and sequenced, thereby eliminating cross-reactivity and background signal issues inherent in direct immunoassay approaches
Solution Approach 2:
The patent creates a nucleic acid copy of the binding event information. The encoding tag on the binder molecule is transferred to or amplified as a nucleic acid recording tag, which serves as a copy that can be detected with high precision through sequencing. This copying mechanism allows accurate reconstruction of binding events without the limitations of direct detection
2Productivity
If traditional assays are used for high-throughput macromolecule analysis, then some level of parallelization can be achieved, but efficiency and throughput remain limited
Solution Approach 1:
The patent merges multiple detection events into a single nucleic acid sequencing readout. By encoding information from numerous binding events into distinct nucleic acid sequences that can be processed simultaneously through amplification and sequencing, the method achieves high parallelization without the time loss associated with sequential traditional assays
Solution Approach 2:
The patent replaces mechanical and chemical detection mechanisms (such as antibody-antigen binding detection in ELISA) with a nucleic acid-based information encoding and sequencing system. This substitution enables scalable parallel processing through established high-throughput sequencing technologies, dramatically increasing throughput while reducing analysis time
3Adaptability or versatility
If affinity agents with detectable labels are used for multiplexing, then recognition of multiple macromolecules can be attempted, but complexity and cross-reactivity issues increase
Solution Approach 1:
The patent employs universal nucleic acid encoding tags that can be attached to various binder molecules (antibodies, aptamers, etc.) and detected through a single sequencing platform. This universal encoding approach enables multiplexing of multiple macromolecule targets without requiring different detection systems for each target, thereby increasing versatility while managing complexity through standardization
Solution Approach 2:
The patent changes the detection parameter from direct optical or chemical signals (prone to cross-reactivity) to nucleic acid sequence information. By encoding binding specificity in the sequence identity rather than in physical signal properties, the system achieves high multiplexing capability with reduced cross-reactivity, as sequence discrimination is far more specific than signal discrimination
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
The method achieves high-throughput, accurate, and sensitive analysis of macromolecules by effectively transferring identifying information, reducing the need for spacers and minimizing DNA-DNA interactions, thereby enhancing the efficiency and accuracy of protein sequencing and characterization.
Implementation Method 1
covalently joining the 5' end of the recording tag to the 3' end of the coding tag by a nucleic acid joining reagent
Implementation Method 2
extending the recording tag using the coding tag as a template by a polymerase, generating a double stranded extended recording tag
Implementation Method 3
cleaving both strands of the double stranded extended recording tag with a double strand nucleic acid cleaving reagent to generate a 3' overhang
Data Source
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AI summary
The present disclosure relates to methods and kits for analyzing a macromolecule. In some embodiments, the present disclosure relates to macromolecule analysis methods which employ barcoding and nucleic acid encoding of molecular recognition events. Also provided herein is a method and related kits for transferring information using a plurality of enzymes, including for performing a ligation, extension, and cleavage reaction with nucleic acid molecules associated with the macromolecule for analysis. In some embodiments, the macromolecule for analysis comprises a peptide, a polypeptide, or a protein.