Nucleic Acid Recording Tags for Macromolecule Sequencing
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Solution Overview
Problem
Current methods for high-throughput macromolecule analysis, such as proteomics, face challenges with limited sensitivity, dynamic range, and cross-reactivity, particularly in multiplexing affinity agents to cognate macromolecules, necessitating improved techniques for efficient, parallelized, accurate, and sensitive protein sequencing and analysis.
Innovation Solution
The use of nucleic acid-encoded binding agents in a cyclical manner, employing hairpin coding and recording tags with double-stranded regions to minimize off-target interactions, combined with nucleic acid joining and cleaving reactions, allows for efficient information transfer and sequencing of macromolecules, including polypeptides, in a high-throughput format.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional immunoassays and mass spectrometry-based methods are used for macromolecule analysis, then protein identification and quantitation can be performed, but sensitivity and dynamic range are limited with potential cross-reactivity and background signals
Solution Approach 1:
The patent introduces nucleic acid recording tags as intermediary molecules that mediate between binding agents and detection systems. These recording tags capture binding events through sequential ligation reactions, converting protein-binding information into nucleic acid sequences that can be amplified and sequenced with high sensitivity, while the modular design reduces cross-reactivity and background signals
Solution Approach 2:
The patent replaces traditional detection mechanisms (such as fluorescent labels and mass spectrometry detection) with a nucleic acid-based information storage and amplification system. By converting binding events into sequencable nucleic acid records, the system achieves higher sensitivity through PCR amplification and more specific detection through sequencing, eliminating many limitations of conventional detection methods
2Productivity
If affinity agents are multiplexed to recognize multiple cognate macromolecules, then high-throughput analysis can be achieved, but cross-reactivity and background signals increase
Solution Approach 1:
The patent segments the detection system into distinct functional modules: binding agents with specific epitope recognition, nucleic acid recording tags for information capture, and sequencing for readout. Each binding agent is assigned a unique recording tag sequence, allowing multiple binding events to be tracked independently through sequential ligation, thereby maintaining specificity while enabling high-throughput multiplexed analysis
Solution Approach 2:
The patent changes the detection parameter from direct signal measurement (fluorescence, mass) to nucleic acid sequence information. By encoding binding events as unique nucleic acid sequences that can be differentially amplified and sequenced, the system can distinguish between multiple binding events with high specificity, even when using multiplexed binding agents with different specificities
3Productivity
If single-stranded nucleic acid tags are used for encoding binding events, then information transfer is efficient, but off-target interactions and noise increase
Solution Approach 1:
The patent uses composite nucleic acid structures combining double-stranded regions for stable, specific hybridization with single-stranded overhangs for efficient ligation and extension. The double-stranded recording tag provides structural stability and reduces off-target interactions through complementary base pairing, while the single-stranded 3' overhang enables efficient primer extension and ligation reactions for information transfer
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 enhances the specificity and sensitivity of macromolecule analysis by reducing noise and background signals, enabling accurate and efficient sequencing of multiple macromolecules simultaneously, thereby overcoming the limitations of existing methods.
Implementation Method 1
extending the recording tag with a polymerase, using the coding tag as a template
Implementation Method 2
using the coding tag as a template
Implementation Method 3
joining the recording tag to the coding tag
Implementation Method 4
cleaving the extended recording tag with a double-stranded nucleic acid cleaving reagent
Data Source
AI summary
The present disclosure relates to macromolecule analysis methods which employ nucleic acid encoding of molecular recognition events in a cyclical manner. Also provided herein are methods and related kits for transferring information using a plurality of enzymes, including for performing joining and cleavage reactions in a cyclical manner with nucleic acid molecules associated with the macromolecule for analysis. In some embodiments, the macromolecule for analysis comprises a polypeptide, and the disclosed methods and related kits are for high-throughput polypeptide analysis, including polypeptide sequencing.