Nucleic Acid-Tagged Polypeptides for Multiplexed Protein Interaction Profiling
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Solution Overview
Problem
Current methods struggle to efficiently identify multiple protein-protein interaction targets in a multiplexed format, particularly in medical diagnostics and personalized medicine applications.
Innovation Solution
The use of nucleic acid-tagged polypeptides in a multiplexed polypeptide affinity assay (MPA) composition, where each prey polypeptide target is chemically linked to a prey nucleic acid, allowing for the identification of protein-protein interaction targets through binding interactions with bait polypeptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If traditional methods are used to identify protein-protein interaction targets, then the process can be performed with simple procedures, but the ability to identify multiple targets in parallel is limited and the process becomes daunting
Solution Approach 1:
The system segments the identification process by assigning unique nucleic acid barcodes to individual prey polypeptide targets. Each bait polypeptide interaction can be independently tracked through its associated barcode, enabling parallel identification of multiple interaction targets without cross-interference. This segmentation transforms a complex multiplexed problem into manageable independent identification channels.
Solution Approach 2:
Nucleic acid barcodes serve as intermediary molecules that bridge the protein-protein interaction detection and the sequencing-based identification system. The barcodes are chemically linked to prey polypeptides but do not participate in the protein interactions themselves, acting as passive carriers of identification information that can be amplified and sequenced independently of the interaction assay.
2Adaptability or versatility
If diverse bait polypeptides are used to increase detection capability, then more interaction targets can be identified, but the complexity of analyzing the mixture increases
Solution Approach 1:
The system creates nucleic acid copies (barcodes) of each prey polypeptide identity. These barcode sequences can be amplified through PCR and sequenced using high-throughput sequencing technologies, converting the protein interaction detection problem into a nucleic acid analysis problem that benefits from exponential amplification and parallel sequencing capabilities.
Solution Approach 2:
The system replaces complex protein-based detection and analysis methods with nucleic acid-based sequencing. Instead of using elaborate protein purification and identification protocols, the invention substitutes a streamlined workflow where nucleic acid barcodes are amplified and sequenced, leveraging the power of next-generation sequencing to simplify the analysis of diverse interaction targets.
3Productivity
If chemically linked nucleic acid tags are attached to each prey polypeptide, then simultaneous identification of multiple targets is enabled, but the manufacturing complexity increases
Solution Approach 1:
The system uses universal nucleic acid barcode sequences that can be chemically attached to different prey polypeptides through standardized chemical linkage methods. Each barcode contains universal regions that facilitate attachment and sequencing, while maintaining a unique identifier portion. This universal design allows the same chemical protocols to be applied across diverse prey polypeptides, simplifying the manufacturing process.
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 enables the simultaneous identification of multiple protein-protein interaction targets, providing a robust method for antigen immunoprofiling and disease-associated antigen identification in complex biological samples.
Implementation Method 1
identifying the many interacting target proteins for multiple protein actors
Implementation Method 2
each PPT in the population is chemically linked to a prey nucleic acid
Data Source
AI summary
Methods and compositions for multiplexed protein-protein interaction profiling (e.g., immunoprofiling), based on nucleic acid tagging of polypeptides (e.g., by RNA display) are described. In some embodiments the described compositions and methods utilize a library of prey polypeptide targets linked to prey RNAs encoding them, and a population of bait polypeptides, e.g., a mixture of antibodies, that bind to one or more of the prey polypeptide targets and are used to isolate and identify the bound prey polypeptide targets by amplification of their associated prey RNAs and sequencing of the corresponding cDNAs. In other embodiments the prey polypeptide targets are linked to DNA Bar Codes, which serve as unique identifiers of the tagged polypeptide.