Nucleic Acid Barcoded Proximity Ligation for Intracellular Mapping
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Solution Overview
Problem
Current methods for determining the spatial proximity of intracellular components are limited in their ability to provide real-time, high-throughput information on interactions between all intracellular components simultaneously, lacking relevance for biochemical functions and requiring separate experiments for each protein, which cannot probe nucleic acids or small molecules effectively.
Innovation Solution
The use of nucleic acid barcoded binding partners with unique nucleotide barcode sequences that ligate upon proximity, allowing for the construction of an intracellular component contact map through PCR amplification and sequencing, enabling the identification of juxtaposed components and their physiological or pharmacological conditions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If separate experiments are conducted for each protein using existing methods, then protein-protein interactions can be detected, but the method cannot probe other cellular components such as nucleic acids and small molecules, and cannot provide high-throughput simultaneous analysis
Solution Approach 1:
The patent employs a universal nucleic acid-based detection platform that can probe multiple types of cellular components (proteins, nucleic acids, small molecules) through a single methodology. Binding partners are designed with universal nucleic acid tags that can hybridize to different target types, enabling one system to perform multiple detection functions simultaneously across diverse cellular components.
Solution Approach 2:
The detection system segments the binding partner into distinct functional modules: a recognition domain specific to the target component and a universal nucleic acid tag domain. This segmentation allows the same nucleic acid tag architecture to be used across different target types while maintaining specific recognition capabilities for each component type through the modular recognition domain.
2Loss of information
If current localization methods are used to determine protein position, then spatial localization information is obtained, but the method provides no information on whether proteins are actively participating in biochemical functions
Solution Approach 1:
The patent merges spatial localization detection with functional interaction detection into a single integrated assay. By using nucleic acid barcoded binding partners that can both localize to specific cellular positions and detect biochemical interactions through ligation events, the system simultaneously obtains both spatial and functional information without requiring separate experimental systems.
Solution Approach 2:
The nucleic acid barcode acts as an intermediary that bridges spatial information and functional information. The barcode sequence itself encodes both the location identity and the interaction status, allowing a single molecular entity to carry multiple information types that would otherwise require separate detection systems.
3Productivity
If high throughput sequencing is utilized, then cost reduction is achieved, but existing methods do not take advantage of this cost reduction to provide comprehensive intracellular interaction data
Solution Approach 1:
The patent changes the detection parameter from traditional readout methods to nucleic acid sequence-based detection, which is directly compatible with high-throughput sequencing technologies. By encoding interaction information in nucleic acid barcode sequences that can be massively parallelized through sequencing, the system leverages the cost and throughput advantages of sequencing to achieve comprehensive detection of numerous intracellular interactions simultaneously.
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 detection and analysis of multiple intracellular components, providing a comprehensive contact map that reflects the spatio-functional status of cells, overcoming the limitations of existing methods by offering real-time, high-throughput data on biochemical interactions.
Implementation Method 1
the spatial proximity of intracellular components may be related to their ability to cooperate in intracellular biochemical reactions. In some embodiments, the present invention contemplates a variety of nucleic acid barcoded binding partners capable of determining the spatial proximity of intracellular components as determined by ligation of their respective nucleotide barcodes.
Implementation Method 2
allowing for the construction of an intracellular component contact map through PCR amplification and sequencing
Implementation Method 3
through PCR amplification and sequencing, enabling the identification of juxtaposed components
Data Source
AI summary
The present invention describes compositions and methods showing that the spatial proximity of intracellular components may be related to their ability to cooperate in intracellular biochemical reactions. In some embodiments, the present invention contemplates a variety of nucleic acid barcoded binding partners capable of determining the spatial proximity of intracellular components as determined by ligation of their respective nucleotide barcodes. As such, an intracellular component contact map may be constructed to fingerprint specific physiological and/or pharmacological intracellular conditions.