DNA Binding Site Mapping via Antibody-Transposase Segmentation
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing technologies for mapping DNA binding sites, such as ChIP-seq and CUT&Tag, face challenges including high background noise, cross-contamination, and the inability to simultaneously map multiple proteins in a single cell effectively.
Innovation Solution
The proposed technology involves a method for identifying DNA binding sites using a tagging composition that includes an antibody or antibody fragment, a heterocyclic compound linked to the antibody, a protein complex, and nucleic acids with barcode sequences. This composition is used to generate tagmented nucleic acid fragments, which are then sequenced to identify binding sites.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional ChIP-seq is used for binding site identification, then binding sites can be mapped, but substantial cell quantity (>1 million cells) is required and notable background noise is introduced
Solution Approach 1:
The patent segments the ChIP-seq process into two distinct phases: (1) antibody-based target recognition and binding in fixed cells, and (2) transposase-based tagmentation and DNA library preparation. This segmentation allows the use of fixed cells (reducing cell quantity requirements) while maintaining binding site identification accuracy through the specific antibody-target interaction followed by precise transposase insertion at binding sites.
Solution Approach 2:
The patent applies preliminary action by fixing cells before the assay to preserve protein-DNA interactions, then performing antibody incubation on fixed cells. This preliminary fixation step allows subsequent processing with fewer live cells while maintaining the integrity of protein-DNA complexes for accurate binding site mapping.
2Quantity of substance
If CUT&Tag is used to reduce cell quantity requirements, then single-cell analysis becomes possible, but dissociation of the transposase-protein A fusion protein and antibody causes spurious tagmentation increasing background noise
Solution Approach 1:
The patent segments the fusion protein into separate components: antibody, protein A, and transposase are provided as separate entities rather than a pre-formed fusion. This segmentation prevents spurious tagmentation because the transposase remains inactive until specifically recruited to the antibody-bound target through protein A-mediated assembly, reducing background noise while enabling single-cell analysis.
Solution Approach 2:
The patent introduces protein A as an intermediary component that mediates the interaction between the antibody and transposase. Protein A binds to the antibody and recruits the transposase to the target site, acting as a controlled bridge that prevents direct, uncontrolled transposase activity and reduces spurious tagmentation background noise.
3Adaptability or versatility
If multiple adaptor-loaded transposase-protein A fusion proteins are used for multiplexing, then multiple targets can be mapped, but swapping of adaptors and antibodies among binding partners produces incorrect signals due to incorrect pairing
Solution Approach 1:
The patent segments the multiplexing system into separate antibody and transposase components with unique identifiers, rather than using pre-assembled fusion proteins. This segmentation eliminates adaptor-swapping errors because each antibody and transposase can be independently tracked and matched through their unique sequences, ensuring correct pairing and maintaining signal accuracy in multiplexed experiments.
Solution Approach 2:
The patent uses unique DNA sequences as copies/identifiers for each antibody-transposase pairing. These unique sequences serve as molecular barcodes that allow bioinformatic matching of antibodies to their corresponding transposase insertion sites, preventing incorrect signal assignment and enabling accurate multiplexed target mapping.
4Adaptability or versatility
If ChIP-seq is performed with multiple rounds of immunoprecipitation to study multiple targets, then comprehensive protein-DNA interactions can be analyzed, but both labor-intensive procedures and substantial initial material quantities are demanded
Solution Approach 1:
The patent merges multiple immunoprecipitation steps into a single simultaneous assay by allowing multiple antibodies to bind to fixed cells at the same time, followed by a single transposase treatment step. This combining of parallel processes into one unified workflow enables comprehensive multi-target analysis while dramatically reducing procedure time and material requirements compared to sequential ChIP-seq experiments.
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 technology enables multiplexed identification of multiple DNA binding sites with reduced background noise and cross-contamination, allowing for the simultaneous mapping of multiple targets in a single cell, thereby improving the accuracy and efficiency of DNA binding site analysis.
Implementation Method 1
contacting the two or more nucleic acids of the tagging composition with a transposase, thereby forming an antibody-barcode-transposase complex, wherein the antibody-barcode-transposase complex generates double stranded breaks in a nucleic acid comprising the nucleic acid binding site
Implementation Method 2
contacting the target that is bound to the nucleic acid binding site with a tagging composition, thereby binding the tagging composition to the target, wherein the tagging composition comprises: (i) an antibody or an antibody fragment that binds to the target
Data Source
AI summary
Provided herein is technology relating to identifying the binding locations of DNA-binding proteins and particularly, but not exclusively, to methods, systems, and kits that use affinity reagent-specific barcodes for simultaneously mapping the binding sites of multiple proteins in the same cell.