Multiplexed Protein-DNA Interaction Detection via Tagmentation
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Solution Overview
Problem
Conventional ChIP-seq protocols face challenges in combining steps for protein-DNA interaction analysis into a single workflow suitable for single-cell analysis, and none of the proposed modifications can simultaneously detect multiple proteins.
Innovation Solution
A method involving fragmenting chromatin, tagging it with a first tag, binding an affinity molecule coupled to a second tag to proteins, and coupling the tags to generate tagged chromatin fragments, allowing for sequencing and simultaneous detection of multiple protein-nucleic interactions in a single cell.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional ChIP-seq protocols are used for protein-DNA interaction analysis, then detection of protein binding sites is achieved, but the protocol cannot be combined into a single workflow suitable for single-cell analysis and cannot simultaneously detect multiple proteins
Solution Approach 1:
The patent divides the detection process into distinct segments: chromatin fragmentation with first tags, affinity molecule binding with second tags, and tag coupling. This segmentation allows multiple affinity molecules targeting different proteins to be introduced simultaneously, each with unique tags that can be independently tracked, thereby enabling multiplexed detection without overwhelming workflow complexity
Solution Approach 2:
The patent employs universal tag structures (first tags on chromatin fragments and second tags on affinity molecules) that can accommodate multiple different affinity molecules targeting different proteins. The tag coupling mechanism is universally applicable regardless of which specific affinity molecules are used, allowing the same workflow to detect multiple protein-DNA interactions simultaneously
2Productivity
If ChIP-seq steps are combined into a single workflow for single-cell analysis, then sensitivity and efficiency are improved, but the ability to interrogate multiple proteins simultaneously is lost
Solution Approach 1:
The patent assigns different local qualities (unique tag combinations) to different affinity molecules within the same workflow. Each affinity molecule carries a specific second tag that can be distinguished from others, allowing the system to maintain high detection efficiency while simultaneously interrogating multiple proteins through localized differentiation of tag identifiers
3Adaptability or versatility
If multiple affinity molecules are introduced to detect different proteins, then multiplexed detection is achieved, but distinguishing distinct events between antibodies becomes difficult
Solution Approach 1:
The patent uses asymmetric tag pairing where first tags are attached to chromatin fragments and second tags are attached to affinity molecules. This asymmetric arrangement creates unique tag combinations for each affinity molecule-chromatin interaction, enabling precise distinction of different protein-DNA binding events even when multiple affinity molecules are present 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
Enables the simultaneous detection of multiple protein-nucleic acid interactions in a single cell context, overcoming the limitations of conventional ChIP-seq protocols.
Implementation Method 1
the tagmentation reaction is performed by a transposase. In some cases, the transposase is selected from the group consisting of a hyperactive Tn5 transposase, a MuA transposase, a Vibhar transposase, and Hermes
Implementation Method 2
binding an affinity molecule to a protein or a peptide associated with at least one tagged chromatin fragment of the plurality of first tagged chromatin fragments, wherein the affinity molecule is coupled to a second tag
Implementation Method 3
annealing a first region of the first oligonucleotide tag to a first region of the second oligonucleotide tag, wherein the first region of the first oligonucleotide tag is complementary to the first region of the second oligonucleotide tag
Implementation Method 4
ligating the first oligonucleotide tag to the second oligonucleotide tag
Data Source
AI summary
The present disclosure provides systems and methods that facilitate, at the single cell level, the linking of information from multiple different antibodies (e.g., antibodies specific for different proteins) to the genomic locations of the protein binding sites, while being able to distinguish these distinct events between antibodies.