CUT&Tag With Mild Cross-Linking for Weak Interaction Detection
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Solution Overview
Problem
Conventional methods for profiling chromatin-associated proteins, such as ChIP and CUT&Tag, require high cell numbers and harsh cross-linking conditions, leading to loss of signal, variability, and inability to detect weak protein-chromatin interactions reliably.
Innovation Solution
A method involving permeabilization of cells, use of a transposome with a transposase in the presence of a crowding agent, and mild cross-linking conditions to detect chromatin binding sites of chromatin-associated factors, allowing for reliable analysis with reduced cell numbers.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If strong crosslinking with formaldehyde is used to stabilize protein-DNA interactions, then weak protein-chromatin interactions can be detected, but epitopes are masked making them less accessible to antibodies and transposase
Solution Approach 1:
The patent applies preliminary action by performing mild crosslinking (0.05-0.2% formaldehyde for 5-15 minutes) before the main CUT&Tag procedure to stabilize protein-DNA interactions, then proceeds with antibody binding and transposase activation. This preliminary stabilization allows detection of weak interactions while maintaining epitope accessibility for subsequent steps.
Solution Approach 2:
The patent changes the parameters of crosslinking by using lower formaldehyde concentrations (0.05-0.2% instead of 1%) and shorter incubation times (5-15 minutes instead of 8-15 minutes), which provides sufficient stabilization without excessive crosslinking that would mask epitopes and prevent antibody binding.
2Measurement precision
If high salt concentrations are used to remove unbound pA-Tn5 during washing, then off-target tagmentation is decreased, but tightly bound proteins may remain bound while weaker interacting proteins are removed
Solution Approach 1:
The patent performs preliminary mild crosslinking to stabilize weak protein-DNA interactions before the high salt washing step, ensuring that even weakly interacting proteins remain bound during the stringent wash conditions that remove unbound pA-Tn5 and reduce off-target tagmentation.
Solution Approach 2:
The patent applies beforehand cushioning by using mild crosslinking to pre-stabilize protein-DNA interactions, creating a protective effect that allows subsequent harsh washing with high salt concentrations to remove non-specifically bound pA-Tn5 without disrupting genuine weak protein-chromatin interactions.
3Quantity of substance
If conventional ChIP requires high number of cells (10^6) as starting material, then sufficient DNA is obtained for sequencing, but the method is not suitable for low cell number samples
Solution Approach 1:
The patent extracts and removes unnecessary steps from conventional ChIP by eliminating chromatin shearing and extensive washing steps, using instead a streamlined CUT&Tag approach with transposase-based tagmentation that directly generates sequencing libraries from immunoprecipitated DNA, enabling sufficient DNA recovery from as few as 1,000 cells.
Solution Approach 2:
The patent changes the parameters of the workflow by replacing sonication-based chromatin fragmentation with transposase-based tagmentation, and reducing the number of washing steps, which minimizes sample loss and enables reliable results with low cell numbers while still obtaining sufficient DNA for sequencing.
4Device complexity
If CUT&Tag does not use chromatin preparation steps, then the workflow is simplified and cell number requirements are reduced, but proteins with weak interactions detach more easily and cannot be reliably detected
Solution Approach 1:
The patent applies preliminary mild crosslinking to stabilize weak protein-DNA interactions before proceeding with the simplified CUT&Tag workflow, ensuring that proteins with weak interactions remain bound throughout the process without requiring complex chromatin preparation steps.
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 reliable detection of a broad range of protein-chromatin interactions with improved data quality and reduced variability, even with low cell inputs, by stabilizing protein binding and minimizing signal loss.
Implementation Method 1
A subsequently added fusion protein consisting of protein A and transposase Tn5 is bound to the antibody. After binding of the protein A part to the antibody and activation of Tn5, the latter cuts the DNA and incorporates sequencing adapters at the site.
Implementation Method 2
the harvested cells are cross-linked with formaldehyde to ensure covalent, reversible cross-linking of the proteins with the DNA
Implementation Method 3
the steps of contacting the permeabilized cell with the transposome and activating the transposase are performed in presence a crowding agent
Implementation Method 4
The recovered DNA is purified and used for enrichment testing by qPCR, library preparation and sequencing
Implementation Method 5
the cross-linking is dissolved by heating to 65°C in the presence of SDS and sodium chloride
Data Source
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AI summary
The present invention provides a novel method for determining at least one chromatin binding site of a chromatin-associated factor of interest in a cell, comprising: (i) permeabilizing the cell; (ii) contacting the permeabilized cell with a first antibody that specially binds the chromatin-associated factor of interest and with a second antibody that specifically binds to the first antibody; (iii) contacting the permeabilized cell with a transposome that is linked to a specific binding agent that specifically binds the first and/or second antibody wherein the transposome comprises a transposase and a first and second DNA molecule; (iv) activating the transposase, thereby excising a DNA segment comprising at least one chromatin binding site of a chromatin-associated factor of interest and tagging the DNA with the first and second DNA molecule; (v) determining the sequence of the excised and tagged DNA segment; and (vi) based on the determined sequence of the excised and tagged DNA segment, determining the at least one chromatin binding site of the chromatin-associated factor of interest in the cell; wherein the steps of contacting the permeabilized cell with the transposome and activating the transposase are performed in presence a crowding agent.