Low-Input CUT&Tag Chromatin Profiling for Weak Protein Binding
Find Innovative SolutionsGenerate Solutions
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, which limit the detection of less abundant or weakly interacting proteins, leading to low data quality and variability.
Innovation Solution
A method involving permeabilization of cells with antibodies and transposomes in the presence of a crowding agent, using mild cross-linking and reduced salt concentrations, allows for the detection of chromatin binding sites of less abundant proteins by excising and tagging DNA segments for sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If strong crosslinking with formaldehyde is used to maintain protein-DNA interactions, then detection reliability of weakly interacting proteins improves, but epitope accessibility deteriorates making antibodies and transposase less effective
Solution Approach 1:
The patent applies preliminary permeabilization of cells before introducing antibodies and transposase. This preliminary action allows reagents to access chromatin without requiring strong crosslinking, thereby maintaining epitope accessibility while ensuring reliable detection of protein-DNA interactions including weakly interacting proteins
Solution Approach 2:
The patent changes the parameter of crosslinking intensity by using no crosslinking or very mild crosslinking conditions instead of strong formaldehyde crosslinking. This parameter change resolves the contradiction by maintaining epitope accessibility while still enabling reliable detection through the modified CUT&Tag protocol
2Measurement precision
If high salt concentrations are used to remove unbound transposase during washing, then off-target tagmentation decreases, but weakly interacting proteins are removed from chromatin
Solution Approach 1:
The patent changes the salt concentration parameter during washing steps to lower concentrations that preserve weakly interacting protein-DNA complexes. This parameter change allows for adequate removal of unbound transposase while maintaining the stability of weakly interacting proteins on chromatin
Solution Approach 2:
The patent uses magnetic beads as an intermediary to capture and retain chromatin-protein complexes during washing steps. This intermediary allows for gentle washing conditions that preserve weak interactions while still effectively removing unbound transposase through the magnetic separation process
3Measurement precision
If conventional ChIP requires high cell numbers (10^6 cells) for sufficient signal, then detection sensitivity improves, but sample consumption and cost increase
Solution Approach 1:
The patent replaces the mechanical sonication-based chromatin shearing with an enzymatic approach using transposase for in situ tagmentation. This substitution dramatically increases efficiency and sensitivity, allowing detection with as few as 10^4 cells while maintaining high detection sensitivity through direct DNA tagging at protein binding sites
Solution Approach 2:
The patent changes the detection sensitivity parameter through the use of transposase-mediated tagmentation, which provides signal amplification by directly incorporating sequencing adapters at binding sites. This parameter change enables high sensitivity detection with minimal cell input, resolving the contradiction between detection sensitivity and sample quantity
4Productivity
If CUT&Tag is used to reduce cell input requirements, then productivity improves, but detection reliability of weakly interacting proteins deteriorates
Solution Approach 1:
The patent modifies several parameters including salt concentration during washing, addition of crowding agents, and optimization of transposase activity conditions. These parameter changes maintain high productivity with low cell input while simultaneously improving detection reliability of weakly interacting proteins by preventing their premature removal
Solution Approach 2:
The patent introduces magnetic beads as an intermediary to enhance the reliability of protein-DNA complex retention during the high-throughput CUT&Tag process. This intermediary ensures that even weakly interacting proteins are reliably captured and retained, maintaining detection reliability while preserving the productivity advantages of CUT&Tag
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 analysis of chromatin binding sites with reduced cell numbers, improving data quality and consistency, particularly for non-histone proteins and weakly interacting transcriptional factors.
Implementation Method 1
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; activating the transposase, thereby excising a DNA segment comprising at least one chromatin binding site
Implementation Method 2
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
Implementation Method 3
a transposome that is linked to a specific binding agent that specifically binds the first and/or second antibody
Data Source
Figure 1
Figure 2
Figure 3
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.