CAP-C Dendrimer Crosslinking for High-Resolution Genomic Proximity Capture
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Current chromosome conformation capture methods, such as Hi-C, are limited by formaldehyde-mediated crosslinking, which masks restriction sites and prevents precise inference of genomic proximity, and struggle to resolve chromatin domains at high resolution, missing finer details of promoter-enhancer interactions and chromatin domains.
Innovation Solution
Chemical-crosslinking Assisted Proximity Capture (CAP-C) uses functionalized scaffolds like dendrimers with defined cross-linkers to expose all potential restriction sites, allowing for high-resolution capture of spatial relationships between genomic sequences, including DNA-DNA, RNA-RNA, and protein interactions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If formaldehyde-mediated crosslinking is used in chromosome conformation capture methods, then chromatin structure can be preserved, but restriction sites are masked and measurement precision of genomic proximity deteriorates
Solution Approach 1:
The patent changes the crosslinking parameter from formaldehyde to UV irradiation-activated photo-crosslinking moieties. This parameter change allows chromatin structure preservation through controlled crosslinking while avoiding the masking of restriction sites, as the photo-crosslinking occurs at specific wavelengths that do not interfere with restriction enzyme recognition and cutting activities.
Solution Approach 2:
The patent introduces photo-crosslinking moieties as intermediary agents between chromatin components. These moieties act as mediators that can be selectively activated by UV irradiation to form crosslinks, providing controlled stabilization without the non-specific masking effects of formaldehyde. The intermediaries enable precise spatial capture while maintaining accessibility of restriction sites.
2Adaptability or versatility
If conventional 3C methods are used, then chromatin organization can be studied, but resolution of chromatin domains is insufficient and finer details of promoter-enhancer interactions are missed
Solution Approach 1:
The patent segments the chromatin analysis into multiple resolution levels by combining UV crosslinking for capturing proximal interactions with restriction enzyme digestion for defined fragment generation. This segmentation allows simultaneous study of both large-scale chromatin organization and fine-scale promoter-enhancer interactions within the same experimental framework, achieving multi-resolution analysis.
Solution Approach 2:
The patent performs preliminary UV irradiation crosslinking before restriction enzyme digestion to stabilize chromatin conformation. This preliminary action preserves the spatial relationships of chromatin elements, ensuring that subsequent enzymatic processing occurs on pre-stabilized structures, thereby maintaining high-resolution information about promoter-enhancer interactions and chromatin domain architecture.
3Reliability
If formaldehyde crosslinking is used, then protein-DNA interactions can be captured, but device complexity increases and ease of operation deteriorates due to extensive covalent linkages
Solution Approach 1:
The patent extracts the crosslinking function from formaldehyde to UV-activatable photo-crosslinking moieties. This extraction allows selective activation of crosslinking only where and when needed, rather than creating extensive non-specific covalent linkages throughout the chromatin. The result is simplified processing, as UV crosslinking creates more controlled and reversible linkages that are easier to manage during subsequent digestion and analysis 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
CAP-C reveals finer details of chromatin domains and promoter-enhancer interactions, providing higher resolution than conventional methods, and can identify previously undetected chromatin structures and interactions, enhancing understanding of chromatin architecture.
Implementation Method 1
DNA in proximity are covalently crosslinked with dendrimers under UV irradiation
Implementation Method 2
The crosslinked chromatin is digested with proteases, releasing the DNA
Implementation Method 3
The DNA is then cleaved with a restriction enzyme
Implementation Method 4
proximal ligation to infer spatial relationships between genomic loci
Data Source
AI summary
Certain embodiments are directed to compositions and methods for capture of elements in physical proximity. In certain aspects the methods comprise (a) contacting a target with a functionalized scaffold or capture agent that comprises activatable cross-linking moieties to form a target/scaffold mixture; (b) exposing the target/scaffold mixture to an activator to activate the cross-linking moieties of the dendrimer and form a cross-linked target/scaffold complex; (c) isolating the target/scaffold complexes; and (d) identify portions of the target or targets that are cross linked with the scaffold.