De-crosslinking Agents for Spatial Gene Expression Analysis
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Solution Overview
Problem
Formaldehyde fixation in biological samples creates crosslinks that are incompatible with gene expression analysis, particularly spatial gene expression analysis, as they hinder the detection and analysis of nucleic acids and proteins, and existing methods to break these crosslinks are often harsh and damaging.
Innovation Solution
A method involving the use of de-crosslinking agents, such as specific compounds, to reverse the crosslinks in fixed biological samples, allowing for the production of de-crosslinked samples that can be analyzed using capture probes with capture domains and spatial barcodes, enabling accurate spatial analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Stability of the object's composition
If formaldehyde fixation is used to preserve biological samples, then sample preservation and stability are improved, but crosslink formation occurs that is incompatible with gene expression analysis
Solution Approach 1:
The patent extracts and removes the harmful crosslinks from the fixed biological sample using de-crosslinking agents. The method involves treating formaldehyde-fixed samples with specific de-crosslinking compounds that break the crosslinks between nucleic acids and proteins, thereby removing the barrier to gene expression analysis while preserving the sample's structural integrity for spatial analysis.
Solution Approach 2:
The patent introduces de-crosslinking agents as intermediary substances that mediate between the fixed sample state and the analytical state required for gene expression detection. These agents temporarily interact with the crosslinked sample to reverse crosslinks, enabling subsequent capture probe hybridization and spatial analysis without permanent damage to the sample architecture.
2Object-generated harmful factors
If heat is used to break crosslinks in fixed tissues, then crosslink removal is achieved, but sample damage and loss of nucleic acid integrity occur
Solution Approach 1:
The patent replaces the mechanical/thermal method (heat treatment) with a chemical method (de-crosslinking agents). Instead of using high temperature to break crosslinks, which causes sample damage, the invention uses specific chemical compounds that selectively break crosslinks under mild conditions, preserving nucleic acid integrity and sample structure for accurate spatial analysis.
Solution Approach 2:
The patent changes the parameters of crosslink removal from high-temperature physical treatment to controlled chemical treatment at physiological temperatures. By using de-crosslinking agents with specific chemical properties, the method achieves crosslink removal at lower temperatures and milder conditions, preventing sample damage while effectively reversing formaldehyde crosslinks.
3Object-generated harmful factors
If traditional crosslink-breaking methods are used, then crosslinks are removed, but assay sensitivity and spatial resolution are reduced due to sample damage
Solution Approach 1:
The patent employs disposable de-crosslinking agents that can be applied and then removed or degraded after use. These chemical agents perform their function of breaking crosslinks and are then discarded, leaving no permanent residue that would interfere with subsequent spatial analysis. This approach ensures that crosslink removal does not compromise the long-term integrity or spatial resolution of the sample.
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 approach allows for the recovery and analysis of nucleic acids and proteins at high spatial resolution, maintaining native spatial context and improving assay sensitivity by effectively reversing crosslinks without causing further damage to the biological samples.
Implementation Method 1
Formaldehyde fixation crosslinks amine functional groups of nucleic acids and proteins through covalent linkage
Implementation Method 2
contacting the fixed biological sample with a substrate including a plurality of capture probes, wherein a capture probe of the plurality of capture probes includes a capture domain
Data Source
AI summary
Provided herein are methods for de-crosslinking fixed biological samples (e.g., fixed biological samples including aminal crosslinks). The compositions and methods disclosed can de-crosslink oligonucleotides (e.g., DNA or RNA) or proteins from fixed biological samples (e.g., fixed biological samples with aminal crosslinks), wherein the de-crosslinked biological sample is compatible with and can be used in spatial gene expression analysis.