Crosslinked Matrix Confining Nucleic Acid Concatemers
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Solution Overview
Problem
Current methods for in situ analysis of nucleic acid molecules in biological samples face challenges in resolving and stabilizing densely packed nucleic acid concatemers, such as rolling circle amplification products, due to their flexibility and expansion, which affects signal resolution and detection.
Innovation Solution
A method involving a crosslinked matrix formed by contacting the biological sample with a crosslinking agent that reacts with endogenous molecules, allowing nucleic acid concatemers to be confined without covalent binding, thereby reducing their size and stabilizing them for improved spatial fidelity and detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If nucleic acid concatemers are allowed to expand freely in the biological sample, then they can be generated with sufficient length for detection, but their spatial occupancy increases and signal resolution deteriorates
Solution Approach 1:
A crosslinking matrix is introduced as an intermediary substance that physically confines nucleic acid concatemers without covalently binding to them. The matrix acts as a mediator between the concatemers and the surrounding environment, restricting their expansion while maintaining their integrity for detection
Solution Approach 2:
The physical state and spatial distribution parameters of nucleic acid concatemers are changed by introducing the crosslinking matrix. This alters the concatemers' effective volume and spatial occupancy without changing their chemical composition or detection capabilities
2Stability of the object's composition
If nucleic acid concatemers are stabilized through covalent crosslinking, then their structural integrity is improved, but they become covalently bound to the matrix which complicates downstream analysis
Solution Approach 1:
The crosslinking matrix serves as a non-covalent intermediary that stabilizes nucleic acid concatemers through physical confinement rather than covalent bonding. This approach maintains concatemer stability while avoiding the complications of covalent attachment to the matrix
Solution Approach 2:
The patent replaces covalent chemical bonding (chemical system) with physical confinement through crosslinking (mechanical system). This substitution achieves stabilization without forming covalent bonds between the concatemers and the matrix
3Stability of the object's composition
If the biological sample is fixed prior to crosslinking, then structural preservation is improved, but the crosslinking efficiency may be reduced
Solution Approach 1:
The biological sample is fixed prior to crosslinking as a preliminary action to preserve its structural integrity. This pre-treatment ensures that the sample maintains its native architecture during the subsequent crosslinking process, even though it may slightly reduce crosslinking efficiency
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
The method effectively reduces the diameter of nucleic acid concatemers and increases the distance between them, enhancing signal resolution and stability during downstream analyses by physically limiting their expansion and compaction within the crosslinked matrix.
Implementation Method 1
contacting the biological sample with a crosslinking agent to form a crosslinked matrix in the biological sample
Data Source
AI summary
The present disclosure in some aspects relates to methods and compositions for confining molecules generated in a biological sample. In particular examples, rolling circle amplification (RCA) products are generated in a fixed biological sample which has been crosslinked prior to RCA, such that the three-dimensional crosslinked matrix generated prior to RCA confines the RCA products, and the RCA products are smaller than they would otherwise be in a fixed biological sample which has not been crosslinked prior to RCA. Confining the RCA products by generating them in a pre-crosslinked matrix in the biological sample may result in compaction of the RCA products and facilitate subsequent in situ analysis. The sample crosslinking can be performed after probe hybridization to nucleic acid molecules in the sample and before RCA.