Porous In Situ Matrix for Spatial Nucleic Acid Detection
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Solution Overview
Problem
Current methods for detecting nucleic acid sequences in tissue samples face limitations due to low signal from endogenous, unamplified sequences and lack control over amplification magnitude, as well as inefficiencies in labeling amplicons and detecting molecular proximity in situ.
Innovation Solution
Development of compositions and methods involving a porous in situ matrix with a low ratio of cross-linking agent to monomer or polymer, allowing for efficient enzymatic activity and precise nucleic acid amplification and detection, enabling high spatial resolution detection of macromolecules and spatial proximity analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If extant in situ amplification methods (e.g., rolling circle amplification) are used, then nucleic acid sequences can be amplified in tissue samples, but control over the magnitude of production is lacking and labeling of amplicons is limited
Solution Approach 1:
The patent applies parameter changes by modifying the cross-linking agent concentration (reducing it to very low levels) and monomer composition in the matrix to enable controlled amplification. This allows precise control over amplification magnitude while maintaining simplicity of the amplification method itself.
Solution Approach 2:
The patent uses composite materials by creating a specialized matrix composed of monomers and cross-linking agents in specific proportions. This composite matrix provides the necessary structural support while allowing controlled enzymatic activity and amplification, resolving the contradiction between control and complexity.
2Strength
If commonly used amounts of cross-linking agents (e.g., bis-acrylamide and acrylamide) are used in polymeric matrices, then matrix structural integrity is achieved, but porosity is insufficient for efficient enzymatic activity
Solution Approach 1:
The patent applies parameter changes by dramatically reducing the cross-linking agent to monomer ratio from commonly used levels to very low concentrations (e.g., 0.01% or lower). This parameter change maintains sufficient matrix integrity while dramatically improving porosity and enzymatic activity efficiency.
Solution Approach 2:
The patent explicitly uses porous materials by designing a matrix with optimized porosity through reduced cross-linking. The porous structure allows efficient diffusion of enzymes and substrates while maintaining structural integrity, directly resolving the contradiction between strength and enzymatic efficiency.
3Stability of the object's composition
If high concentration cross-linking agents are used to form a robust matrix, then matrix stability is improved, but spatial resolution for macromolecule detection deteriorates
Solution Approach 1:
The patent applies parameter changes by optimizing the cross-linking agent concentration to very low levels, which maintains adequate matrix stability while preventing excessive cross-linking that would reduce spatial resolution. This allows precise localization of macromolecules at high spatial resolution.
4Reliability
If endogenous, unamplified nucleic acid sequences are used for detection, then the native state is preserved, but signal strength is insufficient for reliable detection
Solution Approach 1:
The patent applies preliminary action by performing in situ amplification of nucleic acid sequences before detection. This preliminary amplification step increases the quantity of target sequences to detectable levels while maintaining spatial information, thereby improving detection reliability without requiring excessive starting material.
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 precise and efficient nucleic acid amplification and detection at high spatial resolution, facilitating applications such as RNA sequence measurement, copy number variation assessment, and nucleic acid therapy evaluation.
Implementation Method 1
a cross-linking agent including a second monomer or polymer, where the cross-linking agent is capable of crosslinking with the first monomer or linear polymer when the cross-linking agent and the first monomer or linear polymer are combined
Implementation Method 2
a nucleic acid primer or probe having a modification capable of binding or chemically conjugating the primer or probe to the first monomer or linear polymer, to the cross-linking agent, or to both
Data Source
AI summary
The present disclosure relates to compositions and methods for detecting nucleic acid sequences (e.g., coding and non-coding RNAs; nuclear/genomic DNA; mtDNA; pathogen nucleic acids, etc.) in a tissue sample, specifically providing improved matrices and matrix-employing methods for performance of nucleic acid capture and amplification in a tissue sample in situ and/or in a manner that retains spatial location information for captured nucleic acids (including nucleic acid-associated macromolecules).