Matrix-Embedded Primer Exchange Reaction for Spatial Localization
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Solution Overview
Problem
Existing methods for analyzing the identity and spatial localization of analytes in samples are limited by signal amplification, positional stability, and probe penetration in thick tissue samples, which suffer from autofluorescence, light scattering, and optical aberrations.
Innovation Solution
A method involving a primer exchange reaction (PER) in a three-dimensional polymerized matrix, where a nucleic acid molecule with a free 3′ priming region is immobilized, contacted with an initial hairpin molecule and a polymerase having strand displacement activity, and elongated products are detected to preserve spatial localization and amplify signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If in situ hybridization-based approaches are used to analyze analytes in tissue samples, then spatial localization of analytes is preserved, but signal amplification is limited and probe penetration is insufficient in thick tissue samples
Solution Approach 1:
The detection system is segmented into multiple functional components: a first molecule with analyte-binding capability, a second molecule serving as a primer with a free 3' end, and a hairpin molecule with toehold domain. This segmentation allows each component to perform its specific function optimally while working together to achieve both spatial preservation and signal amplification through the matrix-embedded primer exchange reaction
Solution Approach 2:
The matrix-embedded primer exchange reaction acts as an intermediary mechanism that bridges the gap between probe binding and signal detection. The reaction enables information transfer from the analyte-bound first molecule through the second molecule to the hairpin molecule, allowing signal amplification while maintaining the spatial relationship of the original analyte location in the tissue sample
2Loss of information
If conventional probe methods are used in thick tissue samples, then spatial information is maintained, but probe penetration and signal detection are hindered by autofluorescence and light scattering
Solution Approach 1:
The method performs preliminary actions by first embedding the primer system into the matrix before analyte detection. The matrix-embedded primers are pre-positioned and ready to undergo exchange reactions, creating a foundation that enables subsequent signal amplification and detection while preserving spatial information, thereby overcoming the limitations of autofluorescence and light scattering in thick tissues
3Productivity
If signal amplification is increased to improve detection sensitivity, then analyte detection capability is enhanced, but spatial localization accuracy may be compromised
Solution Approach 1:
The amplification process maintains local quality by confining the primer exchange reaction to the immediate vicinity of the analyte-molecule complex. The matrix-embedded architecture ensures that the hairpin molecule and elongation products remain spatially associated with the original analyte location, allowing signal amplification without compromising the spatial localization accuracy of the detected analyte
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 method allows for stable and amplified detection of analytes within the matrix, maintaining spatial information and enabling prolonged information storage and read-out cycles, resistant to diffusion or mechanical stress.
Implementation Method 1
incubating the sample under conditions for polymerization to produce an elongated product of the immobilized nucleic acid molecule
Implementation Method 2
a polymerase having strand displacement activity
Implementation Method 3
immobilizing the nucleic acid molecule to the matrix
Implementation Method 4
a paired stem domain formed by intramolecular nucleotide base pairing between a 3' subdomain of the initial hairpin molecule and a 5' subdomain of the initial hairpin molecule
Data Source
AI summary
The present application provides methods, compositions, and kits for analyzing a biological sample embedded in a three-dimensional polymerized matrix using a primer-exchange reaction (PER). In some embodiments, the methods comprise contacting the sample with a nucleic acid molecule that directly or indirectly binds to an analyte in the sample and immobilizing the nucleic acid molecule in the matrix, wherein the nucleic acid molecule comprises a free 3′ priming region for initiation of PER. In some embodiments, the methods enable sensitive detection of the identity and relative position of analytes in the sample.