Spatial Array Background Reduction Using Diffusion-Restricted Nuclease
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Solution Overview
Problem
Existing methods fail to provide spatially resolved information on the position of single cells within a biological sample, particularly in the context of intact tissues, and suffer from high background analyte binding noise.
Innovation Solution
The use of a diffusion-restricted nuclease to reduce background binding of target nucleic acids on an array by contacting the uncovered area of the array with the nuclease, removing it, and permeabilizing the biological sample to allow capture probes to bind specifically to target nucleic acids, followed by sequencing to determine their location.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional spatial analysis methods are used to provide data for analytes in intact tissue, then spatial information is obtained, but background analyte binding noise is high
Solution Approach 1:
The patent extracts and removes background nucleic acids from the array surface using a diffusion-restricted nuclease that degrades nucleic acids only in uncovered areas. This selective removal eliminates background binding noise while preserving the spatial information captured in the covered areas, directly resolving the contradiction between spatial resolution and background noise.
Solution Approach 2:
The patent applies different treatments to different areas of the array: the covered areas (with biological sample) are protected from nuclease degradation, while uncovered areas are treated with diffusion-restricted nuclease to remove background. This local differentiation allows precise control over where background is removed, maintaining spatial resolution while reducing noise.
2Measurement precision
If diffusion-restricted nuclease is used to reduce background binding, then signal-to-noise ratio improves, but additional processing steps are required
Solution Approach 1:
The patent introduces a diffusion-restricted nuclease as an intermediary agent that selectively degrades background nucleic acids. This mediator enables precise control over background removal by being restricted to uncovered areas only, achieving high signal-to-noise ratio improvement while keeping the process manageable through a single specialized reagent.
3Measurement precision
If permeabilization is performed to allow capture probe binding, then target nucleic acid detection is enabled, but background binding may increase
Solution Approach 1:
The patent performs background removal using diffusion-restricted nuclease before permeabilization and capture probe binding. This preliminary action eliminates background nucleic acids in advance, so when permeabilization occurs later, there is minimal background to bind, thus enabling sensitive detection without increasing background binding.
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 significantly improves the signal-to-noise ratio and sequencing yield by reducing background binding, providing high spatial resolution and accurate location data for target nucleic acids.
Implementation Method 1
contacting the second area of the array with a solution comprising a diffusion-restricted nuclease
Implementation Method 2
diffusion-restricted nuclease
Implementation Method 3
permeabilizing the biological sample, such that the capture domain binds to the target nucleic acid
Data Source
AI summary
Provided herein are methods of determining a location of a target analyte in a non-permeabilized biological sample and methods of reducing background binding of an analyte on an array.