Spatial Gene Expression Libraries With Reversible Probe Blocking
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Solution Overview
Problem
Existing methods fail to provide spatially resolved data on gene expression within tissues, lacking information on the position of single cells within biological samples.
Innovation Solution
A method involving spatial gene expression libraries using arrays with attached first and second probes, where the first probe has a spatial barcode and poly(T) capture domain, and the second probe is reversibly blocked with a poly(GI) capture domain, allowing for the determination of target nucleic acid location through sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Loss of information
If existing techniques are used to analyze gene expression, then analyte data can be obtained, but spatial location information of single cells is lost
Solution Approach 1:
The probe is divided into two separate probes (first probe with poly(T) capture domain and spatial barcode, second probe with poly(GI) capture domain) that can be reversibly blocked and unblocked. This segmentation allows the spatial barcode to be captured on one probe while the target nucleic acid is captured on the other, resolving the contradiction between obtaining spatial information and maintaining method simplicity.
Solution Approach 2:
The reversibly blocked second probe acts as an intermediary mechanism. When blocked, it prevents premature capture of the target nucleic acid. When unblocked, it enables the capture domain to bind the target. This intermediary state allows temporal separation of spatial barcode capture and target nucleic acid capture, solving the information loss problem without requiring complex simultaneous capture mechanisms.
2Measurement precision
If the second probe is reversibly blocked, then spatial resolution is improved, but the complexity of the probe design increases
Solution Approach 1:
The second probe transitions from a blocked state (preventing premature target binding) to an unblocked state (enabling target capture). This dynamic state change allows the same probe design to serve multiple functions at different stages, improving spatial resolution while avoiding the need for entirely separate probe systems.
Solution Approach 2:
The reversibly blocked second probe serves multiple functions: it acts as a placeholder during spatial barcode capture, then becomes an active target capture probe when unblocked. This multi-functionality reduces the need for additional specialized components, balancing measurement precision with design complexity.
3Reliability
If the poly(GI) capture domain is used, then binding specificity is improved, but the difficulty of detecting and measuring increases
Solution Approach 1:
The reversibly blocked design provides feedback control: the blocked state prevents non-specific binding and ensures only the intended target is captured when the probe is activated. This feedback mechanism improves binding specificity while simplifying detection by eliminating off-target signals that would complicate measurement.
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 high-resolution spatial analysis of gene expression in biological samples, retaining native spatial context and providing detailed information on analyte positions.
Implementation Method 1
the first probe comprises in a 5′ to a 3′ direction: a spatial barcode and a capture domain (e.g., a poly(T) capture domain), wherein the poly(T) capture domain binds specifically to the target nucleic acid
Implementation Method 2
adding non-templated cytosines to the 3′ end of the first probe to generate a poly(C) sequence, wherein the poly(C) sequence specifically binds to the poly(GI) capture domain of the second probe
Data Source
AI summary
Provided herein are methods of detecting target nucleic acids and uses of the same.