RNA-Templated Ligation With Spatial Barcodes for FFPE Gene Mapping
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for spatial analysis of analytes in biological samples fail to provide information on the position of single cells within a tissue and are prone to off-target detection due to reliance on common transcript sequences like poly(A) mRNA-like tails, leading to RNA degradation issues in FFPE samples.
Innovation Solution
Targeted RNA capture using spatially barcoded probes that hybridize and ligate to specific analytes, allowing for sensitive measurement of gene expression while retaining spatial context, using enzymatic or chemical ligation and sequencing techniques.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If poly(A) mRNA capture is used for spatial analysis, then high throughput whole transcriptome data can be obtained, but RNA degradation occurs in FFPE samples and off-target detection increases
Solution Approach 1:
The patent extracts only the specific analyte of interest from the complex transcriptome using targeted capture probes, rather than attempting to capture all poly(A) mRNA. This extraction approach isolates the target sequence from degradation-prone regions while maintaining spatial context, resolving the contradiction between comprehensive coverage and reliable detection.
Solution Approach 2:
The patent introduces an intermediary ligation product that bridges the target analyte and the spatial barcode. This intermediary structure allows specific target capture without direct reliance on degraded poly(A) tails, improving detection reliability while maintaining the ability to measure transcriptome-wide expression through systematic application across multiple targets.
2Quantity of substance
If common transcript sequence capture is used, then high number of analytes can be detected, but off-target detection increases and spatial location information is lost
Solution Approach 1:
The patent segments the detection process into distinct functional modules: target-specific capture probes, ligation products, and spatial barcodes. Each segment performs a specific function, allowing high-throughput analyte detection while preserving spatial location information through the dedicated barcode component that remains associated with the captured analyte throughout the process.
Solution Approach 2:
The patent assigns different functional properties to different parts of the detection system: capture probes provide target specificity, ligation products enable signal amplification, and spatial barcodes provide location information. This local differentiation of qualities allows simultaneous achievement of high analyte detection capacity and precise spatial measurement.
3Measurement precision
If targeted RNA capture is used instead of poly(A) capture, then sensitivity for specific genes improves and RNA degradation is reduced, but whole transcriptome coverage may be limited
Solution Approach 1:
The patent creates a universal detection platform where the same core methodology (capture probe + ligation + spatial barcode) can be applied to detect any target analyte. By designing a library of targeted capture probes that can systematically cover the entire transcriptome, the system achieves both high sensitivity for individual genes and flexible whole transcriptome coverage through multi-functional probe sets.
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 determination of analyte location and abundance in biological samples, particularly FFPE tissues, with reduced RNA degradation and improved sensitivity for specific gene detection.
Implementation Method 1
the first probe and the second probe hybridize to the analyte
Implementation Method 2
generating a ligation product by ligating the first probe and the second probe
Data Source
AI summary
Provided herein are methods of detecting an analyte of interest to interrogate spatial gene expression in a sample using RNA-templated ligation.