Spatial Transcriptomics Capture Probes for Reliable Analyte Detection
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Solution Overview
Problem
Existing methods fail to provide spatially resolved data on analyte levels within tissues, and permeabilization conditions for releasing analytes are often unpredictable, affecting downstream capture and sequencing.
Innovation Solution
A method involving contacting a biological sample with capture probes, releasing target analytes, extending the capture probe using the bound analyte as a template, adding homopolynucleotide sequences, and using template switching oligonucleotides to measure fluorescence, allowing for spatial analysis of analytes like RNA or DNA.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If traditional spatial analysis methods are used, then spatial location information is provided, but analyte capture sensitivity and specificity are insufficient
Solution Approach 1:
The capture probe is divided into distinct functional domains: a spatial barcode domain for location identification and a capture domain for analyte binding. This segmentation allows independent optimization of spatial resolution and capture efficiency, resolving the contradiction between providing precise spatial location and ensuring reliable analyte capture.
Solution Approach 2:
The patent introduces an intermediary amplification step using template switching oligonucleotides and rolling circle amplification. This intermediary process enhances the signal from captured analytes, improving detection sensitivity without compromising the spatial location information provided by the barcode domain.
2Quantity of substance
If permeabilization conditions are optimized for one tissue type, then analyte release is improved, but downstream capture and sequencing are affected
Solution Approach 1:
The patent employs a systematic approach to optimize permeabilization parameters (time, temperature, reagent concentration) specifically for each tissue type. By tuning these parameters, the method maximizes analyte release while preserving analyte integrity for subsequent capture and sequencing, resolving the contradiction between release quantity and downstream capture reliability.
Solution Approach 2:
The patent performs preliminary permeabilization optimization for different tissue types before proceeding to capture and sequencing. This preliminary action ensures that the right permeabilization conditions are established beforehand, preventing downstream issues and ensuring reliable capture and sequencing of released analytes.
3Reliability
If multiple permeabilization conditions are tested, then optimal conditions are identified, but time and resources are consumed
Solution Approach 1:
The patent incorporates feedback mechanisms where the results of permeabilization are monitored and used to adjust subsequent capture and sequencing parameters. This feedback loop allows for rapid optimization by learning from previous attempts, reducing the overall time and resources needed to identify optimal conditions for different tissue types.
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
Enhances the sensitivity and specificity of analyte capture and detection, enabling accurate spatial analysis of nucleic acids and proteins within tissues.
Implementation Method 1
measuring presence or absence of fluorescence upon release of the first oligonucleotide from the second oligonucleotide by extension of a 3′ end of the first strand using the second oligonucleotide as a template
Data Source
AI summary
Provided herein are methods of determining efficiencies of spatial transcriptomics methods.