Rolling Circle Amplification Probes for Spatial Transcriptomics
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Solution Overview
Problem
Current methods for in situ nucleic acid detection in biological samples face challenges due to optical and physical crowding, limiting sensitivity and resolution, particularly in achieving high-accuracy transcriptomic imaging.
Innovation Solution
The method involves contacting a biological sample with a detection probe and a ribosomal RNA (rRNA)-targeting probe, which is circularizable, followed by rolling circle amplification to generate a detection rolling circle amplification product, while avoiding detection of a non-detection rolling circle amplification product, thereby reducing optical crowding and enhancing sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If rolling circle amplification is performed to amplify detection signals, then sensitivity is improved, but optical crowding increases and resolution deteriorates
Solution Approach 1:
The patent applies local quality by creating localized expansion zones around specific rRNA targets using circularizable probes. The expansion is confined to regions where probes bind to rRNA, generating local physical expansion that separates detection signals spatially. This localized approach amplifies detection sensitivity in specific regions without causing global optical crowding throughout the entire sample.
Solution Approach 2:
The patent transitions from planar 2D detection to 3D spatial expansion by utilizing the circularizable probe mechanism. The circularization and subsequent rolling circle amplification create three-dimensional expansion of the sample matrix, adding a spatial dimension that separates optical signals and reduces crowding while maintaining detection sensitivity.
2Volume of moving object
If circularizable rRNA-targeting probes are used to expand the sample, then physical expansion is achieved, but probe design complexity increases
Solution Approach 1:
The circularizable rRNA-targeting probes serve multiple functions: they bind to rRNA targets, undergo circularization, and generate rolling circle amplification products that cause physical expansion. This multi-functionality consolidates several operations into a single probe design, reducing overall system complexity while achieving sample expansion.
Solution Approach 2:
The circularizable probes utilize the rRNA target itself as a template for circularization and subsequent amplification. The rRNA sequence serves the dual purpose of being the target for binding and providing the template for probe circularization, eliminating the need for separate circularization reagents or steps and simplifying the overall process.
3Measurement precision
If detection probes are concentrated to improve signal strength, then detection accuracy is improved, but optical crowding increases
Solution Approach 1:
The patent segments the detection process by using multiple circularizable probes targeting different rRNA sequences. Each probe generates its own localized expansion zone with associated detection signals. This segmentation separates optical signals spatially, allowing concentrated detection in each local region while preventing global optical crowding through distributed signal placement.
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 improves the sensitivity and resolution of nucleic acid detection by physically expanding the sample, reducing optical crowding and allowing for more accurate analysis of nucleic acid analytes, including mRNA, without disrupting cellular morphology.
Implementation Method 1
contacting the biological sample with a ribosomal RNA (rRNA)-targeting probe or probe set, wherein the rRNA-targeting probe or probe set hybridizes to a ribosomal RNA (rRNA) target sequence in the biological sample
Implementation Method 2
performing rolling circle amplification of the rRNA-targeting probe or probe set to generate a non-detection rolling circle amplification product (RCP) in the biological sample
Implementation Method 3
contacting the biological sample with a detection probe or probe set, wherein the detection probe or probe set hybridizes to a target nucleic acid in the biological sample
Data Source
AI summary
The present disclosure relates in some aspects to methods and compositions for analyzing a biological sample. In some aspects, provided herein are probes, methods, and kits for analyzing a processing a biological sample, wherein the processing comprises performing rolling circle amplification of a ribosomal RNA (rRNA)-targeting probe, and detecting a detection probe hybridized to a non-rRNA target analyte (e.g., nucleic acid) in the biological sample.