Bridging Oligonucleotides for Spatial Barcoding and mRNA Priming
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Solution Overview
Problem
Existing spatial analysis methods fail to provide comprehensive information on the position of single cells within a biological sample, particularly in the context of intact tissues, and struggle with priming reverse transcription of mRNA due to spatial distribution or design issues with poly(T) oligonucleotides.
Innovation Solution
The use of bridging oligonucleotides with capture-probe-binding and analyte-binding sequences, combined with spatial barcodes, to hybridize and extend analytes for sequencing, allowing determination of their location within a biological sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If poly(T) oligonucleotides are used as primers for reverse transcription, then reverse transcription can be primed, but the method fails when mRNA-capturing oligonucleotides have specific spatial distribution or design that prevents effective priming
Solution Approach 1:
The patent introduces a bridging oligonucleotide as an intermediary component that connects the mRNA-capturing oligonucleotide to the reverse transcription primer. The bridging oligonucleotide includes a first sequence that hybridizes to the mRNA-capturing oligonucleotide and a second sequence that serves as the reverse transcription primer, thereby mediating the interaction between incompatible components and enabling reliable priming across different oligonucleotide designs
Solution Approach 2:
The patent segments the primer function into separate components: the mRNA-capturing oligonucleotide remains dedicated to capture, while the bridging oligonucleotide handles the connection, and the reverse transcription primer is separated as a distinct functional element. This segmentation allows each component to be optimized independently for its specific function while maintaining overall system compatibility
2Quantity of substance
If spatial analysis methods are used to provide data for multiple analytes in intact tissue, then comprehensive spatial information can be obtained, but the methods previously only provided data for a small handful of analytes or failed to provide position information for single cells
Solution Approach 1:
The patent creates a universal spatial analysis system where the same bridging oligonucleotide-based approach can be applied to capture and analyze multiple different analytes (mRNA, DNA, proteins) while maintaining spatial position information. The universal bridging mechanism enables the system to handle diverse analyte types without sacrificing spatial precision
Solution Approach 2:
The patent uses spatial barcodes as copies or identifiers that are attached to or associated with captured analytes. These barcodes serve as molecular copies that encode spatial position information, allowing the system to track the location of individual analytes or cells through the analysis process while maintaining comprehensive multi-analyte capability
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 efficiency and sensitivity of spatial analysis by increasing the number of interactions between capture probes and analytes, providing high-resolution spatial data on multiple analytes and their interactions.
Implementation Method 1
hybridizing the analyte to the capture domain, thereby generating a capture analyte; contacting the captured analyte to a bridging oligonucleotide
Implementation Method 2
extending the bridging oligonucleotide using the analyte as a template to generate an extended capture probe
Data Source
AI summary
Disclosed herein are methods of amplifying an analyte in a biological sample using a bridging oligonucleotide that hybridizes to a captured analyte. The methods disclosed herein include steps of (a) contacting a biological sample with a substrate having capture probes comprising a capture domain and a spatial barcode; (b) hybridizing the analyte to the capture domain; and (c) contacting the analyte to a bridging oligonucleotide comprising (i) a capture-probe-binding sequence, and (ii) an analyte-binding sequence; (d) extending the bridging oligonucleotide; and (e) determining (i) all or a part of the sequence of the analyte, or a complement thereof, and (ii) the spatial barcode, or a complement thereof, and using the determined sequence of (i) and (ii) to determine the location of the analyte in the biological sample.