Circular Oligonucleotide Probes for Spatial RNA Sequencing
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Solution Overview
Problem
Current single-cell technologies face challenges in accurately profiling the genome, epigenome, and transcriptome of individual cells, particularly in resolving spatial gene and protein co-expression patterns, which is crucial for understanding tissue development and disease progression such as cancer, and in achieving precise molecular-level identification and monitoring.
Innovation Solution
A method involving the hybridization of oligonucleotides with specific sequences, including probe hybridization sequences and barcode sequences, followed by extension and ligation to generate a circular oligonucleotide, which is then amplified to produce multiple complements of the complementary barcode sequence, enabling precise detection and sequencing of RNA molecules within cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If single-cell technologies are used to profile genome, epigenome, and transcriptome, then cellular composition can be quantified, but precise sequencing information and spatial gene co-expression patterns cannot be obtained
Solution Approach 1:
The invention divides the complex task of single-cell profiling into separate functional modules: oligonucleotides with target hybridization sequences for specific binding, barcode sequences for identification, and probe hybridization sequences for detection. This segmentation allows each component to perform its function independently, achieving precise sequencing information while managing complexity through modular design
Solution Approach 2:
The invention introduces oligonucleotide probes as intermediary molecules that bridge the target polynucleotide and the detection system. These probes contain barcode sequences that carry spatial and identity information, enabling precise sequencing and spatial gene co-expression pattern detection without requiring direct complex interactions between cells and sequencing equipment
2Loss of information
If oligonucleotide probes with barcode sequences are used, then spatial gene co-expression patterns can be resolved, but the process requires multiple hybridization and ligation steps
Solution Approach 1:
The invention combines multiple functions into single oligonucleotide structures: target hybridization sequences and barcode sequences are merged into one oligonucleotide, while probe hybridization sequences are integrated into detection probes. This merging reduces the number of separate operations needed while maintaining the ability to resolve spatial gene co-expression patterns
Solution Approach 2:
The invention performs preliminary hybridization of oligonucleotides to target polynucleotides before detection, pre-assembling the information-carrying structures. The barcode sequences are already attached to the target-bound oligonucleotides before the actual sequencing or detection process, enabling spatial pattern resolution while streamlining subsequent steps
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 allows for the precise detection and sequencing of RNA molecules within cells, overcoming the limitations of existing technologies by enabling accurate profiling and monitoring of cellular components, facilitating better understanding of tissue development and disease mechanisms.
Implementation Method 1
hybridizing a first oligonucleotide to a target polynucleotide, wherein the first oligonucleotide includes a first target hybridization sequence
Implementation Method 2
extending the second oligonucleotide probe along the barcode sequence with a polymerase to generate a complementary barcode sequence
Implementation Method 3
ligating the complementary barcode sequence to the first oligonucleotide probe, and ligating the first oligonucleotide probe to the second oligonucleotide probe, thereby generating a circular oligonucleotide
Data Source
AI summary
Disclosed herein, inter alia, are oligonucleotides, methods, and kits useful for amplifying and detecting targets such as nucleic acids, proteins, and carbohydrates.