Circularizable Oligonucleotide Probes for Spatial Transcriptomics
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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 like cancer, and in achieving precise sequencing for molecular-level identification and treatment.
Innovation Solution
A method involving the use of oligonucleotides with specific hybridization sequences and a circularizable oligonucleotide to generate an amplification product through hybridization, extension, and ligation, allowing for the amplification of circular oligonucleotides with complementary barcode sequences, enabling precise detection and sequencing of RNA molecules within cells.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional single-cell technologies are used for profiling genome, epigenome, and transcriptome, then basic cellular composition can be obtained, but spatial gene and protein co-expression patterns cannot be accurately resolved
Solution Approach 1:
The method segments the cellular analysis into distinct spatial zones by using oligonucleotide probes that hybridize to specific RNA molecules at their native locations within the cell. Each probe carries a unique barcode that identifies both the RNA type and its spatial position, enabling resolution of co-expression patterns without disrupting spatial information.
Solution Approach 2:
The patent introduces oligonucleotide probes with barcodes as intermediary elements that bridge the gap between RNA molecules and detection systems. These probes hybridize to target RNAs in situ and serve as mediators that transfer spatial and identity information to the amplification and sequencing systems, preserving spatial relationships while enabling molecular identification.
2Productivity
If fluorescence signals are used to detect multiple RNAs in the same volume, then detection capability is provided, but spatial overlap of fluorescence signals creates measurement ambiguity
Solution Approach 1:
The method creates a molecular copy of the spatial information by incorporating unique barcodes into the amplification products. Instead of relying on overlapping fluorescence signals from multiple RNA types, the system generates amplified copies of each RNA-bound probe, each carrying its unique barcode that preserves the original spatial and identity information, allowing unambiguous identification even when signals overlap.
Solution Approach 2:
The patent employs barcode sequences that can be detected through sequencing, effectively replacing the ambiguous color/fluorescence channel system with a high-resolution digital barcode identification system. Each barcode acts as a unique molecular identifier that can be read through sequencing technology, providing precise distinction between different RNA molecules regardless of their spatial overlap in fluorescence microscopy.
3Ease of manufacture
If linear oligonucleotide probes are used for RNA detection, then hybridization can occur, but circularization capability for amplification is lost
Solution Approach 1:
The oligonucleotide probe design incorporates conditional circularization capability. The probes remain linear during hybridization to maintain ease of manufacture and binding flexibility, but can be circularized after hybridization to enable rolling circle amplification. This dynamic transition from linear to circular form allows the system to benefit from both the manufacturing simplicity of linear probes and the amplification efficiency of circular templates.
Solution Approach 2:
The method performs hybridization of linear probes to target RNAs as a preliminary action before circularization and amplification. This sequence allows the linear probes to easily hybridize to their targets, after which the circularizable ends can be joined to create amplifiable circular structures, combining the advantages of linear probe simplicity with circular template amplification power.
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 enables the precise amplification and detection of RNA molecules within cells, resolving spatial overlap of fluorescence signals and allowing for the quantitative measurement of multiple RNAs in each resolved volume, thereby improving the understanding of cellular heterogeneity and disease mechanisms.
Implementation Method 1
contacting a target polynucleotide with a first oligonucleotide including a first target hybridization sequence, a first hybridization sequence, a barcode sequence, and a second hybridization sequence
Implementation Method 2
extending the circularizable oligonucleotide along the barcode sequence with a polymerase to generate a complementary barcode sequence
Implementation Method 3
ligating the complementary barcode sequence to the circularizable oligonucleotide, thereby generating a circular oligonucleotide
Implementation Method 4
amplifying the circular oligonucleotide, thereby generating an amplification product including multiple complements of the complementary barcode sequence
Data Source
AI summary
Disclosed herein, inter alia, are oligonucleotides, methods, and kits useful for amplifying and detecting target nucleic acids.