Oligonucleotide Barcoding for Single-Cell Spatial Gene Co-Expression
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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, barcodes, and circularizable oligonucleotides to generate amplification products through hybridization, extension, and ligation, allowing for the amplification and detection of target polynucleotides within cells, enabling precise sequencing and identification.
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 patent implements nested barcoding where a first barcode is incorporated into a second barcode through sequential hybridization and ligation steps. The outer barcode contains an inner barcode sequence, creating a hierarchical structure that preserves spatial information while enabling multi-level identification of nucleic acid molecules within single cells
Solution Approach 2:
The patent uses adapter oligonucleotides as intermediaries that facilitate the incorporation of barcodes into target nucleic acids. These adapters serve as mediators between the target molecules and sequencing platforms, enabling precise spatial mapping without directly modifying the target sequences
2Manufacturing precision
If conventional amplification methods are used, then nucleic acid can be amplified, but precise sequencing information for molecular-level identification cannot be achieved
Solution Approach 1:
The patent performs preliminary barcode incorporation and circularization of oligonucleotides before amplification. By pre-assembling the complete barcode structure on the target nucleic acid and forming circular intermediates, the method ensures that sequencing information is established prior to amplification, preventing information loss or contamination
Solution Approach 2:
The patent divides the amplification process into distinct segments: first amplifying the circularized oligonucleotide intermediate, then using the amplified product as template for final target amplification. This segmented approach maintains precision by keeping barcode information separate from bulk amplification processes
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 method enables precise amplification and detection of target nucleic acids within cells, facilitating the resolution of spatial gene and protein co-expression patterns and improving the accuracy of single-cell profiling, thereby aiding in understanding cellular development and disease mechanisms.
Implementation Method 1
contacting a target polynucleotide with an oligonucleotide including a target hybridization sequence
Implementation Method 2
extending the second sequence along the barcode sequence with a polymerase to generate a complementary barcode sequence
Implementation Method 3
ligating the complementary barcode sequence to the first sequence, 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.