In Situ Tissue Sequencing Using Circular Oligonucleotide Amplification
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Solution Overview
Problem
Existing single-cell technologies struggle to accurately profile the genome, epigenome, and transcriptome of individual cells, particularly in understanding spatial gene and protein co-expression patterns and their influence on tissue development and disease spread, such as cancer, and lack precise sequencing methods for molecular identification and treatment.
Innovation Solution
A method involving hybridization of oligonucleotide primers to target nucleic acids, circularization, amplification using strand-displacing polymerase, and sequencing of extension products to obtain precise sequencing information.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If existing single-cell technologies are used to profile genome, epigenome, and transcriptome, then basic cellular composition can be obtained, but precise sequencing information and spatial co-expression patterns cannot be accurately determined
Solution Approach 1:
The method segments the sequencing process into distinct stages: in situ hybridization of oligonucleotide primers to target nucleic acids within intact cells, circularization of primers to generate circular oligonucleotides, amplification through rolling circle replication, and sequencing. This segmentation allows preservation of spatial information during hybridization while enabling sufficient signal amplification for precise sequencing, thereby resolving the contradiction between measurement precision and information loss.
Solution Approach 2:
The method performs preliminary in situ hybridization and circularization of oligonucleotide primers to target nucleic acids before amplification and sequencing. This preliminary action captures spatial information within intact cells prior to any processing that might disrupt spatial relationships, enabling subsequent precise sequencing while retaining spatial co-expression pattern data.
2Quantity of substance
If conventional amplification methods are used, then signal strength can be increased, but spatial information and molecular precision are lost
Solution Approach 1:
The method uses circular oligonucleotides as intermediaries between the target nucleic acids in situ and the amplification process. The circular oligonucleotides are generated through rolling circle replication, providing a stable intermediate that can be amplified while preserving the spatial information encoded in the original hybridization pattern, thus enabling signal amplification without losing spatial precision.
Solution Approach 2:
The method transitions from direct linear amplification of target nucleic acids to amplification of circular oligonucleotide intermediaries. This dimensional change in the molecular structure (from linear to circular) enables robust signal amplification through rolling circle replication while maintaining the spatial information that was captured during in situ hybridization, resolving the contradiction between quantity and precision.
3Loss of information
If in situ sequencing is performed directly on target nucleic acids, then spatial information is preserved, but sufficient signal for accurate sequencing cannot be achieved
Solution Approach 1:
The method performs preliminary in situ hybridization of oligonucleotide primers to target nucleic acids and circularization to generate circular oligonucleotides before amplification. This preliminary action captures spatial information while creating a structure suitable for robust amplification, enabling subsequent sequencing with sufficient signal accuracy without losing spatial co-expression pattern information.
Solution Approach 2:
The method creates multiple copies of circular oligonucleotide intermediaries through rolling circle amplification. These copies are generated from the in situ hybridized primers, preserving the spatial information of the original target nucleic acids while providing sufficient signal intensity for accurate sequencing, thereby resolving the contradiction between information preservation and measurement precision.
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
Enables precise sequencing and identification of agent-mediated nucleic acid sequences, allowing for the detection of cells responding to genetically modifying agents and identifying the agents, thereby facilitating molecular-level monitoring and treatment.
Implementation Method 1
hybridizing an oligonucleotide primer to the target nucleic acid, wherein the oligonucleotide primer includes a first region at a 3′ end that hybridizes to a first complementary region of the target nucleic acid
Implementation Method 2
amplifying the circular oligonucleotide by extending an amplification primer hybridized to the circular oligonucleotide with a strand-displacing polymerase, wherein the amplification primer extension generates an extension product including multiple complements of the circular oligonucleotide
Data Source
AI summary
Disclosed herein, inter alia, are compositions and methods of use thereof for interrogating a tissue sample.