In Situ Gene Sequencing with Hydrogel-Embedded Amplicons
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Solution Overview
Problem
Current in situ sequencing methods face challenges in implementing enzymatic reactions in dense, complex tissue environments, particularly in the mammalian brain, due to low efficiency, sensitivity, fidelity, and scalability issues, limiting the retrieval of high-content gene-expression information while retaining 3D positional anatomy at cellular resolution.
Innovation Solution
The method involves contacting fixed and permeabilized intact tissue with oligonucleotide primers, performing rolling circle amplification, embedding amplicons in hydrogel subunits, and using a pair of primers for ligation to achieve in situ gene sequencing, enabling spatially-resolved transcriptomics with improved sensitivity and scalability.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current in situ sequencing methods are used, then sequencing can be performed in tissue, but efficiency and sensitivity are low
Solution Approach 1:
The sequencing process is divided into multiple discrete steps: (1) hybridization of padlock probes to target RNA, (2) ligation to form circular DNA, (3) rolling circle amplification, (4) hydrogel embedding, and (5) sequencing by ligation. This segmentation allows optimization of each step independently, improving overall efficiency and sensitivity.
Solution Approach 2:
The method performs preliminary actions outside the cell before sequencing: RNA is extracted and converted to cDNA, padlock probes are designed to target specific genes, and amplification is performed before embedding. This allows optimization of molecular biology steps separately from the tissue context.
2Ease of operation
If enzymatic reactions are implemented in dense tissue environment, then in situ sequencing is possible, but fidelity decreases
Solution Approach 1:
Hydrogel serves as an intermediary medium between the tissue and the sequencing reaction. It provides a controlled microenvironment that concentrates reagents, maintains structural integrity, and facilitates enzymatic reactions while preserving tissue architecture and cellular context.
Solution Approach 2:
The method changes physical and chemical parameters to optimize reactions in tissue: using fixed and permeabilized tissue to control access, adjusting hydrogel composition and crosslinking density, optimizing ligation and amplification conditions, and controlling imaging parameters to maintain fidelity.
3Measurement precision
If high-content gene-expression information is retrieved, then cellular resolution is achieved, but scalability for throughput is limited
Solution Approach 1:
The hydrogel-embedded amplicon platform serves multiple functions simultaneously: it preserves tissue architecture, enables high-resolution imaging, facilitates amplification, and allows sequencing. This multi-functionality eliminates the need for separate processing steps, improving throughput.
Solution Approach 2:
Rolling circle amplification creates multiple copies of the target sequence from a single template, enabling signal amplification without increasing the physical footprint. This allows detection of low-abundance transcripts while maintaining spatial resolution and scalability.
4Shape
If 3D volumes of intact tissue are sequenced, then spatial anatomy is preserved, but sensitivity and scalability are insufficient
Solution Approach 1:
The method embeds amplicons within hydrogel that is integrated into the tissue matrix, creating nested structures where molecular information is preserved within the 3D tissue architecture. This maintains spatial anatomy while enabling sensitive detection through the amplified signal.
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 high-content gene-expression analysis with cellular resolution, identifying diverse cell types and quantifying gene expression in 3D volumes, providing a platform for molecular phenotyping and spatially-resolved gene detection.
Implementation Method 1
contacting a fixed and permeabilized intact tissue with at least a pair of oligonucleotide primers under conditions to allow for specific hybridization
Implementation Method 2
adding ligase to ligate the second oligonucleotide and generate a closed nucleic acid circle
Implementation Method 3
performing rolling circle amplification in the presence of a nucleic acid molecule, wherein the performing comprises using the second oligonucleotide as a template and the first oligonucleotide as a primer for a polymerase to form one or more amplicons
Implementation Method 4
embedding the one or more amplicons in the presence of hydrogel subunits to form one or more hydrogel-embedded amplicons
Implementation Method 5
contacting the one or more hydrogel-embedded amplicons having the barcode sequence with a pair of primers under conditions to allow for ligation, wherein the pair of primers comprise a third oligonucleotide and a fourth oligonucleotide, wherein the ligation only occurs when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon
Data Source
AI summary
Provided herein are devices, methods, and systems for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue. Methods of screening a candidate agent to determine whether the candidate agent modulates gene expression of a nucleic acid in a cell in an intact tissue are also provided herein.