mRNA Spatial Sequencing via Photo-Cleavable Barcodes
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Solution Overview
Problem
Current methods for sequencing and spatial decoding of mRNA molecules in tissues face challenges in achieving subcellular resolution, as they are limited to multicellular levels and struggle with single-molecule decoding, relying on feature sizes of spots on arrays and methods used for DNA barcode decoding.
Innovation Solution
A method involving a solid surface with fiducial markers, anchor molecules with photo-cleavable linkers and adapters, and scaffolding molecules to create random barcodes during sequencing-by-synthesis, allowing simultaneous detection of barcode sequences and spatial locations, enabling high-resolution mRNA sequencing and spatial decoding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If standard in vitro NGS sequencing techniques are used with spatial identifiers pre-spotted on an array, then sequencing can be performed, but the spatial resolution is limited to multicellular level due to feature size of spots
Solution Approach 1:
The invention segments the spatial identification process by moving from pre-spotted array identifiers to single-molecule DNA barcode sequences that are directly integrated with each mRNA molecule. This segmentation allows each molecule to carry its own spatial information rather than relying on bulk spot identifiers, achieving subcellular resolution
Solution Approach 2:
The invention transitions from two-dimensional array spot positioning to three-dimensional single-molecule spatial coordinates within tissue sections. By embedding DNA barcodes directly in mRNA molecules and using imaging-based spatial decoding, the system achieves resolution in the nanometer scale rather than being constrained by array spot dimensions
2Quantity of substance
If DNA barcodes are used for spatial transcriptomes, then highly multiplexed analyses can be performed, but achieving single-molecule decoding remains challenging
Solution Approach 1:
The invention performs preliminary action by incorporating DNA barcodes directly into mRNA molecules during reverse transcription before tissue sectioning and imaging. This preliminary barcode integration simplifies subsequent detection by allowing direct imaging of barcode sequences in situ without complex decoding procedures
Solution Approach 2:
The invention uses copying by creating multiple copies of the barcode sequence through PCR amplification after in situ imaging, or by using rolling circle amplification to generate signal amplification from single-molecule barcodes, making single-molecule detection feasible
3Loss of information
If spatial identifiers are pre-spotted on arrays, then spatial information can be maintained, but the position resolution is dependent on spot feature size
Solution Approach 1:
The invention merges spatial information and molecular identity by integrating DNA barcodes directly into mRNA molecules. This merging eliminates the need for separate spatial identifier spots, as each mRNA molecule carries both its spatial coordinates (through in situ position) and molecular identity (through embedded barcode) in a unified structure
Solution Approach 2:
The invention uses DNA barcodes as intermediaries that bridge spatial information and sequencing data. These barcodes are incorporated into mRNA during reverse transcription, allowing the mRNA to serve as both the target of interest and the carrier of spatial information, eliminating the need for separate spatial tagging intermediaries
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 achieves spatial resolution of 50 to 300 nm, enabling the precise sequencing and spatial identification of mRNA molecules at a single-molecule level, improving upon existing limitations by integrating barcode generation and decoding concomitantly.
Implementation Method 1
remove c-DNA strands from the solid surface by cleaving the photo-cleavable linker of the anchor molecules
Data Source
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AI summary
The invention is directed to a method to obtain the spatial location and sequence information of an m-RNA target sequence on a tissue sample comprising the steps a. providing a solid surface having at least with at least one fiducial marker b. attaching a plurality of anchor molecules comprising a photo cleavable linker and an adapter unit to the solid surface c. Binding scaffolding molecules comprising a unit capable of binding to the adapter unit of an anchor molecule, a poly-inosine unit having 5 to 30 inosine bases and a poly-adenine unit having 10 to 50 adenine bases to the anchor molecules d. randomly incorporating adenine, guanine, cytosine and thymine as nucleic bases to the anchor molecule complementing the inosine bases of the scaffolding molecules thereby creating barcodes on the anchor molecules, wherein the nucleic bases are provided with a photo-detectable unit and wherein the sequence of the barcodes and their spatial location relative to the fiducial marker is detected simultaneously as spatial information. e. incorporating thymine to the anchor molecules complementing the poly-adenine unit of the scaffolding molecules thereby creating a poly-T unit f. removing the scaffolding molecules from the anchor molecules g. providing a tissue sample comprising at least one m-RNA strand wherein at least one m-RNA strand of the sample binds to a poly-T unit of at least one anchor molecule h. reverse transcription of the m-RNA strand creating a c-DNA strand attached to the solid surface i. remove c-DNA strands from the solid surface by cleaving the photo-cleavable linker of the anchor molecules j. obtaining the sequence information of the c-DNA strands and linking the spatial information with the sequence information of the c-DNA strands.