Spatial Barcoding via Photocleavable Light Activation
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Solution Overview
Problem
Current methods for spatially barcoding biological molecules in tissues face limitations such as low intensity image signals, auto-fluorescence, background noise, and the inability to decode abundant molecules, as well as inefficiencies in reverse transcription and high costs associated with solid supports, leading to incomplete and imprecise data.
Innovation Solution
A novel image-free method using light to guide the assembly of spatial barcodes onto nucleic acid molecules, allowing for high-throughput sequencing and precise localization of biological molecules with single-molecule sensitivity, utilizing common instruments and standard tissue slides, and incorporating photocleavable groups for controlled barcode assembly.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If image-based methods are used for gene expression measurement, then spatial information can be obtained, but the image signals have low intensity and are difficult to discriminate due to auto-fluorescence and background noise
Solution Approach 1:
The patent replaces optical detection mechanisms with nucleic acid sequencing mechanisms. Instead of using fluorescence imaging to detect spatial information, the method uses DNA barcodes that are sequenced to determine the location and identity of RNA molecules, thereby eliminating the problems of low signal intensity, auto-fluorescence, and background noise associated with optical detection
Solution Approach 2:
The patent introduces DNA barcodes as an intermediary between the RNA molecules and the detection system. These barcodes serve as detectable proxies that can be easily sequenced, allowing indirect detection of RNA spatial information without directly imaging the RNA molecules themselves, thus avoiding the signal intensity and noise problems
2Adaptability or versatility
If repeated cycles of fluorescence imaging are used to identify multiple RNA molecules, then multiple genes can be detected, but the images require exact alignment to within a few nanometres which is technically challenging
Solution Approach 1:
The patent replaces the mechanical image alignment process with a molecular sequencing process. Instead of requiring precise spatial alignment of multiple images, the method uses DNA barcoding and sequencing to identify RNA molecules, where the sequencing process inherently provides precise molecular identification without needing nanometre-level spatial alignment
Solution Approach 2:
The patent segments the detection process into discrete steps: first assigning unique DNA barcodes to different RNA molecules, then sequencing these barcodes to identify the molecules. This segmentation allows multiple genes to be detected independently through the sequencing process, eliminating the need for precise alignment across multiple images
3Measurement precision
If high magnification is used to achieve single-molecule resolution, then spatial detail can be obtained, but the time required for analysis increases and only very small areas of tissue can be imaged
Solution Approach 1:
The patent replaces high-magnification optical imaging with nucleic acid sequencing. Instead of using high magnification to resolve individual molecules, the method sequences DNA barcodes that have been assigned to molecules, allowing rapid identification of multiple molecules across large tissue areas without the time and area limitations of high-magnification imaging
Solution Approach 2:
The patent creates a copy of the spatial and molecular information through DNA barcodes. These barcode copies contain encoded information about the location and identity of RNA molecules, allowing the original spatial data to be preserved and analyzed through sequencing without requiring continuous high-magnification imaging, thereby increasing throughput
4Ease of manufacture
If solid support is used for spatial DNA barcode array, then barcoding can be performed, but the support is expensive to produce, fragile, and results in low spatial resolution
Solution Approach 1:
The patent eliminates the need for expensive, fragile solid supports by using a disposable glass slide with integrated microfluidic channels. This simple, inexpensive substrate allows DNA barcodes to be delivered to tissue sections without requiring complex solid support structures, thereby reducing costs and improving spatial resolution
Solution Approach 2:
The patent uses a microfluidic delivery system as an intermediary to transport DNA barcodes to the tissue section. This fluid-based delivery mechanism replaces the need for solid support arrays, allowing precise barcode placement without the manufacturing and resolution limitations of solid substrates
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 efficient, cost-effective, and high-resolution spatial analysis of transcripts and proteins, providing detailed molecular information without the limitations of existing techniques, allowing for simultaneous analysis of multiple molecules and improved data accuracy.
Implementation Method 1
Illuminating a location of interest on the substrate to be spatially barcoded, wherein the illumination cleaves or alters the photocleavable group of the or each root molecule present within the location
Data Source
AI summary
The present invention relates to a method of spatially barcoding a given location on a substrate, and further to spatially barcoding detection probes present in a sample such as a biological tissue specimen for the purposes of analysing molecular features present in the tissue. Such analysis may include: i) the spatial expression of one or more biological molecules, specifically; ii) the spatial analysis of the transcriptome and/or iii) the spatial analysis of the proteome, including post-translational protein modifications. The invention further relates to various component products for performing such methods that include reagents kits, instrumentation and software.