Spatial Nucleic Acid Tagging with Simultaneous Probe Release and Extension
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Solution Overview
Problem
Existing spatial transcriptomic and genomic methods are limited by factors such as hybridization efficiency, extension reaction efficiency, probe density, and non-specific extension, leading to under-representation of low abundance transcripts.
Innovation Solution
A method combining the release and extension steps of capture probes from a solid substrate using a cleavage enzyme in a single reaction mixture, allowing nucleic acids to hybridize and extend simultaneously, thereby increasing the number and diversity of captured nucleic acids.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If separate release and extension steps are used, then reaction conditions can be optimized for each step, but the number of captured nucleic acid molecules is limited
Solution Approach 1:
The patent combines the release step and extension step into a single simultaneous reaction process. The cleavage enzyme and extension enzyme are both present in the same reaction mixture, allowing capture probes to be released from the solid substrate and extended in one unified step, thereby increasing the number of captured molecules without proportionally increasing process complexity
2Quantity of substance
If capture probe density is increased, then more nucleic acids can be captured, but hybridization efficiency decreases
Solution Approach 1:
The patent performs the release action before extension is complete by using a cleavage enzyme that acts on the cleavage domain while the extension reaction is ongoing. This preliminary release prevents steric hindrance from accumulating, maintaining hybridization efficiency even at higher probe densities
3Loss of information
If extension reaction is performed while probes are immobilized, then spatial information is preserved, but extension efficiency is reduced
Solution Approach 1:
The patent segments the capture probe into multiple functional domains: a cleavage domain containing the spatial barcode, a linker domain, and a capture domain. This segmentation allows the spatial information (barcode) to remain associated with the probe during extension, while the flexible linker enables the probe to adopt conformations that facilitate efficient extension even when immobilized
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 approach enhances the sensitivity of spatial transcriptomic and genomic methods by improving the capture of unique nucleic acid molecules, resulting in a more representative spatially tagged transcriptome.
Implementation Method 1
contacting said solid substrate with an aqueous reaction mixture comprising: (i) a polymerase enzyme capable of extending the capture probes; (ii) a cleavage enzyme capable of releasing the capture probes from the surface of the solid substrate
Implementation Method 2
under conditions that allow nucleic acids of the biological specimen to hybridise to the capture domains of the capture probes
Implementation Method 3
a polymerase enzyme capable of extending the capture probes using the nucleic acid hybridised to the capture probes as an extension template to produce extended probes
Data Source
Figure 1
Figure 2A~2E
Figure 3A~3B
AI summary
The present invention relates to methods for spatial tagging of nucleic acid molecules in a biological specimen and in particular to a method comprising: (a) providing a solid substrate on which multiple species of capture probes are immobilized such that each species occupies a distinct position on the solid substrate, wherein said probes are for a primer extension reaction and wherein each species of said capture probes comprise a nucleic acid molecule comprising: (i) a cleavage domain for releasing the capture probe from the surface of the solid substrate, (ii) a positional domain that corresponds to the position of the capture probe on the solid substrate, and (iii) a capture domain; (b) contacting said solid substrate with a biological specimen; and (c) releasing said capture probes from the surface of the solid substrate under conditions that allow nucleic acids of the biological specimen to hybridise to the capture domain in said capture probes and simultaneously and/or subsequently extending said capture probes using the nucleic acid molecules hybridised to the capture probes as extension templates to produce extended probes thereby spatially tagging the nucleic acids of the biological specimen, wherein step (c) comprises contacting said solid substrate with an aqueous reaction mixture comprising: (i) a polymerase enzyme capable of extending said capture probes using the nucleic acid molecules hybridised to the capture probes as extension templates; and (ii) means for releasing said capture probes from the surface of the solid substrate.