Spatial Analysis of Genomic DNA Using DNA Templated Ligation
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Solution Overview
Problem
Current spatial analysis techniques fail to effectively analyze genomic DNA (gDNA) molecules, particularly in the context of spatial heterogeneity within tissues, as they lack methods to adapt RNA-templated ligation (RTL) principles for gDNA, limiting the ability to determine the position and abundance of gDNA analytes in biological samples.
Innovation Solution
The method involves using oligonucleotide probes that hybridize to adjacent complementary genomic DNA sequences, incorporating a 5′ FLAP sequence that can be cleaved and detected without requiring single-stranded DNA, allowing for the detection of gDNA analytes or genetic variants by hybridizing the cleaved 5′ FLAP to a capture domain and determining its sequence.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional RTL principles are used for RNA analysis, then spatial resolution is enhanced, but the method cannot be adapted to genomic DNA (gDNA) analysis
Solution Approach 1:
The patent modifies the RTL method by changing the chemical parameters: using DNA probes instead of RNA probes, and employing a DNA flap endonuclease instead of an RNAse. This parameter change allows the method to work with gDNA templates while maintaining the spatial resolution benefits of RTL
Solution Approach 2:
The method segments the detection process into distinct functional domains: a capture domain for hybridizing to the gDNA target, a spacer region for structural separation, and a flap sequence for enzymatic cleavage and signal generation. This segmentation enables the adaptation of RTL principles to gDNA analysis
2Measurement precision
If probe ligation and endoribonuclease treatment are used in RTL, then spatial analysis can be performed, but the process complexity and cost increase
Solution Approach 1:
The patent extracts and removes the RNA-specific endoribonuclease step from the traditional RTL protocol. By eliminating this specialized enzymatic step and replacing it with a DNA flap endonuclease that acts on the DNA probe structure, the method simplifies the overall process while maintaining spatial analysis precision
Solution Approach 2:
The patent introduces a DNA flap sequence as an intermediary element that mediates between the hybridized DNA probe and the detection system. This flap sequence serves as a recognizable substrate for the DNA flap endonuclease, enabling signal generation without requiring probe ligation or RNA-specific enzymes
3Loss of information
If methods provide data for a small handful of analytes in intact tissue, then spatial context is preserved, but the number of detectable analytes is limited
Solution Approach 1:
The patent creates a universal detection platform that can detect multiple different gDNA analytes using the same fundamental methodology. By designing probes with modular domains (capture domain, spacer, flap sequence) that can be customized for different targets, the system can analyze numerous different genomic regions while maintaining spatial context through the capture probe barcoding system
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 enables the precise detection and localization of gDNA analytes and genetic variants within biological samples, enhancing spatial resolution and avoiding the need for probe ligation and endoribonuclease treatment, thus improving the efficiency and cost-effectiveness of gDNA analysis.
Implementation Method 1
oligonucleotide probes that hybridize to adjacent complementary genomic DNA sequences
Implementation Method 2
an oligonucleotide (e.g., a second RTL probe) that hybridizes to the gDNA analyte includes a 5′ FLAP sequence that can be removed and detected upon an enzyme-mediated cleavage event
Data Source
AI summary
Provided herein are methods of detecting an analyte of interest to interrogate spatial gene expression in a sample using DNA templated ligation. For example, provided herein are methods for detecting an gDNA analyte in a biological sample where (i) the first RTL probe and second RTL probe hybridize to adjacent sequences on the gDNA analyte, (ii) enzyme-mediate cleavage of a 5′ FLAP results in release of the 5′ FLAP, and (iii) the sequence of the 5′ FLAP is determined and used to detect the gDNA analyte in the biological sample.