Spatially Barcoded Padlock Probes for Microbial Nucleic Acid Mapping
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Solution Overview
Problem
Existing spatial analysis methods fail to capture non-polyadenylated nucleic acids, such as exogenous target nucleic acids from microbes, limiting the understanding of host-microbial interactions and spatially resolved analyses.
Innovation Solution
The use of padlock probes with capture probes that include spatially barcoded capture domains to hybridize, ligate, and cleave target nucleic acids, allowing for the determination of their location within a biological sample.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If poly(A) sequence-based capture probes are used for spatial analysis, then endogenous nucleic acid capture is improved, but exogenous target nucleic acid capture deteriorates
Solution Approach 1:
The capture probe is designed with a universal capture mechanism that can bind to both polyadenylated endogenous nucleic acids and non-polyadenylated exogenous nucleic acids. The probe includes a capture domain that hybridizes to target sequences while maintaining compatibility with both types of nucleic acids, enabling a single probe design to serve multiple detection purposes.
Solution Approach 2:
The invention modifies the capture probe design by removing the dependency on poly(A) sequences and instead using direct hybridization to target-specific sequences. This parameter change in the capture mechanism allows the probe to effectively bind to exogenous nucleic acids that lack polyadenylation while maintaining detection of endogenous nucleic acids.
2Measurement precision
If spatial analysis methods are optimized for host nucleic acids, then host gene expression analysis is improved, but microbial target detection deteriorates
Solution Approach 1:
The method segments the detection process into distinct functional components: a capture probe with a capture domain for target binding, a padlock probe for specific target recognition and circularization, and spatial barcoding for location identification. This segmentation allows each component to be optimized for its specific function while working together to detect both host and microbial nucleic acids with high spatial resolution and reliability.
Solution Approach 2:
The padlock probe serves as an intermediary between the capture probe and the final detection system. It specifically recognizes and binds to target sequences (including microbial nucleic acids), undergoes circularization to generate a stable signal, and enables subsequent spatial barcoding. This intermediary mechanism ensures reliable detection of microbial targets while preserving the spatial information necessary for host-microbial interaction analysis.
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-resolution spatial analysis of both endogenous and exogenous nucleic acids, providing insights into microbial presence and host responses, aiding in elucidating infection mechanisms and potential treatments.
Implementation Method 1
b) hybridizing the padlock probe to the target nucleic acid
Implementation Method 2
d) ligating a first end of the extended padlock probe to a second end of the extended padlock probe, thereby generating a ligated padlock probe
Data Source
AI summary
Provided herein are methods, compositions, and kits for determining the spatial location of target nucleic acids, including endogenous and exogenous target nucleic acids, in a biological sample using padlock probes and substrates with spatially barcoded capture probes. Also disclosed herein are methods for determining a presence and/or location of a microbe (e.g., archaea, fungi, bacteria) in a biological sample, e.g., by determining the presence and/or location of a microbial target nucleic acid in the biological sample.