Spatial Nucleic Acid Capture for 5' and 3' End Mapping
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Solution Overview
Problem
Existing spatial array analysis techniques are biased towards capturing 3' end sequences of nucleic acid analytes, failing to provide comprehensive information on the position of single cells within a tissue and lacking multiplex capture of analytes from biological samples.
Innovation Solution
A method involving reverse transcription with primers complementary to target nucleic acids, incorporation of non-templated polynucleotide sequences, and hybridization to capture probes with spatial barcodes, combined with ligation and sequencing techniques to capture and determine the location of both 5' and 3' end sequences of nucleic acid analytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If spatial array analysis is used to capture nucleic acid analytes, then spatial location information can be obtained, but capture is biased towards 3' end sequences and 5' end sequences are not captured
Solution Approach 1:
The patent divides the capture process into two separate pathways: one for capturing 3' end sequences using poly(dT) capture probes, and another for capturing 5' end sequences using template switching oligonucleotides (TSOs) and primers. This segmentation allows each pathway to be optimized for its specific target region, resolving the bias towards 3' end capture while preserving 5' end information.
Solution Approach 2:
The patent introduces template switching oligonucleotides (TSO) as an intermediary element that facilitates the capture of 5' end sequences. The TSO contains a sequence complementary to the 5' end of the cDNA and a functional domain that enables template switching during reverse transcription, acting as a mediator to capture information that would otherwise be lost in conventional spatial array analysis.
2Ease of manufacture
If conventional capture techniques are used, then simple capture processes can be maintained, but multiplex capture of multiple analyte types cannot be achieved
Solution Approach 1:
The patent implements a universal capture platform where a single spatial array can simultaneously capture multiple types of analytes including full-length transcripts, 3' end sequences, and 5' end sequences using different capture probes and primers. The system uses universal functional domains that can be combined with different target-specific sequences, allowing one array to perform multiple capture functions without requiring separate arrays for each analyte type.
3Productivity
If only 3' end sequences are captured, then capture efficiency is high, but comprehensive gene expression information is lost
Solution Approach 1:
The patent merges multiple capture strategies into a single integrated workflow: poly(dT) capture for 3' end sequences, TSO-based capture for 5' end sequences, and probe-based capture for specific gene targets. All these methods are performed simultaneously on the same spatial array using the same basic protocol, combining the high efficiency of 3' end capture with the comprehensive information content of 5' end capture without requiring separate experiments.
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 the determination of the precise location of multiple nucleic acid analytes within a biological sample by capturing and sequencing both 5' and 3' end sequences, providing comprehensive spatial information for gene expression analysis.
Implementation Method 1
hybridizing the first probe and the second probe to the first target nucleic acid
Implementation Method 2
hybridizing the first capture sequence of the ligation product to the first capture domain on an array
Implementation Method 3
hybridizing the first primer to the second target nucleic acid and extending the first primer using the second target nucleic acid as a template to generate an extension product
Implementation Method 4
generating a ligation product by ligating the first probe to the second probe
Data Source
Figure 1
Figure 2A~2B
Figure 3
AI summary
Provided herein are methods, compositions, and kits for the spatial analysis of target nucleic acids, or complements thereof, by their 5' end.