In Situ Multiplexed Detection Using Photocleavable Linkers
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Solution Overview
Problem
Current immunohistochemical and in situ hybridization methods are limited in their ability to simultaneously detect and quantify multiple protein or nucleic acid targets, typically allowing for detection of only six to ten targets at most, with high background noise and limited resolution, and are not adaptable for use with existing sequencing technologies.
Innovation Solution
A method involving in situ synthesis of nucleic acid sequences in tissue samples using probes with target-binding and target-identification domains, where nucleotides are ligated and extended using nucleotide-polymerase complexes with photocleavable linkers, allowing for spatially-resolved profiling of target analytes through photocleavage and sequencing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Quantity of substance
If standard immunohistochemical and in situ hybridization methods are used, then detection of multiple targets is achieved, but the number of simultaneous targets is limited to six to ten with high background noise
Solution Approach 1:
The method segments the detection process into multiple sequential rounds, where each round detects a subset of targets. By dividing the full set of targets into smaller groups detected in separate rounds, the method enables detection of many more targets (over 100) while maintaining low background noise, as each round processes fewer targets simultaneously.
Solution Approach 2:
The method employs periodic action through sequential detection rounds, where targets are detected in repeated cycles across different rounds. Each round performs detection, imaging, and then the process repeats with additional targets in subsequent rounds, enabling multiplexed detection beyond the simultaneous capacity of traditional methods.
2Quantity of substance
If standard immunohistochemical and in situ hybridization methods are used, then detection of multiple targets is achieved, but spatial resolution is limited
Solution Approach 1:
The method segments both the target set and spatial detection into manageable rounds. By detecting targets in sequential rounds with repeated imaging of the same tissue section, the method maintains high spatial resolution while expanding the number of detectable targets, as each round focuses on a subset of targets without compromising image quality.
Solution Approach 2:
The method performs preliminary actions by sequentially detecting and imaging targets in ordered rounds before proceeding to the next round. This preliminary detection approach allows systematic accumulation of data across many targets while preserving spatial information, enabling high-resolution mapping of numerous targets that would be impossible to detect simultaneously.
3Adaptability or versatility
If standard immunohistochemical and in situ hybridization methods are used, then detection is achieved, but adaptability to existing sequencing technologies is limited
Solution Approach 1:
The method achieves universality by designing a detection system that outputs data compatible with existing sequencing technologies. The sequential detection rounds generate standardized data formats that can be processed by sequencing platforms, enabling the system to serve multiple functions: detecting hundreds of targets while interfacing with established sequencing infrastructure for data analysis.
Solution Approach 2:
The method introduces an intermediary data format that bridges the detection process and sequencing technologies. By generating output data that can be processed by sequencing platforms, the method enables seamless integration between the detection system and existing sequencing infrastructure, allowing analysis of hundreds of targets through familiar sequencing workflows.
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 simultaneous, multiplexed detection and quantification of protein and nucleic acid expression with higher resolution and reduced background noise, compatible with existing sequencing technologies, allowing for detailed analysis of multiple targets in user-defined regions of a tissue or cell.
Implementation Method 1
illuminating the first location of the tissue sample with light sufficient to cleave the photocleavable linker of the at least one nucleotide-polymerase complex, thereby releasing the polymerase and exposing a free 3'-OH moiety on the extended at least one bound probe
Data Source
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AI summary
The present disclosure is based in part on probes, compositions, methods, and kits for simultaneous, multiplexed spatial detection and quantification of protein and/or nucleic acid expression in a user-defined region of a tissue, user-defined cell, and/or user-defined subcellular structure within a cell.