Photoremovable Cage Oligonucleotides for Spatial Cell Marker Generation
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Solution Overview
Problem
In 3D cell culture, particularly with spheroids, it is challenging to visually identify and track specific cell clusters among a large number of uniform clusters due to their similarity, making it difficult to distinguish and follow them over time.
Innovation Solution
The method involves introducing oligonucleotide constructs with a promoter, nucleic acid sequence encoding a fluorescent protein, and a photoremovable cage molecule into biological samples, followed by selective uncaging with spatially constrained light beams to generate unique markers, allowing for precise identification of specific regions within the samples.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional visual identification methods are used for cell clusters, then the process is simple and requires minimal equipment, but it becomes impossible to distinguish and track specific cell clusters among large numbers of uniform spheroids
Solution Approach 1:
The invention divides the identification system into modular components: oligonucleotide constructs with photoremovable cage molecules that can be selectively activated. Each cell cluster can be assigned a unique marker through spatially controlled uncaging, enabling precise identification without requiring complex imaging systems for all clusters simultaneously.
Solution Approach 2:
The oligonucleotide constructs are introduced into cell clusters in advance with photoremovable cage molecules attached. The actual marker activation is then triggered by light exposure at specific time points and locations, allowing pre-prepared clusters to be selectively marked without complex real-time identification equipment.
2Reliability
If photoremovable cage molecules are used for selective uncaging, then specific cell clusters can be precisely labeled and tracked, but the process requires complex optical equipment and multiple treatment steps
Solution Approach 1:
The photoremovable cage molecule acts as an intermediary between the oligonucleotide construct and the fluorescent marker. It blocks the marker's function until light exposure triggers its removal, providing a simple on/off switch mechanism that maintains operational ease while enabling reliable selective tracking.
Solution Approach 2:
The invention replaces complex mechanical tracking systems with an optical activation mechanism. Instead of physically tracking or mechanically distinguishing clusters, the system uses light-triggered chemical changes to activate markers, simplifying the operational process while improving tracking reliability.
3Adaptability or versatility
If multiple fluorescent markers are used to label different cell clusters, then each cluster can be uniquely identified, but the complexity of managing multiple markers and their corresponding light parameters increases
Solution Approach 1:
The photoremovable cage molecule system provides a universal platform for marker activation. The same cage structure and uncaging mechanism can be applied to multiple different fluorescent markers, allowing versatile labeling of numerous cell clusters while managing complexity through a standardized activation approach rather than requiring unique mechanisms for each marker.
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 efficient and accurate labeling of biological samples, allowing for distinct identification and tracking of cell clusters through fluorescent markers, even when numerous clusters are present, by utilizing different fluorescent proteins and light parameters for selective uncaging.
Implementation Method 1
The photoremovable cage molecule is photoremovable from the oligonucleotide construct by a spatially constrained, in particular focused, light beam
Implementation Method 2
exposing, in particular scanning, at least a first region of the biological sample with a first spatially constrained, in particular focused, light beam to form uncaged first oligonucleotide constructs
Implementation Method 3
a first nucleic acid sequence encoding a first fluorescent protein
Data Source
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AI summary
A method is provided for generating a marker in a biological sample (300, 302, 304) comprising a plurality of cells by means of oligonucleotide constructs (100). The method comprises the following steps: introducing at least a plurality of first oligonucleotide constructs (100) into the biological sample (300, 302, 304), wherein the first oligonucleotide constructs (100) comprise a first promoter (102), a first nucleic acid sequence (104) encoding a first fluorescent protein (106), and a first photoremovable cage molecule (108); and exposing, at least a first region of the biological sample (300, 302, 304) with a first spatially constrained light beam to form uncaged first oligonucleotide constructs (112) in order to enable synthesis of first fluorescent proteins (106) from the first nucleic acid sequence (104) and generate at least a part of the marker in the first region of the biological sample (300, 302, 304). In further aspects, an oligonucleotide construct and a system for generating a marker in a biological sample is provided.