DNA Construct with Quencher for Cell Surface Protein Labeling
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Solution Overview
Problem
Current methods for labeling cell surface proteins in native cells are costly, laborious, and lack efficiency, particularly in detecting small-molecule interactions, due to difficulties in maintaining binding affinity and requiring excessive probe concentrations, which complicates multiplexed protein detection and signal enhancement.
Innovation Solution
A DNA construct comprising oligonucleotides covalently bound to fluorescent dyes and quenching agents, allowing for reversible modification and binding to specific cell surface proteins, enabling efficient and versatile labeling without the need for extensive washing or probe excess.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If small-molecule ligands are conjugated to fluorescent dyes to label cell surface proteins, then the ability to track small-molecule interactions is enabled, but binding affinity decreases and requires large excess of probes
Solution Approach 1:
The probe is divided into separate functional components: a fluorescent dye, a quenching agent, and a small-molecule ligand. The quenching agent is initially positioned close to the fluorescent dye to suppress background signal, but upon binding to the cell surface protein, the probe structure allows separation of the fluorophore from the quencher, enabling fluorescence activation while maintaining binding affinity through the ligand component.
Solution Approach 2:
The quenching agent acts as an intermediary that modulates the fluorescence signal. It is covalently linked to the probe structure and serves to suppress background fluorescence while allowing signal activation upon target binding, thereby enabling sensitive detection without requiring excessive probe concentrations that would compromise binding affinity.
2Reliability
If large excess of probes is used to compensate for reduced binding affinity, then labeling can be achieved, but background signal increases
Solution Approach 1:
The quenching agent, which initially suppresses fluorescence and could be considered a harmful factor, is converted into a beneficial signal control mechanism. By positioning the quencher close to the fluorophore in the unbound state, background signal is suppressed. Upon binding to the target, the probe conformational change separates the fluorophore from the quencher, activating fluorescence specifically where needed, thus converting the potential harm of the quencher into a useful signal activation switch.
3Object-generated harmful factors
If washing is performed to remove excess probes, then background signal is reduced, but probe-POI complex dissociates
Solution Approach 1:
The probe is designed with intrinsic properties that enable selective binding and signal activation before washing is required. The quenching agent is pre-positioned to suppress background signal, and the probe structure is pre-configured to activate fluorescence upon binding. This preliminary setup allows specific labeling to occur without requiring extensive washing that would disrupt the probe-POI complex, as the binding affinity is maintained through the ligand-quencher-fluorophore assembly.
4Illumination intensity
If multiple fluorescent dyes are integrated into a single probe to enhance signal intensity, then detection sensitivity improves, but probe complexity and manufacturing difficulty increase
Solution Approach 1:
Multiple functional elements (fluorescent dye, quenching agent, ligand) are merged into a single probe molecule through covalent linkage. This integration allows the probe to perform multiple functions simultaneously: binding to the target protein, suppressing background signal, and activating fluorescence upon binding. The merged structure simplifies delivery and reduces the need for separate reagents, thereby reducing overall system complexity while maintaining high signal intensity through the coordinated action of the integrated components.
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 provides a cost-effective, efficient method for labeling cell surface proteins with high binding affinity, allowing for precise control over protein interactions and concentrations, enhancing signal intensity and reducing background noise.
Implementation Method 1
A DNA construct comprising oligonucleotides covalently bound to fluorescent dyes and quenching agents, allowing for reversible modification and binding to specific cell surface proteins
Implementation Method 2
A DNA construct comprising oligonucleotides covalently bound to fluorescent dyes and quenching agents
Implementation Method 3
A DNA construct comprising oligonucleotides covalently bound to fluorescent dyes and quenching agents, allowing for reversible modification and binding to specific cell surface proteins, enhancing signal intensity and reducing background noise
Data Source
AI summary
The disclosure presented herein provides DNA constructs, recombinant cells comprising thereof, system comprising thereof, bacterial probes, and/or a recombinant cell decorated with various labels and/or synthetic agents, wherein said labels and/or synthetic agents can be reversibly modified or removed from the cells. Also disclosed herein are methods for decorating and/or modifying cells, preferably bacteria cells, and methods for using thereof.