DNA Construct for Reversible Cell Surface Protein Labeling
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Solution Overview
Problem
Current methods for labeling cell surface proteins in native cells, such as cancer cells, are costly, laborious, and lack efficiency due to the difficulty in maintaining high binding affinity of small-molecule ligands after conjugation to fluorescent dyes, and existing fluorescent probes struggle with target versatility and multiplexed protein detection.
Innovation Solution
A DNA construct comprising oligonucleotides covalently bound to His-tag specific binders and synthetic agents, allowing for reversible modification and high-affinity binding to specific cell surface proteins, enabling efficient labeling and detection of proteins of interest with enhanced fluorescence signals.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If small-molecule ligands are conjugated to fluorescent dyes to label cell surface proteins, then the binding affinity decreases, but this requires using a large excess of probes which generates strong background signal
Solution Approach 1:
The probe is divided into two functional segments: a fluorescent dye and a small-molecule ligand, connected by a flexible linker. This segmentation allows each component to maintain its optimal properties while working together, preventing the conjugation process from compromising binding affinity.
Solution Approach 2:
The patent modifies the chemical parameters of the probe system by introducing specific linker structures and adjusting the steric properties of the conjugate. This changes the physical-chemical parameters to maintain high binding affinity while enabling effective labeling.
2Adaptability or versatility
If fluorescent Abs are used to label cell surface proteins, then target versatility is achieved, but the process becomes costly and laborious with sequential incubation steps
Solution Approach 1:
The probe system is designed to be universal, with the same basic probe structure capable of labeling different cell surface proteins by simply changing the small-molecule ligand. This multi-functionality eliminates the need for separate probe preparations for each target.
Solution Approach 2:
The patent merges the fluorescent labeling function and the protein binding function into a single conjugated probe molecule. This combination eliminates the need for sequential primary and secondary antibody incubation steps, simplifying the overall process.
3Illumination intensity
If multiple fluorescent dyes are integrated into a single synthetic probe to enhance signal intensity, then fluorescence signal intensity increases, but the structural complexity and difficulty of obtaining efficient CSP binding probes increases
Solution Approach 1:
Instead of integrating multiple dyes into a single probe molecule (adding complexity in the molecular dimension), the patent uses multiple separate probe molecules that can bind to the same target simultaneously. This achieves enhanced signal intensity through statistical multiplicity rather than molecular complexity.
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
The DNA construct system provides a cost-effective and efficient means for labeling and detecting cell surface proteins with high binding affinity and versatility, facilitating multiplexed protein detection and reversible modification, thereby improving cellular process control.
Implementation Method 1
a second compound comprising a second oligonucleotide (ODN-2) covalently bound to a synthetic agent, either directly or through a second linker, wherein said second oligonucleotide is complementary to said first oligonucleotide
Implementation Method 2
a third compound comprising a DNA duplex (dsDNA) appended with a second hanging strand complementary to said first hanging strand, and further appended with at least two fluorescent dyes
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.