Polymer-Nucleic Acid Conjugates for Bright Fluorescent Tags
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Solution Overview
Problem
Current fluorescent labeling methods for biological detection face challenges in enhancing detectable signals for low-expression proteins without compromising specificity and binding affinity, often resulting in reduced fluorescence due to self-quenching with high degrees of substitution.
Innovation Solution
A composition comprising a polymer with extending chains or branches, where one or more strands of nucleic acid are attached to the side chains, forming double strands that are complexed with complementary strands, allowing for the association of multiple fluorescent compounds via intercalation or covalent bonding, enabling energy transfer and increased brightness without dissociation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Illumination intensity
If the number of fluorophores is increased to enhance fluorescence signals, then the detectable signal is improved, but the protein precipitates and loses solubility
Solution Approach 1:
The patent introduces a polymer intermediary with multiple attachment points that serves as a mediator between the fluorophores and the target protein. This polymer scaffold allows multiple fluorophores to be displayed in a controlled manner without direct conjugation to the protein, thereby maintaining protein solubility while achieving high fluorescence signal through the polymer-mediated organization of multiple fluorophore units.
2Illumination intensity
If the number of dyes per antibody is increased to amplify signal, then the fluorescence intensity is improved, but self-quenching occurs and reduces fluorescence output
Solution Approach 1:
The patent segments the fluorophore loading from the antibody structure by using a separate polymer scaffold. This segmentation allows the fluorophores to be distributed across multiple polymer chains rather than clustered on a single antibody, reducing inter-fluorophore interactions that cause self-quenching while maintaining high overall fluorescence intensity through the collective emission of multiple fluorophore units on the polymer assembly.
3Illumination intensity
If more than four to six fluorophores are attached per protein, then the signal amplification is improved, but binding affinity and specificity are reduced
Solution Approach 1:
The polymer acts as an intermediary that decouples the fluorophore loading from the antibody binding function. The antibody conjugated to the polymer maintains its binding affinity and specificity because the fluorophores are attached to the polymer scaffold rather than directly to the antibody. This intermediary structure allows high fluorophore loading (signal amplification) without compromising the antibody's binding reliability.
4Illumination intensity
If catalytic reporter deposition methods are used to generate high density labeling, then the signal brightness is improved, but precise timing control is required to achieve quantitative and reproducible results
Solution Approach 1:
The patent employs preliminary action by pre-synthesizing polymers with a high density of attachment points before conjugation to the antibody. This pre-prepared polymer scaffold eliminates the need for complex in situ catalytic deposition timing control, as the high fluorophore capacity is already built into the polymer structure. The conjugation step is simplified and does not require precise timing control to achieve high signal brightness, making the process more reproducible and easier to execute.
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 approach results in a significantly brighter fluorescent signal, maintaining specificity and binding affinity, with the polymer-nucleic acid assemblies providing a stable platform for hundreds or thousands of fluorescent dye molecules, outperforming commercially available antibody tags by at least an order of magnitude in sensitivity and brightness.
Implementation Method 1
At least one fluorescent compound is intercalated with the double strand of nucleic acid on each of the plurality of the side chains
Implementation Method 2
allowing for the association of multiple fluorescent compounds via intercalation or covalent bonding, enabling energy transfer and increased brightness
Implementation Method 3
One of a plurality of a second strand of nucleic acid, which is complementary (that is, partially of full complementary) to the first strand of nucleic acid, is complexed to each of the plurality of the first strand of nucleic acid to form a double strand of nucleic acid
Data Source
AI summary
A composition includes a polymer including extending chains, side chains, or branches. One (or more) of a plurality of a first strand of nucleic acid is attached to each of a plurality of the side chains. One (or more) of a plurality of a second strand of nucleic acid, which is complementary to the first strand of nucleic acid, is complexed to each of the plurality of the first strand of nucleic acid to form a double strand of nucleic acid on each of the plurality of the side chains. At least one fluorescent compound is associated with the double strand of nucleic acid on each of the plurality of the side chains.