Single-Stranded DNA Aptamers for NF-kB RelA Quantification
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Solution Overview
Problem
Current methods lack effective and specific tools for recognizing and quantifying activated NF-κB/RelA, which is crucial for understanding and managing cellular inflammation and immune responses.
Innovation Solution
Development of single-stranded DNA aptamers that specifically bind to activated NF-κB/RelA, enabling its quantification, isolation, and modulation, using techniques like affinity binding and mass spectrometry for precise measurement and analysis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional methods are used for detecting NF-κB/RelA, then general protein detection is possible, but specificity and sensitivity for activated RelA quantification are insufficient
Solution Approach 1:
The patent introduces single-stranded DNA aptamers as intermediary molecules that specifically bind to activated RelA. These aptamers serve as mediators between the target protein (activated RelA) and the detection system, enabling highly specific recognition and quantification through affinity binding assays and mass spectrometry-based methods.
Solution Approach 2:
The patent employs parameter changes by modifying the detection approach from general protein detection to aptamer-based specific binding. The aptamers are selected and optimized to bind activated RelA with high affinity, changing the binding parameter (Kd) to achieve superior sensitivity and specificity for quantification.
2Reliability
If aptamers are developed for specific binding, then recognition specificity improves, but development time and resource investment increase
Solution Approach 1:
The patent applies preliminary action by performing in vitro selection (SELEX) to pre-screen and identify aptamers with specific binding activity against activated RelA before actual applications. This preliminary selection process ensures that only high-affinity, specific binders are advanced to validation and use, reducing subsequent optimization time.
Solution Approach 2:
The aptamer selection process utilizes self-service principles where the aptamer pool automatically enriches for high-affinity binders through multiple rounds of selection, washing, and amplification. The system self-optimizes to identify specific binders without requiring extensive manual intervention at each step.
3Measurement precision
If high affinity binding is achieved, then quantification sensitivity increases, but cross-reactivity with other proteins may increase
Solution Approach 1:
The patent applies local quality by designing aptamers that recognize specific local features or epitopes on activated RelA rather than general protein structures. The aptamers are selected to bind to unique conformational or sequence features present only on activated RelA, providing localized specificity that reduces cross-reactivity while maintaining high affinity.
Solution Approach 2:
The patent replaces traditional mechanical or chemical affinity tags with biologically derived aptamer binding. The aptamers provide specific recognition through molecular complementarity rather than physical tagging, enabling high sensitivity detection while maintaining protein native state and reducing non-specific interactions.
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 aptamers effectively inhibit NF-κB/RelA-mediated inflammation, enhance sensitivity and accuracy in quantification, and provide a tool for modulating immune responses, offering a versatile method for both diagnostic and therapeutic applications.
Implementation Method 1
single stranded DNA aptamers that specifically bind to activated NF-κB/RelA, enabling its quantification, isolation, and modulation
Implementation Method 2
The aptamers effectively inhibit NF-κB/RelA-mediated inflammation
Data Source
AI summary
DNA aptamers are high affinity ligands selected by genetic enrichment techniques to bind to specific protein targets. Because these represent chemically stable and reproducible molecules, they have application as affinity reagents and/or therapeutic drugs to affect the target protein's actions. NF-kB is an important mediator of the innate immune response and mediator of tissue inflammation. Although RNA and double stranded DNA aptamers have been identified to bind to the NF-kB family of proteins, the present invention represents the first identification of single stranded DNA aptamers that recognize NFkB RelA. The aptamers disclosed herein bind to several distinct regions of RelA and may be useful to antagonize the DNA binding of RelA as an inhibitor of cellular inflammation, visualize the location or amount of RelA in tissues from pathological conditions, or to quantitatively measure the activated state of RelA by affinity binding.