Dynamic DNA Hybridization for High-Affinity Complex Identification
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Solution Overview
Problem
Current methods for identifying high-affinity complexes of two ligands and a receptor in DNA-encoded chemical libraries face challenges in improving the signal-to-noise ratio, requiring numerous time-consuming and costly parallel selection experiments, especially when the binding affinities of the entire library for the target protein are unknown, and are inefficient in distinguishing high-affinity binders from weak or non-binding compounds.
Innovation Solution
A method involving dynamic hybridization and dissociation of single-stranded DNA or RNA ligands at room temperature or through controlled heating and cooling, allowing for the specific dissociation of high-affinity ligands to re-form ligand complexes, thereby enriching high-affinity ligand-receptor complexes and increasing the sensitivity of detection, using a microfluidic device and a self-assembling chemical library with ligands of varying complementary base lengths.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If parallel selection experiments with different stringencies are conducted to identify high-affinity complexes, then the selection conditions can be optimized, but the time and cost required increase significantly
Solution Approach 1:
The patent applies dynamics by implementing dynamic hybridization and dissociation cycles at room temperature, allowing the system to continuously equilibrate and enrich high-affinity complexes without requiring multiple static experimental conditions. This dynamic approach replaces numerous parallel selection experiments with a single time-resolved process.
Solution Approach 2:
The patent changes the temperature parameter dynamically during the selection process, alternating between hybridization and dissociation conditions to enrich high-affinity complexes. This parameter modulation allows optimization of signal-to-noise ratio without conducting multiple parallel experiments at different fixed conditions.
2Stability of the object's composition
If ligands are hybridized to form stable complexes, then the complexes can be maintained, but high-affinity ligands may be trapped in low-affinity complexes and not available for receptor binding
Solution Approach 1:
The patent creates a dynamic equilibrium between hybridized and dissociated ligands through repeated cycles of hybridization and dissociation. This dynamic process allows high-affinity ligands to be released from low-affinity complexes and available for receptor binding, while maintaining overall system stability through controlled equilibrium.
Solution Approach 2:
The patent employs periodic hybridization and dissociation cycles that repeatedly form and break ligand complexes. This periodic action ensures that high-affinity ligands are continuously released and available for receptor interaction, preventing permanent trapping in low-affinity complexes while maintaining system stability through cyclic equilibrium.
3Measurement precision
If heating and cooling cycles are applied to achieve dynamic hybridization and dissociation, then the sensitivity of identification is improved, but the complexity of the procedure increases
Solution Approach 1:
The patent changes the temperature parameter to achieve dynamic hybridization and dissociation, improving detection sensitivity. By controlling temperature variations, the system can selectively form and break complexes to enrich high-affinity binders.
4Stability of the object's composition
If the length of complementary DNA or RNA bases is increased to improve hybridization stability, then the ligand complexes are more stable, but the specificity of high-affinity binding decreases
Solution Approach 1:
The patent optimizes the length of complementary DNA or RNA bases and controls temperature parameters to achieve the right balance between hybridization stability and binding specificity. By carefully tuning these parameters, the system maintains sufficient complex stability while preserving the ability to discriminate high-affinity from low-affinity interactions through dynamic enrichment.
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 enhances the sensitivity of identifying high-affinity ligand-receptor complexes by quantitatively converting high-affinity ligands into ligand-receptor complexes, improving the signal-to-noise ratio and reducing the need for extensive experimental conditions, allowing for more efficient identification of high-affinity binders without trapping ligands in low-affinity complexes.
Implementation Method 1
more than 10 bases of the single-stranded DNA or RNA from a first part of the ligands are complementary to bases of the single-stranded DNA or RNA from a second part of the ligands, and wherein the ligands are complexed to form ligand complexes via hybridization of the DNA or RNA
Implementation Method 2
which is achieved even at room temperature for the embodiment of the invention, but requires a cycle of heating and cooling for a non-inventive embodiment
Implementation Method 3
which is achieved even at room temperature for the embodiment of the invention, but requires a cycle of heating and cooling for a non-inventive embodiment
Data Source
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AI summary
The present invention relates to a method for the sensitive identification of high-affinity complexes consisting of two ligands (2, 3, 4, 5, 6, 7) and a receptor (1). In this method, a plurality of different ligands (2, 3, 4, 5, 6, 7) from a chemical library are contacted with at least one receptor (1) in a solution. The ligands of the library have single-stranded DNA (8, 9) or RNA with a base length of 2 to 10 bases or alternatively more than 10 bases. Furthermore, the solution is incubated for a specific period of time, and complexes consisting of two ligands (2, 3, 4, 5, 6, 7) and a receptor (1) are identified.