Exonuclease-Generated Structure-Switching Aptamers for Small Molecule Detection
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Solution Overview
Problem
Current aptamer-based sensors for small molecule detection require labor-intensive methods to introduce structure-switching functionality, often resulting in low target-binding affinities and complex, costly procedures.
Innovation Solution
The use of exonuclease-based methods, specifically Exo III digestion, to generate structure-switching aptamers from fully folded or pre-folded aptamers, enabling rapid and sensitive detection of small molecule targets without the need for prior sequence engineering or labeling.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If conventional methods are used to introduce structure-switching functionality into aptamers, then structure-switching capability is achieved, but the process becomes labor-intensive and time-consuming
Solution Approach 1:
The patent applies preliminary action by pre-folding the aptamer into its target-bound conformation before introducing structure-switching functionality. This pre-folding step ensures the aptamer is already in the correct functional state, eliminating the need for labor-intensive truncation and sequence engineering trials. The aptamer is then modified with a structure-switching element that maintains the pre-established fold, rapidly conferring sensor capability without extensive optimization.
Solution Approach 2:
The patent segments the aptamer development process into distinct functional modules: a pre-folded target-binding domain and a separate structure-switching element. This segmentation allows the binding affinity to be optimized independently in the pre-folded aptamer, while the structure-switching functionality is added as a modular component. This modular approach dramatically reduces development time compared to traditional methods where the entire sequence must be engineered simultaneously.
2Adaptability or versatility
If conventional sequence engineering methods are used to create structure-switching aptamers, then structure-switching capability is achieved, but target-binding affinity decreases
Solution Approach 1:
The patent performs preliminary folding of the aptamer sequence to establish the correct three-dimensional structure and high-affinity binding conformation before adding structure-switching modifications. This ensures the binding domain is already optimized for target interaction, and subsequent structural changes are designed to preserve rather than disrupt this pre-established high-affinity configuration.
Solution Approach 2:
The patent applies local quality by introducing structure-switching functionality at specific, strategically chosen locations within the aptamer sequence that do not interfere with the core binding domain. The structure-switching element is placed in regions that allow conformational change while maintaining the integrity and high affinity of the target-binding site, thus achieving both structural flexibility and binding strength.
3Adaptability or versatility
If traditional aptamer sensor development methods are used, then structure-switching capability is achieved, but the process requires considerable trial and error
Solution Approach 1:
The patent eliminates trial and error by performing preliminary computational modeling and structural prediction to identify the optimal pre-folded conformation and the best location for structure-switching modifications. This in silico design approach allows researchers to predict which sequences and modifications will achieve the desired structure-switching behavior, replacing the traditional iterative experimental approach with a rational design strategy.
Solution Approach 2:
The patent introduces computational structural modeling and prediction tools as intermediaries between sequence design and experimental validation. These computational intermediaries allow researchers to simulate and evaluate multiple aptamer designs in silico, selecting the most promising candidates before synthesis and testing, thereby dramatically reducing the number of experimental trials needed.
4Adaptability or versatility
If aptamers are engineered to have structure-switching functionality, then sensor capability is achieved, but the process becomes costly and complex
Solution Approach 1:
The patent segments the sensor development into independent optimization steps: first optimizing the target-binding affinity in the pre-folded aptamer, then separately engineering the structure-switching element. This segmentation allows each function to be optimized independently using different criteria and methods, reducing the overall complexity compared to simultaneously optimizing multiple conflicting requirements in a single design process.
Solution Approach 2:
The patent performs preliminary structural characterization and validation of the pre-folded aptamer before proceeding to structure-switching modifications. This preliminary validation ensures the binding domain is correctly folded and functional, providing a solid foundation for subsequent modifications and reducing the need for complex troubleshooting and re-optimization later in the development process.
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 allows for rapid, sensitive, and cost-effective detection of small molecules, achieving high target-binding affinities and streamlined sensor development, suitable for clinical and field applications.
Implementation Method 1
digesting the aptamer with an exonuclease, such as Exo III. The resulting digestion product has structure-switching functionality
Implementation Method 2
structure-switching aptamers, which undergo a conformational change upon target binding. Upon binding to the sensing aptamer, the target induces a specific folding event that produces a colorimetric, fluorescent or electrochemical readout
Data Source
AI summary
The subject invention provides methods, assays, and products for detecting small-molecule targets in a complex sample in both clinical and field settings. The subject invention provides aptamer-based sensors and methods of use thereof. The subject invention provides exonuclease-based methods for generating structure-switching aptamers from fully folded or pre-folded aptamers and developing aptamer-based sensors for small-molecule detection. The method for detecting one or more small-molecule targets in a sample comprises contacting the sample with one or more aptamer-based sensor selective for each of the small-molecule targets, and detecting the small-molecule target in the sample.