X-Aptamer Selection via Bead-Based Split-Pool Synthesis
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Solution Overview
Problem
Current methods for selecting aptamers with desired chemical substituents are limited, as modifications often disrupt the three-dimensional structure essential for binding, and traditional SELEX methods struggle with nuclease stability and enzymatic amplification of chemically modified sequences.
Innovation Solution
A bead-based next-generation aptamer selection scheme using split-pool synthesis allows for the introduction of chemical modifications at any position in the aptamer sequence, enabling the creation of X-aptamers with enhanced chemical functionality and nuclease resistance through the use of monothioate and dithioate backbone substitutions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If chemical modifications are introduced into the aptamer sequence to enhance functionality, then chemical functionality and nuclease resistance are improved, but the three-dimensional structure essential for binding is disrupted
Solution Approach 1:
The patent divides the aptamer into functional segments: a core binding region that maintains the essential three-dimensional structure, and modular chemical modification sites at terminal regions or specific positions that can be independently optimized. This segmentation allows chemical functionality to be enhanced without disrupting the core binding structure.
Solution Approach 2:
Chemical modifications are applied locally at specific positions rather than uniformly throughout the aptamer sequence. The patent introduces chemical substituents at predetermined positions (e.g., 5-position of pyrimidines, 3-position of purines) or at terminal regions, allowing local enhancement of chemical functionality while preserving the global three-dimensional binding structure.
2Ease of manufacture
If traditional SELEX methods are used to select modified aptamers, then selection process is simplified, but enzymatic amplification of chemically modified sequences fails
Solution Approach 1:
Chemically modified nucleotides are pre-incorporated into the aptamer library during solid-phase synthesis before the selection process begins. This preliminary introduction of modifications eliminates the need for post-selection modification steps and allows direct selection of fully modified aptamers that maintain enzymatic amplification capability.
Solution Approach 2:
The patent uses unmodified complementary strands as templates for PCR amplification, while the modified aptamers serve as the selected product. This copying strategy allows enzymatic amplification to proceed efficiently using standard polymerases on unmodified templates, while the desired chemically modified aptamers are recovered and characterized.
3Measurement precision
If aptamers are selected to bind acidic proteins, then target specificity is achieved, but binding affinity is limited due to lack of cationic groups
Solution Approach 1:
The patent systematically varies the chemical substituent parameters at defined positions to introduce cationic character. By selecting appropriate chemical groups (e.g., amines, ammonium salts, or basic side chains) at specific positions, the electrostatic properties of the aptamer are modified to enhance interaction with acidic protein surfaces while maintaining sequence-specific recognition.
4Measurement precision
If iterative SELEX cycles are performed to select aptamers, then binding specificity is improved, but the process time increases significantly
Solution Approach 1:
A diverse library of chemically modified aptamers is synthesized in advance with modifications incorporated during solid-phase synthesis. This preliminary creation of a pre-modified library eliminates the need for multiple iterative rounds of modification and re-selection, allowing direct selection of high-specificity aptamers in fewer cycles.
Solution Approach 2:
The patent replaces the iterative biological selection-amplification mechanism with a direct chemical synthesis approach. Instead of repeatedly cycling through selection and enzymatic amplification, chemically modified nucleotides are directly incorporated during solid-phase synthesis, substituting the iterative mechanical process with a more efficient chemical manufacturing approach.
Data Source
AI summary
Provided herein are methods for a novel bead-based next-generation “X-aptamer” selection scheme that extends aptamer technology to include X-modified bases, thus resulting in X-aptamers, at any position along the sequence because the aptamers are chemically synthesized via a split-pool scheme on individual beads. Also provides are application to a wide range of commonly used DNA modifications, including, but not limited to, monothioate and dithioate backbone substitutions. This new class of aptamer allows chemical modifications introduced to any of the bases in the aptamer sequence as well as the phosphate backbones and can be extended to other carbohydrate-based systems.