Aptamer Selection via Click Chemistry Modifications
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Solution Overview
Problem
Traditional SELEX methods face limitations in introducing chemical diversity into nucleic acids due to compatibility issues with enzymatic steps and limited commercial availability of chemically modified nucleotides, restricting the use of chemical modifications in aptamer selection.
Innovation Solution
A method involving click chemistry to introduce azide-alkyne modifications into nucleic acids, allowing for the use of alkyne-modified nucleobases that undergo 1,3 dipolar cycloaddition to form azide-alkyne modified nucleobases, enabling the incorporation of diverse chemical entities during aptamer selection without disrupting enzymatic processes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If chemically modified nucleotides are used to increase chemical diversity, then the ability to address difficult targets is improved, but compatibility with enzymatic steps such as PCR is lost
Solution Approach 1:
The patent introduces chemically modified nucleotides (with azide or alkyne groups) at specific positions in the nucleic acid sequence before the SELEX process begins. These modifications are strategically placed in regions that will not interfere with polymerase binding or extension, allowing chemical diversity to be introduced in advance without compromising subsequent enzymatic steps.
Solution Approach 2:
The chemical modifications are applied locally at specific positions within the nucleic acid sequence rather than uniformly throughout. The modified nucleotides are placed in randomized regions that are less critical for enzymatic recognition, while maintaining standard nucleotides in regions essential for polymerase function, thus achieving local chemical diversity without global compatibility loss.
2Adaptability or versatility
If chemically modified nucleotide-triphosphates are used, then chemical diversity is increased, but commercial availability and ease of use are reduced
Solution Approach 1:
The patent uses standard, commercially available nucleotide-triphosphates that have been modified with small chemical groups (azide or alkyne). These modifications maintain the fundamental chemical properties needed for enzymatic recognition while adding the desired chemical diversity. The modified nucleotides are designed to be as commercially accessible as standard nucleotides.
3Adaptability or versatility
If larger chemical entities are introduced into DNA libraries, then chemical diversity is enhanced, but compatibility with PCR amplification is reduced
Solution Approach 1:
Larger chemical entities are introduced at specific, localized positions within the nucleic acid sequence rather than throughout the entire molecule. The modifications are concentrated in randomized regions that tolerate chemical diversity, while critical regions for PCR amplification maintain standard nucleotide structure, preserving enzymatic compatibility.
Solution Approach 2:
The chemical modifications are introduced into the nucleic acid library before the SELEX and amplification processes begin. This preliminary introduction allows the chemical entities to be in place for target binding while ensuring that the modifications are positioned to minimize interference with subsequent PCR steps.
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 chemical diversity of DNA libraries, allowing for the introduction of larger entities and improving compatibility with PCR, enabling the selection of aptamers that bind targets with high specificity and affinity.
Implementation Method 1
the modified nucleotide comprises an alkyne-modified nucleobase that is further modified via a 1,3 dipolar cycloaddition of an azide to yield an azide-alkyne modified nucleobase
Data Source
Figure 1~2
Figure 3A~4
AI summary
The invention relates to a method of identifying or producing an aptamer, and a reagent comprising a nucleic acid ligand capable of binding a target sample, wherein the nucleic acid ligand comprises at least one nucleobase modified to contain an azide-alkyne chemical group.