Aptamer Selection Without Target Immobilization
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Solution Overview
Problem
Current methods for selecting aptamers, such as SELEX and capillary electrophoresis, face limitations when dealing with small molecules and complex mixtures, as they require immobilization of target analytes, which can alter their structure and disrupt molecular complexes, and are not effective for selecting aptamers that bind to multiple epitopes or small molecules without affecting their structure.
Innovation Solution
A method involving a library of oligonucleotides with a random region flanked by primer recognition sites, combined with blocker oligonucleotides, exposed to an immobilization field of random antisense oligonucleotides, allowing for selection without immobilization of target analytes, enabling direct selection for binding and conformational changes, and counter-selection against non-binding sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Manufacturing precision
If target analytes are immobilized on solid support for partitioning, then oligonucleotides bound to target can be separated from unbound oligonucleotides, but the immobilization process alters the chemical structure and electronic properties of small molecule targets
Solution Approach 1:
The patent uses an antibody specific to the target analyte as an intermediary mediator. The antibody is immobilized on the solid support, while the target analyte remains in solution in its native state. This allows bound oligonucleotides to be captured via the antibody-target complex without direct immobilization of the target, preserving its chemical structure and electronic properties.
2Measurement precision
If washing steps are performed to increase selection stringency, then non-specifically bound oligonucleotides are removed, but molecular complexes may be disrupted
Solution Approach 1:
The antibody serves as a stable intermediary that strengthens the complex. The oligonucleotide binds to the target analyte, which is held by the immobilized antibody, creating a multi-component complex that is more resistant to wash-induced disruption than simple oligonucleotide-target pairs.
Solution Approach 2:
The patent employs a multi-layered selection process with multiple washing steps at increasing stringency, creating a robust selection funnel that maintains complex integrity while removing non-specific binders.
3Productivity
If conventional SELEX methods are used for small molecules, then aptamers can be selected, but the method requires immobilization of target which is not suitable for small molecules that pass through nitrocellulose
Solution Approach 1:
The patent creates a universal selection system using immobilized antibodies that can capture and retain various small molecule targets on the solid support, enabling SELEX to be applied to any small molecule that forms a complex with a suitable antibody, regardless of its size or ability to pass through nitrocellulose.
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 the selection of aptamers that bind specifically to target molecules, including small molecules and complexes, without disrupting their structure, and can be applied to complex mixtures, increasing the stringency and specificity of the selection process.
Implementation Method 1
aptamers are oligonucleotides that mimic antibodies in their ability to act as ligands and bind to analytes
Implementation Method 2
Single stranded oligonucleotides will pass through nitrocellulose while many analyte molecules, in particular protein analytes, will not
Implementation Method 3
there is a need to separate those oligonucleotide sequences that have bound to the target from those that have not
Data Source
Figure 1~2
AI summary
The present invention relates to a method for the selection of aptamers that does not require immobilization of the target or the oligonucleotide library. The method comprises: (i) combining a selection library of oligonucleotides that contain a random region flanked by primer recognition sites with blocker oligonucleotides that anneal to the primer recognition sites; (ii) exposing the blocked selection library to an immobilization field comprising random oligonucleotides and removing unbound selection library oligonucleotides; (iii) recovering the selection library oligonucleotides that bound to the immobilization field and combining with a target analyte of interest; (iv) exposing the selection library oligonucleotides-target analyte mixture to an immobilization field; and (v) recovering those selection library oligonucleotides that did not bind to the immobilization field.