Porous Filter Aptamer Selection Reduces Off-Target Binding
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Solution Overview
Problem
The SELEX process for generating nucleic acid aptamers has a low success rate due to challenges in selecting target-bound aptamers from fluid phase contaminants and often results in off-target binding, leading to the loss of desired aptamers.
Innovation Solution
A method involving contacting a solid-phase analyte with a fluid-phase library of affinity reagents, using a porous filter to separate the reagents, transferring the analyte to a second vessel, and washing to remove contaminants, while maintaining target-bound reagents, along with the use of specific polypeptides with flanking moieties to enhance epitope binding.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional SELEX process is used to select target-bound aptamers, then aptamers can be enriched from fluid phase, but contamination by off-target binding aptamers occurs and success rate remains low
Solution Approach 1:
The selection process is divided into distinct phases: initial binding phase where aptamers bind to target analyte, separation phase using porous filter to isolate solid-phase bound aptamers from fluid phase contaminants, washing phase to remove non-specific binders, and elution phase to recover selected aptamers. This segmentation allows each phase to be optimized independently, improving overall reliability while reducing contamination.
Solution Approach 2:
The harmful fluid phase contaminants are extracted and removed from the system through the porous filter during the separation phase. Only the desired solid-phase bound aptamers are retained, effectively taking out the harmful elements that cause off-target binding and improve the purity of the selected aptamer population.
2Manufacturing precision
If iterative selection steps are performed to enrich target-bound aptamers, then specificity can be improved, but time consumption increases substantially
Solution Approach 1:
The porous filter enables continuous washing of the solid-phase analyte with fluid phase buffer, maintaining continuous contact between the analyte and washing solution to thoroughly remove contaminants. This continuous action achieves high specificity without requiring multiple discrete iterative steps, thereby reducing time consumption while maintaining purification effectiveness.
3Object-generated harmful factors
If washing steps are performed to remove contaminant aptamers, then purity of selected aptamers increases, but loss of desired aptamers may occur
Solution Approach 1:
The porous filter provides localized separation at the solid-liquid interface, allowing washing of only the solid-phase bound aptamers while leaving the fluid phase contaminants behind. This localized quality of separation protects the desired aptamers from being washed away while effectively removing contaminants, achieving high purity with minimal loss.
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 improves the enrichment of desired aptamers by reducing contamination and off-target binding, increasing the specificity and yield of aptamers during the selection process.
Implementation Method 1
passing the fluid-phase library of affinity reagents through the porous filter to separate affinity reagents of the library from the solid-phase analyte
Data Source
AI summary
The present disclosure provides methods, compositions and apparatus for generating probes having affinity for analytes of interest, such as polypeptides. Also provided are methods, compositions and apparatus for evaluating and characterizing probes.