High-Throughput Aptamer Screening With Sequence-Linked Affinity Measurement
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Solution Overview
Problem
The process of generating non-natural aptamers is challenging due to the need for costly and time-consuming polymerase engineering, labor-intensive selection processes, and lengthy characterization methods, which limits the exploration of chemical space and results in aptamers with low specificity, especially in complex samples.
Innovation Solution
A method involving solid support clusters of sequence-identified adaptor end-linked aptamers with non-natural nucleotides, allowing for rapid screening and correlation of binding affinity with nucleotide sequence using a throughput approach, incorporating machine learning for aptamer library enrichment.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional SELEX-style selection processes are used to generate non-natural aptamers, then aptamers can be obtained, but the process is labor intensive and results in aptamers with low specificity
Solution Approach 1:
The patent replaces traditional mechanical/manual SELEX selection processes with automated high-throughput screening using flow cytometry and automated liquid handling systems. This substitution eliminates labor-intensive manual operations while maintaining or improving selection quality, directly resolving the contradiction between automation and selection effectiveness.
Solution Approach 2:
The patent changes the selection parameters by performing selections in physiological buffer conditions rather than simple buffer, and by using fluorescently labeled targets with flow cytometry detection. These parameter changes enable rapid automated screening while maintaining high specificity, resolving the time-specificity contradiction.
2Reliability
If buffer-based selection is used to avoid unintended selection of aptamers against interferents, then false positives are reduced, but aptamers exhibit poor specificity in complex samples such as cell lysate or serum
Solution Approach 1:
The patent performs preliminary negative selection steps against interferents present in complex samples before final selection against the target. This preliminary action removes aptamers that would bind to interferents, ensuring that selected aptamers maintain high specificity when applied to complex biological samples.
Solution Approach 2:
The patent uses fluorescently labeled targets and interferents as intermediaries in flow cytometry-based screening. These labeled intermediaries enable automated detection and differentiation of specific versus non-specific binding, allowing rapid identification of high-specificity aptamers without manual intervention.
3Measurement precision
If individual aptamer characterization is performed using traditional methods, then binding affinity can be measured, but the process is lengthy and labor intensive, limiting the number of selected aptamers that can be tested
Solution Approach 1:
The patent merges multiple individual aptamer characterization steps into a single high-throughput flow cytometry assay. By combining binding affinity measurement, specificity assessment, and ranking into one automated workflow, the system achieves both precise measurement and high productivity, resolving the contradiction between measurement quality and screening throughput.
Solution Approach 2:
The patent replaces manual, low-throughput characterization methods with automated flow cytometry and computational analysis. This mechanical substitution enables simultaneous characterization of hundreds of aptamers with precise binding affinity measurements, directly increasing productivity without sacrificing measurement precision.
4Adaptability or versatility
If polymerase engineering is performed to enable processing of modified nucleotides, then non-natural aptamers can be generated, but the process is costly and time-consuming
Solution Approach 1:
The patent uses a universal polymerase system that can process multiple types of modified nucleotides without requiring separate engineering for each modification. This multi-functional approach enables exploration of diverse chemical spaces using standard polymerases, eliminating the time-consuming iterative engineering process while maintaining versatility.
Data Source
AI summary
The disclosure is directed to methods and compositions for screening a library of aptamers for aptamers having a binding affinity to a target molecule. The methods and compositions described herein utilize a throughput approach that is able to simultaneously measure binding affinity and link the binding affinity to the identity (e.g., sequence) of the aptamer.