Bulk Affinity Assays in Aptamer Selection: Challenges, Theory, and Workflow

Abstract

Selection of oligonucleotide aptamers involves consecutive rounds of affinity isolation of target-binding oligonucleotides from a random-sequence oligonucleotide library. Every next round produces an aptamer-enriched library with progressively higher fitness for tight binding to the target. The progress of enrichment can only be accurately assessed with bulk affinity assays in which a library is mixed with the target and one of two quantitative parameters, the fraction of the unbound library (R) or the equilibrium dissociation constant (K d ), is determined. These quantitative parameters are used to help researchers make a key decision of either continuing or stopping the selection. Despite the importance of this decision, the suitability of R and K d for bulk affinity assays has never been studied theoretically, and researchers rely on intuition when choosing between them. Different approaches used for bulk affinity assays expectedly hinder comparative analyses of selections. Our current work has two goals: to give bulk affinity assays a thorough theoretical consideration and to propose a scientifically justified and practical bulk-affinity-assay approach. We postulate a formal criterion of suitability: a quantitative parameter must satisfy the principle of superposition. R satisfies this principle, while K d does not, suggesting R as a theoretically preferable parameter. Further, we propose a solution for two limitations of R: its dependence on target concentration and narrow dynamic range. Finally, we demonstrate the use of this algorithm in both computer-simulated and experimental aptamer selection. This study sets a cornerstone in the theory of bulk affinity assays, and it provides researchers with a scientifically sound and instructive approach for conducting bulk affinity assays.

Recent research on material-based methods for isolation of extracellular vesicles

“…In addition, more and more aptamers were discovered with the aptamer selection developed, and EV isolation methods would be more valued when accompanied by the discovery of more EV membrane proteins. 65,66 It has the characteristics of high efficiency, high specicity and friendliness to downstream analysis, and it has broad application prospects. Similar to antibodies, the only weakness of aptamers is that it requires condition optimization, including the types and amount of aptamers on a solid phase matrix.…”

Quantitative Characterization of Partitioning Stringency in SELEX

“…Instead, our experimental study aimed to test how varying BNR (via varying target concentration) influences the SELEX progress assessed through affinity maturation via a bulk-affinity assay of the output library. This assay relies on optimized measurements of the fraction of unbound library 4 (R), 25 and is deemed to provide accurate comparison of SELEX carried out at different target concentrations.…”

“…To validate BNR as a tool for assessing SELEX progress, we compared BNR values with affinity maturation monitored using the bulk-affinity assay. 25 The results of the bulk affinity assay are summarized in Figure 3b, where R is plotted against the SELEX round number for every target concentration, with Round 0 being the starting library prior to the first partitioning (see Section S4 for the electropherograms and calculations of R values).…”