Quantitative Characterization of Partitioning Stringency in SELEX

Maintaining stringent conditions in SELEX (Systematic Evolution of Ligands by EXponential enrichment) is crucial for obtaining high-affinity aptamers; however, excessive stringency greatly increases the risk of SELEX failure. The control of stringency remains a technical challenge reliant solely on intuition, largely due to the absence of a measure of stringency. Essentially, researchers increase or decrease stringency through its influencers without defining and quantitating it. This study was motivated by our insight that while stringency may not be easily definable via its multiple influencers, it can be delineated by its effect: increasing stringency leads to a decrease in the normalized quantity of binders at the output of partitioning. Building on this insight, we introduce a measure of stringency called the Binder-to-Nonbinder Ratio (BNR) and derive an expression for its experimental determination using a single experimental tool: quantitative PCR. The outcomes of our theoretical analysis and the result of SELEX experiments targeting three distinct protein targets underscore the importance of maintaining a BNR significantly greater than zero to avoid SELEX failure due to excessive stringency – a principle that we term the SELEX non-failure criterion. Utilizing BNR as a measure of stringency alongside this criterion will enable researchers to rationally control SELEX progress.