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. Controlling stringency has remained a technical challenge, largely dependent on intuition, due to the absence of a clear, quantitative measure of stringency. This study was motivated by our insight that, while stringency is influenced by multiple factors, it can be quantified by its effect: increasing stringency reduces the quantity of binders normalized to that of nonbinders after partitioning. Based on this insight, we propose measuring stringency using the binder-to-nonbinder ratio (BNR), where a lower BNR indicates higher stringency. We derive an experimental method for determining BNR via quantitative PCR. Our theoretical analysis and SELEX experiments using two distinct proteins as selection targets underscore the importance of maintaining a BNR significantly greater than zero to avoid failure, a principle we call the SELEX nonfailure criterion. By employing inverse BNR to quantify stringency and applying this criterion, researchers can more rationally control SELEX progress. The quantitative stringency measure and nonfailure criterion can also be applied to other artificial evolution methods, provided that selected binders are quantifiable.