Le An T H, Teclemichael Eden, Krylova Svetlana M, Krylov Sergey N
Department of Chemistry, York University, 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada.
Centre for Research on Biomolecular Interactions, York University, 4700 Keele Street, Toronto, Ontario M3J 1P3, Canada.
JACS Au. 2024 Dec 9;4(12):4910-4920. doi: 10.1021/jacsau.4c00890. eCollection 2024 Dec 23.
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.
在指数富集配体系统进化技术（SELEX）中维持严格条件对于获得高亲和力适配体至关重要。然而，过度严格会大大增加SELEX失败的风险。由于缺乏明确、定量的严格度衡量标准，控制严格度一直是一项技术挑战，很大程度上依赖于直觉。本研究的灵感来源于我们的洞察：虽然严格度受多种因素影响，但可以通过其效果进行量化，即增加严格度会降低分区后结合物与非结合物数量的归一化值。基于这一洞察，我们提出使用结合物与非结合物比率（BNR）来衡量严格度，其中较低的BNR表示较高的严格度。我们推导了一种通过定量PCR测定BNR的实验方法。我们的理论分析以及以两种不同蛋白质为选择靶标的SELEX实验强调了维持BNR显著大于零以避免失败的重要性，我们将这一原则称为SELEX非失败标准。通过采用反向BNR来量化严格度并应用这一标准，研究人员可以更合理地控制SELEX进程。只要所选结合物可量化，这种定量严格度衡量方法和非失败标准也可应用于其他人工进化方法。
