Quantitative Characterization of Partitioning Stringency in SELEX

IF 8.5 Q1 CHEMISTRY, MULTIDISCIPLINARY
An T. H. Le, Eden Teclemichael, Svetlana M. Krylova and Sergey N. Krylov*, 
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引用次数: 0

Abstract

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.

SELEX中分区严格性的定量表征
在SELEX(配体的指数富集系统进化)中保持严格的条件对于获得高亲和力的适体至关重要。但是,过于严格会大大增加SELEX失败的风险。控制严格程度仍然是一项技术挑战,很大程度上取决于直觉,因为缺乏明确的严格程度的定量衡量。本研究的动机是我们的见解,虽然严格性受到多种因素的影响,但可以通过其效果来量化:增加严格性减少了划分后归一化为非粘合剂的粘合剂的数量。基于这一见解,我们建议使用粘合剂与非粘合剂的比率(BNR)来测量严格性,BNR越低表明严格性越高。我们推导了一种通过定量PCR测定BNR的实验方法。我们的理论分析和SELEX实验使用两种不同的蛋白质作为选择目标,强调了保持BNR显著大于零以避免失败的重要性,我们称之为SELEX非失败准则。利用逆BNR量化严格性,并应用该准则,可以更合理地控制SELEX进程。定量的严格度量和不失效准则也可以应用于其他人工进化方法,只要所选择的粘合剂是可量化的。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
CiteScore
9.10
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0.00%
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审稿时长
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