Department of Molecular Biology and Genetics, Cornell University, Biotechnology Building, Ithaca, NY 14853,USA.
Nucleic Acids Res. 2013 Aug;41(14):7167-75. doi: 10.1093/nar/gkt477. Epub 2013 Jun 3.
The non-specific binding of undesired ligands to a target is the primary factor limiting the enrichment of tight-binding ligands in affinity selection. Solution-phase non-specific affinity is determined by the free-energy of ligand binding to a single target. However, the solid-phase affinity might be higher if a ligand bound concurrently to multiple adjacent immobilized targets in a cooperative manner. Cooperativity could emerge in this case as a simple consequence of the relationship between the free energy of binding, localization entropy and the spatial distribution of the immobilized targets. We tested this hypothesis using a SELEX experimental design and found that non-specific RNA aptamer ligands can concurrently bind up to four bead-immobilized peptide targets, and that this can increase their effective binding affinity by two orders-of-magnitude. Binding curves were quantitatively explained by a new statistical mechanical model of density-dependent cooperative binding, which relates cooperative binding to both the target concentration and the target surface density on the immobilizing substrate. Target immobilization plays a key role in SELEX and other ligand enrichment methods, particularly in new multiplexed microfluidic purification devices, and these results have strong implications for optimizing their performance.
非特异性配体与靶标的结合是非特异性结合，是限制亲和选择中强结合配体富集的主要因素。溶液相中无特异性亲和性取决于配体与单个靶标的结合自由能。然而，如果配体以协同的方式同时与多个相邻固定化的靶标结合，则固相中亲和性可能会更高。在这种情况下，协同作用可能是结合自由能、定位熵和固定化靶标空间分布之间关系的简单结果。我们使用 SELEX 实验设计测试了这一假设，发现非特异性 RNA 适体配体可以同时结合多达四个珠粒固定化肽靶标，并且这可以将其有效结合亲和力提高两个数量级。通过一种新的密度依赖性协同结合统计力学模型对结合曲线进行了定量解释，该模型将协同结合与靶标浓度和固定化基质上靶标表面密度联系起来。靶标固定化在 SELEX 和其他配体富集方法中起着关键作用，特别是在新的多路复用微流控纯化设备中，这些结果对优化其性能具有重要意义。
