Laboratory of Physicochemistry of Electrolytes, Colloids and Analytical Sciences, Ecole Nationale Supérieure de Chimie de Paris (Chimie ParisTech), Paris, France.
Electrophoresis. 2010 Jan;31(3):546-55. doi: 10.1002/elps.200900387.
Binding between an aptamer and its target is highly dependent on the conformation of the aptamer molecule, this latter seeming to be affected by a variety of cations. As only a few studies have reported on the interactions of monovalent or divalent cations with aptamers, we describe herein the use of ACE in its mobility shift format for investigating interactions between various monovalent (Na+, K+, Cs+ or divalent (Mg2+, Ca2+, Ba2+) cations and a 30-mer lysozyme-binding aptamer. This study was performed in BGEs of different natures (phosphate and MOPS buffers) and ionic strengths. First, the effective charges of the aptamer in 30 mM ionic strength phosphate and MOPS (pH 7.0) were estimated to be 7.4 and 3.6, respectively. Then, corrections for ionic strength and counterion condensation effects were performed for all studies. The effective mobility shift was attributed not only to these effects, but also to a possible interaction with the buffer components (binary or ternary complexes) as well as possible conformational changes of the aptamer. Finally, apparent binding constants were calculated for divalent cations with mathematical linearization methods, and the influence of the nature of the BGE was evidenced.
适配体与靶标之间的结合高度依赖于适配体分子的构象，而后者似乎受到各种阳离子的影响。由于只有少数研究报告了单价或二价阳离子与适配体的相互作用，我们在此描述了使用 ACE 在其迁移率变化格式中研究各种单价（Na+、K+、Cs+）或二价（Mg2+、Ca2+、Ba2+）阳离子与 30 个碱基对溶菌酶结合适配体之间的相互作用。该研究在不同性质的 BGE（磷酸盐和 MOPS 缓冲液）和离子强度下进行。首先，在 30 mM 离子强度的磷酸盐和 MOPS（pH 7.0）中，适配体的有效电荷分别估计为 7.4 和 3.6。然后，对所有研究都进行了离子强度和抗衡离子凝聚效应的校正。有效迁移率变化不仅归因于这些效应，还归因于与缓冲成分（二元或三元配合物）的可能相互作用以及适配体的可能构象变化。最后，使用数学线性化方法计算了二价阳离子的表观结合常数，并证明了 BGE 性质的影响。
