Okholm Kasper R, Nooteboom Sjoerd W, Vinther Johan Nygaard, Lamberti Vincenzo, Dey Swayandipta, Andersen Ebbe Sloth, Zijlstra Peter, Sutherland Duncan S
Interdisciplinary Nanoscience Center, Aarhus University, Gustav Wieds Vej 14, Aarhus C 8000, Denmark.
The Centre for Cellular Signal Patterns (CELLPAT), Gustav Wieds Vej 14, Aarhus C 8000, Denmark.
ACS Nano. 2024 Dec 31;18(52):35429-35442. doi: 10.1021/acsnano.4c12600. Epub 2024 Dec 16.
Multivalency as an interaction principle is widely utilized in nature. It enables specific and strong binding by multiple weak interactions through enhanced avidity and is a core process in immune recognition and cellular signaling, which is also a current concept in drug design. Here, we use the high signals from plasmon-enhanced fluorescence of nanoparticles to extract binding kinetics and dynamics of multivalent interactions on the single-molecule level and in real time. We study mono-, bi-, and trivalent binding interactions using a DNA Holliday Junction as a model construct with programmable valency and introduce a step-binding model for binding kinetics relevant for structured macromolecules by including an experimentally extractable binding restriction term ω to quantify the effects from conformation, steric effects, and rigidity. We used this approach to explore how length and flexibility of the DNA ligands affect binding restriction and binding strength, where the overall binding strength decreased with spacer length. For trivalent systems, increasing spacer length additionally activated binding in the trivalent state, giving insight into the design of multivalent drug or targeting moieties. By systematically changing the receptor density, we explored the binding super selectivity of the multivalent HJ at the single-molecule level. We find a polynomial behavior of the trivalent binding strength that clearly shows receptor-density-dependent selective binding. Interestingly, we could exploit the rapidly decaying near fields of the plasmon that induce a strong dependence of the signal on the position of the dye to observe binding dynamics during single multivalent binding events.
多价性作为一种相互作用原理在自然界中被广泛应用。它通过增强亲和力，利用多个弱相互作用实现特异性和强结合，是免疫识别和细胞信号传导中的核心过程，也是药物设计中的一个当前概念。在此，我们利用纳米粒子的等离子体增强荧光产生的高信号，在单分子水平上实时提取多价相互作用的结合动力学和动力学过程。我们以具有可编程价态的DNA霍利迪连接体作为模型构建物，研究单价、二价和三价结合相互作用，并通过纳入一个可通过实验提取的结合限制项ω来引入一个与结构化大分子相关的结合动力学的逐步结合模型，以量化构象、空间位阻效应和刚性的影响。我们采用这种方法来探究DNA配体的长度和灵活性如何影响结合限制和结合强度，其中总体结合强度随间隔长度的增加而降低。对于三价系统，增加间隔长度还会激活三价状态下的结合，从而为多价药物或靶向部分的设计提供了思路。通过系统地改变受体密度，我们在单分子水平上探究了多价霍利迪连接体的结合超选择性。我们发现三价结合强度呈现多项式行为，清楚地表明了受体密度依赖性的选择性结合。有趣的是，我们可以利用等离子体快速衰减的近场，其诱导信号对染料位置有强烈依赖性，从而在单个多价结合事件中观察结合动力学。
