Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive West, Lethbridge, AL T1K 3M4, Canada.
Department of Chemistry and Toxicology, University of Guelph, Guelph, ON N1G 2W1, Canada.
Molecules. 2019 Aug 10;24(16):2908. doi: 10.3390/molecules24162908.
Aptamers are functional nucleic acids that bind to a range of targets (small molecules, proteins or cells) with a high affinity and specificity. Chemically-modified aptamers are of interest because the incorporation of novel nucleobase components can enhance aptamer binding to target proteins, while fluorescent base analogues permit the design of functional aptasensors that signal target binding. However, since optimally modified nucleoside designs have yet to be identified, information about how to fine tune aptamer stability and target binding affinity is required. The present work uses molecular dynamics (MD) simulations to investigate modifications to the prototypical thrombin-binding aptamer (TBA), which is a 15-mer DNA sequence that folds into a G-quadruplex structure connected by two TT loops and one TGT loop. Specifically, we modeled a previously synthesized thymine (T) analog, namely 5-furyl-2'-deoxyuridine (5FurU), into each of the six aptamer locations occupied by a thymine base in the TT or TGT loops of unbound and thrombin bound TBA. This modification and aptamer combination were chosen as a proof-of-principle because previous experimental studies have shown that TBA displays emissive sensitivity to target binding based on the local environment polarity at different 5FurU modification sites. Our simulations reveal that the chemically-modified base imparts noticeable structural changes to the aptamer without affecting the global conformation. Depending on the modification site, 5FurU performance is altered due to changes in the local environment, including the modification site structural dynamics, degree of solvent exposure, stacking with neighboring bases, and interactions with thrombin. Most importantly, these changes directly correlate with the experimentally-observed differences in the stability, binding affinity and emissive response of the modified aptamers. Therefore, the computational protocols implemented in the present work can be used in subsequent studies in a predictive way to aid the fine tuning of aptamer target recognition for use as biosensors (aptasensors) and/or therapeutics.
适体是与一系列靶标（小分子、蛋白质或细胞）具有高亲和力和特异性结合的功能性核酸。化学修饰的适体很有研究价值，因为新型核苷碱基成分的掺入可以增强适体与靶蛋白的结合，而荧光碱基类似物则允许设计出能够发出靶标结合信号的功能性适体传感器。然而，由于最佳修饰核苷的设计尚未确定，因此需要了解如何微调适体的稳定性和靶标结合亲和力。本工作使用分子动力学 (MD) 模拟研究了对原型凝血酶结合适体 (TBA) 的修饰，该适体是一个由 15 个碱基组成的 DNA 序列，折叠成一个由两个 TT 环和一个 TGT 环连接的 G-四链体结构。具体来说，我们在无结合和凝血酶结合 TBA 的 TT 或 TGT 环中每个由胸腺嘧啶 (T) 占据的适体位置模拟了之前合成的胸腺嘧啶 (T) 类似物，即 5-糠基-2'-脱氧尿苷 (5FurU)。这种修饰和适体组合被选为一个原理证明，因为之前的实验研究表明，TBA 根据不同 5FurU 修饰位点的局部环境极性显示出对靶标结合的发光敏感性。我们的模拟表明，化学修饰的碱基会对适体产生明显的结构变化，而不会影响全局构象。根据修饰位置的不同，5FurU 的性能会因局部环境的变化而改变，包括修饰位置的结构动力学、溶剂暴露程度、与相邻碱基的堆积以及与凝血酶的相互作用。最重要的是，这些变化与实验观察到的修饰适体的稳定性、结合亲和力和发光响应的差异直接相关。因此，本工作中实施的计算方案可以以预测的方式用于后续研究，以帮助微调适体的靶标识别，用于作为生物传感器（适体传感器）和/或治疗剂。
