Troisi Romualdo, Riccardi Claudia, Pérez de Carvasal Kévan, Smietana Michael, Morvan François, Del Vecchio Pompea, Montesarchio Daniela, Sica Filomena
Department of Chemical Sciences, University of Naples Federico II, 80126 Naples, Italy.
Institut des Biomolécules Max Mousseron, Université de Montpellier, CNRS, ENSCM, 34293 Montpellier, France.
Mol Ther Nucleic Acids. 2022 Nov 15;30:585-594. doi: 10.1016/j.omtn.2022.11.007. eCollection 2022 Dec 13.
Despite their unquestionable properties, oligonucleotide aptamers display some drawbacks that continue to hinder their applications. Several strategies have been undertaken to overcome these weaknesses, using thrombin binding aptamers as proof-of-concept. In particular, the functionalization of a thrombin exosite I binding aptamer (TBA) with aromatic moieties, e.g., naphthalene dimides (N) and dialkoxynaphthalenes (D), attached at the 5' and 3' ends, respectively, proved to be highly promising. To obtain a molecular view of the effects of these modifications on aptamers, we performed a crystallographic analysis of one of these engineered oligonucleotides (TBA-NNp/DDp) in complex with thrombin. Surprisingly, three of the four examined crystallographic structures are ternary complexes in which thrombin binds a TBA-NNp/DDp molecule at exosite II as well as at exosite I, highlighting the ability of this aptamer, differently from unmodified TBA, to also recognize a localized region of exosite II. This novel ability is strictly related to the solvophobic behavior of the terminal modifications. Studies were also performed in solution to examine the properties of TBA-NNp/DDp in a crystal-free environment. The present results throw new light on the importance of appendages inducing a -cyclic charge-transfer structure in nucleic acid-based ligands to improve the interactions with proteins, thus considerably widening their potentialities.
尽管寡核苷酸适配体具有毋庸置疑的特性，但它们仍存在一些缺点，这些缺点继续阻碍着它们的应用。已经采取了几种策略来克服这些弱点，以凝血酶结合适配体作为概念验证。特别是，分别在5'和3'末端连接芳香基团，如萘二酰亚胺（N）和二烷氧基萘（D）的凝血酶外位点I结合适配体（TBA）的功能化，被证明是非常有前景的。为了从分子层面了解这些修饰对适配体的影响，我们对其中一种与凝血酶复合的工程化寡核苷酸（TBA-NNp/DDp）进行了晶体学分析。令人惊讶的是，所研究的四个晶体结构中的三个是三元复合物，其中凝血酶在外位点II以及外位点I结合一个TBA-NNp/DDp分子，这突出了这种适配体与未修饰的TBA不同，还能够识别外位点II的一个局部区域的能力。这种新能力与末端修饰的疏溶剂行为密切相关。还在溶液中进行了研究，以检查TBA-NNp/DDp在无晶体环境中的性质。目前的结果为附属物在基于核酸的配体中诱导α-环状电荷转移结构以改善与蛋白质的相互作用的重要性提供了新的线索，从而大大拓宽了它们的潜力。
