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Probing the Nitroindole-Modified Central Loop of Thrombin Aptamer HD1 as a Recognition Site.
Nucleic Acid Therapeutics ( IF 4 ) Pub Date : 2019-03-13 , DOI: 10.1089/nat.2018.0757
Natalia A Kolganova 1 , Vladimir B Tsvetkov 2, 3, 4 , Igor P Smirnov 2 , Edward N Timofeev 1
Affiliation  

Thrombin-binding aptamer HD1 is a DNA-based thrombin inhibitor that features an antiparallel G-quadruplex (GQ) structure. We recently reported a single-nucleotide G8 to 5-nitroindole (NI) modification of HD1 (N8) that notably improves the anticoagulant properties and binding affinity of the aptamer. Based on molecular modeling and binding studies, it was originally proposed that N8 may acquire the ability to bind thrombin by a modified central loop. To verify this possibility, in this study, we report new variations of the N8 aptamer with intact or damaged TT loops. Anomeric alpha-thymidine was used as a "damaging" residue to disable the primary recognition site of N8. Biophysical characterization of modified aptamers supports the formation of HD1-like antiparallel GQs with varying stability by all studied variants. Binding experiments showed that N8 variants with impaired TT loops lost the ability to bind thrombin, suggesting the primary role of thymines in TT loops for the thrombin-N8 interaction. Aptamer N8α(7/9) bearing NI at position 8 and damaged thymidines 7 and 9 retained thrombin affinity, which was intermediate between N8 and HD1. Fluorescence polarization studies suggest 1:1 stoichiometry for thrombin complexes with either HD1, N8, or N8α(7/9). Further molecular dynamics (MD) study of complexes formed by these three aptamers with thrombin disproves the idea of direct interaction between central loop residues and the protein. Based on MD results, the origin of the NI tuning effect is associated with its ability to promote the formation of compact and rigid structures through hydrophobic interactions with the GQ core and loop thymines.

中文翻译:

探测硝基吲哚修饰的凝血酶适体HD1中央环作为识别位点。

凝血酶结合适体HD1是一种基于DNA的凝血酶抑制剂,具有反平行的G-四链体(GQ)结构。我们最近报道了HD1(N8)的单核苷酸G8到5-硝基吲哚(NI)修饰,可显着改善适体的抗凝血特性和结合亲和力。基于分子建模和结合研究,最初提出N8可以通过修饰的中央环获得结合凝血酶的能力。为了验证这种可能性,在这项研究中,我们报告了具有完整或损坏的TT环的N8适体的新变化。端粒的α-胸腺嘧啶核苷被用作“破坏性”残基来禁用N8的主要识别位点。修饰的适体的生物物理表征支持通过所有研究的变体以不同的稳定性形成类似HD1的反平行GQ。结合实验表明,TT环受损的N8变异体失去了结合凝血酶的能力,表明胸腺嘧啶在TT环中对于凝血酶-N8相互作用的主要作用。适体N8α(7/9)在第8位带有NI,受损的胸苷7和9保留了凝血酶亲和力,介于N8和HD1之间。荧光偏振研究表明,与HD1,N8或N8α(7/9)形成的凝血酶复合物的化学计量比为1:1。由这三个适体与凝血酶形成的复合物的进一步分子动力学(MD)研究证明了中心环残基与蛋白质之间直接相互作用的想法。根据MD结果,NI调节作用的起源与其通过与GQ核心和环胸腺嘧啶的疏水相互作用促进形成致密和刚性结构的能力有关。
更新日期:2019-11-01
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