具有增强蛋白质结合亲和力的末端炔烃修饰 DNA 适体,ACS Chemical Biology

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具有增强蛋白质结合亲和力的末端炔烃修饰 DNA 适体
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2023-08-02 , DOI: 10.1021/acschembio.3c00183
Eric M Kohn ₁ , Kirill Konovalov _{1,

2} , Christian A Gomez ₁ , Gillian N Hoover ₁ , Andrew Kai-Hei Yik _{1,

2} , Xuhui Huang _{1,

2} , Jeffrey D Martell _{1,

3}

Affiliation

基于核酸的受体（称为适体）的发现和制造速度相对较快，但缺乏蛋白质受体（例如抗体）的多种功能组。DNA适体的结合特性可以通过附加非生物官能团来增强；例如，萘等芳香族基团会缓慢地从蛋白质中解离。尽管末端炔烃是一种富含π电子的官能团，已被用于小分子药物中以通过非共价相互作用增强与蛋白质的结合，但它在增强DNA适体结合亲和力方面仍未得到探索。在这里，我们展示了末端炔烃在改善 DNA 与蛋白质结合方面的效用。我们制备了一个包含 256 个带有末端炔烃的 HD22 变体的文库，HD22 是一种以纳摩尔亲和力结合蛋白质凝血酶的 DNA 适体。经过一步凝血酶结合选择后，发现了一种含有两个炔烃的高亲和力适体，其凝血酶结合比相应的未修饰序列紧密3.2倍。更紧密的结合归因于与凝血酶的解离速率较慢（比 HD22 慢 5.2 倍）。通过溶质回火复制交换 (REST2) 增强采样的分子动力学模拟表明，富含 π 电子的炔与凝血酶上的天冬酰胺侧链 N-H 基团相互作用，形成稳定适体-蛋白质界面的非共价相互作用。总体而言，这项工作代表了末端炔烃增强适体结合特性的第一个案例，并强调了末端炔烃作为原子经济的富含π电子的官能团的效用，以最小的空间扰动增强结合亲和力。

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Terminal Alkyne-Modified DNA Aptamers with Enhanced Protein Binding Affinities

Nucleic acid-based receptors, known as aptamers, are relatively fast to discover and manufacture but lack the diverse functional groups of protein receptors (e.g., antibodies). The binding properties of DNA aptamers can be enhanced by attaching abiotic functional groups; for example, aromatic groups such as naphthalene slow dissociation from proteins. Although the terminal alkyne is a π-electron-rich functional group that has been used in small molecule drugs to enhance binding to proteins through noncovalent interactions, it remains unexplored for enhancing DNA aptamer binding affinity. Here, we demonstrate the utility of the terminal alkyne for improving the binding of DNA to proteins. We prepared a library of 256 terminal-alkyne-bearing variants of HD22, a DNA aptamer that binds the protein thrombin with nanomolar affinity. After a one-step thrombin-binding selection, a high-affinity aptamer containing two alkynes was discovered, exhibiting 3.2-fold tighter thrombin binding than the corresponding unmodified sequence. The tighter binding was attributable to a slower rate of dissociation from thrombin (5.2-fold slower than HD22). Molecular dynamics simulations with enhanced sampling by Replica Exchange with Solute Tempering (REST2) suggest that the π-electron-rich alkyne interacts with an asparagine side chain N–H group on thrombin, forming a noncovalent interaction that stabilizes the aptamer–protein interface. Overall, this work represents the first case of terminal alkynes enhancing the binding properties of an aptamer and underscores the utility of the terminal alkyne as an atom economical π-electron-rich functional group to enhance binding affinity with minimal steric perturbation.

更新日期：2023-08-02

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