In silico selection of an aptamer to estrogen receptor alpha using computational docking employing estrogen response elements as aptamer-alike molecules.
In silico selection of an aptamer to estrogen receptor alpha using computational docking employing estrogen response elements as aptamer-alike molecules.
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DOI：
10.1038/srep21285
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发表时间：
2016-02-22
影响因子：
4.6
通讯作者：
Nahar P
中科院分区：
文献类型：
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作者：
Ahirwar R;Nahar S;Aggarwal S;Ramachandran S;Maiti S;Nahar P
核酸适体是传统抗体的化学抗体替代品，主要是通过SELEX技术进行多轮选择和富集发现的。绕过传统的方法，在这里，我们报告了一种利用人类雌激素反应元件（EREs）的RNA类似物对雌激素受体α (ERα)的适体的硅选择。ERE的反向重复性质和形成稳定发夹的能力被用作获得适体相似序列的标准。对选定的单链EREs的近天然RNA类似物进行建模，并使用AutoDock Vina、HADDOCK和PatchDock对接检测它们作为ERα适体出现的可能性。通过等温滴定量热法测量ERα -RNA相互作用的热力学参数，验证了这些计算机预测。基于实验和体外结果，我们选择了一个结合常数（Ka）为1.02±0.1 × 108 M−1的候选RNA （ERaptR4; 5’-GGGGUCAAGGUGACCCC-3’）作为er α-适配体。通过细胞化学和固相免疫分析证实了所选ERaptR4适体的靶特异性。此外，稳定性分析发现ERaptR4在ERα存在时对血清和RNase A降解具有抗性。综上所述，一种高效的ERα-RNA适体是使用非selex适体选择程序鉴定的。具有高亲和力和特异性，可用于乳腺癌及相关疾病的ERα检测。
Aptamers, the chemical-antibody substitute to conventional antibodies, are primarily discovered through SELEX technology involving multi-round selections and enrichment. Circumventing conventional methodology, here we report an in silico selection of aptamers to estrogen receptor alpha (ERα) using RNA analogs of human estrogen response elements (EREs). The inverted repeat nature of ERE and the ability to form stable hairpins were used as criteria to obtain aptamer-alike sequences. Near-native RNA analogs of selected single stranded EREs were modelled and their likelihood to emerge as ERα aptamer was examined using AutoDock Vina, HADDOCK and PatchDock docking. These in silico predictions were validated by measuring the thermodynamic parameters of ERα -RNA interactions using isothermal titration calorimetry. Based on the in silico and in vitro results, we selected a candidate RNA (ERaptR4; 5′-GGGGUCAAGGUGACCCC-3′) having a binding constant (Ka) of 1.02 ± 0.1 × 108 M−1 as an ERα-aptamer. Target-specificity of the selected ERaptR4 aptamer was confirmed through cytochemistry and solid-phase immunoassays. Furthermore, stability analyses identified ERaptR4 resistant to serum and RNase A degradation in presence of ERα. Taken together, an efficient ERα-RNA aptamer is identified using a non-SELEX procedure of aptamer selection. The high-affinity and specificity can be utilized in detection of ERα in breast cancer and related diseases.