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首页> 外文期刊>RSC Advances >In silico post-SELEX screening and experimental characterizations for acquisition of high affinity DNA aptamers against carcinoembryonic antigen
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In silico post-SELEX screening and experimental characterizations for acquisition of high affinity DNA aptamers against carcinoembryonic antigen

机译：在计算机上进行SELEX筛选并获得针对癌胚抗原的高亲和力DNA适体的实验表征

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DNA aptamers against carcinoembryonic antigen (CEA) have been identified through the systematic evolution of ligands by exponential enrichment (SELEX) technique, but their affinity needs to be improved. In this study, an in silico approach was firstly used to screen the mutation sequences of a reported DNA aptamer (the parent aptamer, denoted as P) against CEA. The affinities of several high-score DNA mutants were determined by the biolayer interferometry technique. Finally, the newly obtained aptamers were verified in an aptasensor application. For the in silico approach, Mfold and RNA Composer were combined to generate the 3D RNA structures of the DNA mutants. The RNA structures were then modified to 3D DNA structures with the Write program. The docking model and binding ability of the 3D DNA structures with CEA were simulated and predicted with the ZDOCK program. Two mutation sequences (P-ATG and GAC-P) exhibited significantly higher ZDOCK scores than P. The dissociation constant of P-ATG and GAC-P to CEA was determined to be 4.62 and 3.93 nM respectively, obviously superior to that of P (6.95 nM). The detection limit of the P-ATG and GAC-P based aptasensors was 1.5 and 1.2 ng mL ~(?1) , respectively, markedly better than that based on P (3.4 ng mL ~(?1) ). The consistency between the in silico and the experimental results indicates that the developed in silico post-SELEX screening approach is feasible for improving DNA aptamers. The P-ATG and GAC-P aptamers found in this study could be used for future CEA aptasensor design and fabrication, promisingly applicable for highly sensitive CEA detection and early cancer diagnosis.
机译：已通过指数富集（SELEX）技术对配体进行系统进化，确定了针对癌胚抗原（CEA）的DNA适体，但需要提高其亲和力。在这项研究中，首先采用计算机方法来筛选报道的针对CEA的DNA适体（母体适体，表示为P）的突变序列。通过生物层干涉技术确定了几个高分DNA突变体的亲和力。最后，在aptasensor应用程序中验证了新获得的适体。对于计算机方法，将Mfold和RNA Composer组合在一起以生成DNA突变体的3D RNA结构。然后使用Write程序将RNA结构修改为3D DNA结构。使用ZDOCK程序模拟和预测了3D DNA结构与CEA的对接模型和结合能力。两个突变序列（P-ATG和GAC-P）的ZDOCK得分明显高于P.P-ATG和GAC-P与CEA的解离常数分别为4.62和3.93 nM，明显优于P（ 6.95 nM）。基于P-ATG和GAC-P的适体传感器的检出限分别为1.5和1.2 ng mL〜（？1），明显优于基于P的（3.4 ng mL〜（？1））。计算机和实验结果之间的一致性表明，开发的计算机后SELEX筛选方法对于改善DNA适体是可行的。在这项研究中发现的P-ATG和GAC-P适体可用于将来的CEA适体传感器的设计和制造，有望应用于高度敏感的CEA检测和早期癌症诊断。

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