Staggered Target SELEX, a novel approach to isolate non-cross-reactive aptamer for detection of SEA by apta-qPCR
Staggered Target SELEX, a novel approach to isolate non-cross-reactive aptamer for detection of SEA by apta-qPCR
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DOI：
10.1016/j.jbiotec.2018.09.006
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发表时间：
2018-11-20
影响因子：
4.1
通讯作者：
Fooladi, Abbas Ali Imani
中科院分区：
文献类型：
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作者：
Sedighian, Hamid;Halabian, Raheleh;Fooladi, Abbas Ali Imani
背景和目的：适体或化学抗体是能够以高特异性和亲和力结合各种靶点的寡核苷酸（DNA或RNA），例如通过称为SELEX的体外方法分离的毒素。迄今为止，有许多用于分离针对蛋白质的新型适体的SELEX方法。然而，并非所有修饰的SELEX都适用于基于序列同源性的类似蛋白质，例如葡萄球菌肠毒素。A型葡萄球菌肠毒素（SEA）是世界范围内引起葡萄球菌食物中毒（SFP）的最常见毒素。SEA与葡萄球菌D型肠毒素（SED）和E型肠毒素（SEE）的同源性分别为50%和83%。本研究以SEA为模型蛋白，采用交错靶点选择性扩增（Staggered Target SELEX，ST-SELEX）技术，从SEA中分离出无交叉反应的特异性核酸适体，应用apta-qPCR技术对SEA进行检测。用SEE对SEA蛋白进行计算机分析，找到SEA蛋白表面的特异区域后，分两步进行ST-SELEX（经典SELEX和第二步SELEX）。最后，在分离高特异性适体后，apta-qPCR用于SEA的检测。该技术将抗SEA多克隆抗体固定在G蛋白琼脂糖凝胶珠上。然后，用多克隆抗体捕获SEA蛋白作为固定在琼脂糖凝胶珠上的靶标。将分离的适配子结合在Ab-PGs捕获的SEA蛋白表面。最后，通过qPCR扩增热释放的适配子。结果：我们的研究表明，通过基于ST-SELEX程序的十轮筛选过程，在体外产生了解离常数（K-D）值为7.44 +/- 0.6 nM，检测限（LOD）为146.67 fM的适配子。与其他SELEX方法相比，ST-SELEX的优势是选择针对两种或多种具有高序列同源性的蛋白质的特异性非交叉反应性适体。这些适体可用于灵敏的检测方法，如apta-qPCR。
Background and objective: Aptamers or chemical antibodies are oligonucleotides (DNA or RNA) that are able to bind to various targets with high specificity and affinity such as toxins which are isolated by an in vitro method known as SELEX. To date, there are many SELEX procedures for the isolation of novel aptamers against proteins. However not all modified SELEX are suitable for similar protein based on sequence homology such as staphylococcal enterotoxins. Staphylococcal enterotoxin type A (SEA) is the most prevalent toxin involved in staphylococcal food poisoning (SFP) worldwide. SEA is homologous to Staphylococcal enterotoxin type D (SED) and Staphylococcal enterotoxin type E (SEE) about 50% and 83%, respectively. Here, we have developed Staggered Target SELEX (ST-SELEX) as a novel designed SELEX procedure to acquire specific non-cross-reactive aptamers against SEA as a model protein.Methods: In this study, isolated ssDNA aptamers by ST-SELEX were used for detection of SEA via apta-Real time PCR (apta-qPCR). After in silico analysis of SEA protein with SEE and finding the specific region on the surface of protein, ST-SELEX was carried out in two steps (classical SELEX and Second SELEX). Finally, after isolating high specific aptamers, the apta-qPCR was used for the detection of the SEA. In this technique, poly-clonal antibody against SEA was immobilized on protein G sepharose beads (Ab-PGs). Then, the SEA protein was captured by poly clonal antibody as the target that immobilized on sepharose beads. The isolated aptamers were bound on the surface of SEA protein that captured by Ab-PGs. Finally, the heat-released aptamers were amplified by qPCR.Result: Our investigation showed that the aptamers were generated in vitro by a ten-round selection process based on ST-SELEX procedure with dissociation constant (K-D) value 7.44 +/- 0.6 nM and limit of detection (LOD) of 146.67 fM.Discussion and conclusion: The advantage of ST-SELEX compared to other SELEX methods was to select a specific non cross-reactive aptamer against two or more proteins with high sequence homology. These aptamers can be used in sensitive detection methods such as apta-qPCR.