Development of simultaneous screening methods of aptamers which specifically recognize marker proteins for diseases

我们开发了基于靶特异性的适体筛选方法，利用竞争蛋白筛选适体。由于传统的SELEX方法仅根据适配体的亲和力筛选适配体，因此难以根据特异性筛选适配体。在我们的选择方法中，文库与目标蛋白和其他蛋白作为竞争对手同时孵育。我们用适体印迹法对筛选的文库进行评估，该方法可以可视化寡核苷酸与蛋白质的结合。在本研究中，我们通过这种方法仅经过几轮筛选就鉴定出了两个针对Tau蛋白和荧光素酶的DNA适体。所分离的适体对目标蛋白具有高亲和力，不与竞争蛋白结合。这些结果表明，我们的选择方法可能有助于有效地选择针对目标蛋白的特异性适配体。此外，我们应用该方法筛选与靶组织中特定未表征蛋白结合的适体，而无需纯化。常规的组织蛋白体外筛选方法不能应用于非特征蛋白的筛选，或者在筛选过程中不确定是否获得靶蛋白的适体。我们通过可视化寡核苷酸与靶组织中每个蛋白的结合，并从检测到的条带中分离结合的寡核苷酸，开发了靶组织中未表征蛋白的适体选择方法。这种方法将使我们能够选择靶组织中任何蛋白质的适体。因此，我们相信这种方法将有助于选择适体来分析未表征的蛋白质。

We have developed the screening method of aptamer based on target specificity using protein competitors. It is difficult to screen aptamers according to specificity by conventional SELEX, since such methods screen aptamers only based on their affinity. In our selection method, the library is simultaneously incubated with the target protein and other proteins used as competitors. We evaluated the screened library with the aptamer blotting method, which visualizes the binding of oligonucleotides to proteins. In this study, we identified two DNA aptamers against Tau protein and luciferase after only several rounds of selection by this method. The isolated aptamers had high affinity for the target protein, and did not bind to the competitor protein. These results suggest that our selection method may be useful for efficiently selecting specific aptamers against target proteins.Moreover, we applied this method to screen the aptamers that bind to a specific uncharacterized protein in target tissue without purification. The conventional in vitro selection methods against proteins in tissue cannot be applied to the selection against uncharacterized proteins or it is not certain whether aptamers to target proteins are obtained during selection. We developed the selection method of aptamers to an uncharacterized protein in target tissue by visualizing the bindings of oligonucleotide to each protein in target tissue and isolating bound oligonucleotides from detected bands. This method would enable us to select aptamers to any protein in target tissue. We therefore believe that this method would be useful in selecting aptamers for analyses of uncharacterized proteins.