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

我们利用蛋白质竞争对手建立了基于靶标特异性的适体筛选方法。用传统的SELEX方法很难根据特异性筛选适配子，因为这种方法只根据它们的亲和力来筛选适配子。在我们的选择方法中，文库与目标蛋白和其他用作竞争对手的蛋白同时孵育。我们用适配子印迹法对筛选的文库进行了评估，该方法直观地显示了寡核苷酸与蛋白质的结合。在本研究中，我们仅经过几轮筛选，就鉴定出了两个针对Tau蛋白和荧光素酶的DNA适配子。分离的适配子与目的蛋白有较高的亲和力，不与竞争蛋白结合。这些结果表明，我们的选择方法对于有效地选择针对目标蛋白的特定适配子是有用的。此外，我们应用该方法来筛选与靶组织中特定的未表征蛋白结合的适配子，而无需进行纯化。传统的针对组织中蛋白质的体外选择方法不能用于针对未鉴定的蛋白质的选择，或者在选择过程中不能确定是否获得了与靶蛋白的适体。我们通过可视化寡核苷酸与靶组织中每个蛋白质的结合，并从检测到的条带中分离结合的寡核苷酸，发展了靶组织中未鉴定蛋白质的适体选择方法。这种方法将使我们能够选择目标组织中任何蛋白质的适配子。因此，我们相信这种方法在选择适配子用于分析未鉴定的蛋白质方面是有用的。

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.