Département de Pharmacochimie Moléculaire UMR 5063, Institut de Chimie Moléculaire de Grenoble FR 2607, CNRS-Université Grenoble I (Joseph Fourier), 38041 Grenoble cedex 9, France.
Anal Chem. 2012 Aug 21;84(16):7203-11. doi: 10.1021/ac301552e. Epub 2012 Aug 1.
Here, we describe a new fluorescence polarization aptamer assay (FPAA) strategy which is based on the use of the single-stranded DNA binding (SSB) protein from Escherichia coli as a strong FP signal enhancer tool. This approach relied on the unique ability of the SSB protein to bind the nucleic acid aptamer in its free state but not in its target-bound folded one. Such a feature was exploited by using the antiadenosine (Ade)-DNA aptamer (Apt-A) as a model functional nucleic acid. Two fluorophores (fluorescein and Texas Red) were introduced into different sites of Apt-A to design a dozen fluorescent tracers. In the absence of the Ade target, the binding of the labeled aptamers to SSB governed a very high fluorescence anisotropy increase (in the 0.130-0.200 range) as the consequence of (i) the large global diffusion difference between the free and SSB-bound tracers and (ii) the restricted movement of the dye in the SSB-bound state. When the analyte was introduced into the reaction system, the formation of the folded tertiary structure of the Ade-Apt-A complex triggered the release of the labeled nucleic acids from the protein, leading to a strong decrease in the fluorescence anisotropy. The key factors involved in the fluorescence anisotropy change were considered through the development of a competitive displacement model, and the optimal tracer candidate was selected for the Ade assay under buffer and realistic (diluted human serum) conditions. The SSB-assisted principle was found to operate also with another aptamer system, i.e., the antiargininamide DNA aptamer, and a different biosensing configuration, i.e., the sandwich-like design, suggesting the broad usefulness of the present approach. This sensing platform allowed generation of a fluorescence anisotropy signal for aptamer probes which did not operate under the direct format and greatly improved the assay response relative to that of the most previously reported small target FPAA.
在这里，我们描述了一种新的荧光偏振适体分析（FPAA）策略，该策略基于使用来自大肠杆菌的单链 DNA 结合（SSB）蛋白作为一种强大的 FP 信号增强工具。这种方法依赖于 SSB 蛋白结合游离状态下的核酸适体而不是其靶标结合折叠状态下的核酸适体的独特能力。这种特性通过使用抗腺嘌呤（Ade）-DNA 适体（Apt-A）作为模型功能核酸来利用。将两个荧光团（荧光素和 Texas Red）引入 Apt-A 的不同位置，设计了十几个荧光示踪剂。在没有 Ade 靶标的情况下，标记适体与 SSB 的结合导致非常高的荧光各向异性增加（在 0.130-0.200 范围内），这是由于（i）自由和 SSB 结合示踪剂之间的大全局扩散差异，以及（ii）在 SSB 结合状态下染料的受限运动。当分析物被引入反应体系中时，折叠的 Ade-Apt-A 复合物的形成触发标记的核酸从蛋白质中释放，导致荧光各向异性强烈降低。通过开发竞争置换模型考虑了涉及荧光各向异性变化的关键因素，并选择了最佳示踪剂候选物用于在缓冲液和实际（稀释人血清）条件下进行 Ade 测定。发现 SSB 辅助原理也适用于另一种适体系统，即抗精氨酸酰胺 DNA 适体，以及另一种生物传感配置，即夹心样设计，表明本方法具有广泛的用途。该传感平台允许为不适于直接格式的适体探针产生荧光各向异性信号，并相对于先前报道的最小目标 FPAA 大大提高了测定的响应。
