Raman Scattering Reveals Ion-Dependent G-Quadruplex Formation in the 15-mer Thrombin-Binding Aptamer upon Association with α-Thrombin
Raman Scattering Reveals Ion-Dependent G-Quadruplex Formation in the 15-mer Thrombin-Binding Aptamer upon Association with α-Thrombin
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DOI：
10.1021/acs.analchem.3c02751
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发表时间：
2023-10-23
影响因子：
7.4
通讯作者：
Harris，Joel M.
中科院分区：
文献类型：
--
作者：
Myres，Grant J.;Kitt，Jay P.;Harris，Joel M.
结合生物分子靶标的 DNA 适体的发现推动了生物传感领域的重大创新。适体形成二级结构，与其结合伙伴表现出选择性的高亲和力相互作用。适体与其靶标的结合通常伴随着构象变化，并且适体的传感通常依赖于这些变化来提供来自外在标签的读出信号以检测靶标关联。许多生物传感应用涉及固定在表面的适体，但缺乏表征固定适体构象及其原位响应的方法。为了应对这一挑战，我们开发了一种结构信息丰富的拉曼光谱方法来确定固定在多孔二氧化硅表面上的 15 聚体凝血酶结合适体 (TBA) 的构象。 TBA 之所以令人感兴趣，是因为它与 α-凝血酶的结合取决于形成反平行 G-四联体的适体，这被认为驱动信号变化，从而允许检测到凝血酶结合。然而，即使在没有其蛋白质靶点的情况下，特定的金属阳离子也能稳定适体的 G-四链体构象。为了更深入地了解 TBA 的构象响应，我们利用拉曼光谱来量化金属阳离子 K+（稳定）和 Li+（非稳定）对 G-四链体与 TBA 未折叠群体的影响。然后，在 K+ 或 Li+ 溶液中，我们检测 α-凝血酶与固定适体的关联，这可以在结合蛋白的拉曼散射中观察到。结果表明，K+ 溶液中 α-凝血酶的缔合不会产生可检测到的适体构象变化，而在结合其靶标之前和之后，均以 G-四链体形式发现适体构象的变化。然而，在 Li+ 溶液中，TBA 在 α-凝血酶缔合之前展开，蛋白质结合发生在适体形成 G-四链体时。
The discovery of DNA aptamers that bind biomolecular targets has enabled significant innovations in biosensing. Aptamers form secondary structures that exhibit selective high-affinity interactions with their binding partners. The binding of its target by an aptamer is often accompanied by conformational changes, and sensing by aptamers often relies on these changes to provide readout signals from extrinsic labels to detect target association. Many biosensing applications involve aptamers immobilized to surfaces, but methods to characterize conformations of immobilized aptamers and theirin situresponse have been lacking. To address this challenge, we have developed a structurally informative Raman spectroscopy method to determine conformations of the 15-mer thrombin-binding aptamer (TBA) immobilized on porous silica surfaces. The TBA is of interest because its binding of α-thrombin depends on the aptamer forming an antiparallel G-quadruplex, which is thought to drive signal changes that allow thrombin-binding to be detected. However, specific metal cations also stabilize the G-quadruplex conformation of the aptamer, even in the absence of its protein target. To develop a deeper understanding of the conformational response of the TBA, we utilize Raman spectroscopy to quantify the effects of the metal cations, K+(stabilizing) and Li+(nonstabilizing), on G-quadruplex versus unfolded populations of the TBA. In K+or Li+solutions, we then detect the association of α-thrombin with the immobilized aptamer, which can be observed in Raman scattering from the bound protein. The results show that the association of α-thrombin in K+solutions produces no detectable change in aptamer conformation, which is found in the G-quadruplex form both before and after binding its target. In Li+solutions, however, where the TBA is unfolded prior to α-thrombin association, protein binding occurs with the formation of a G-quadruplex by the aptamer.