Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, Utah 84112-0850, United States.
Anal Chem. 2023 Nov 7;95(44):16160-16168. doi: 10.1021/acs.analchem.3c02751. Epub 2023 Oct 23.
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 their response 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.
DNA 适体的发现使得生物传感领域有了重大创新。适体形成二级结构，与它们的结合伙伴表现出选择性的高亲和力相互作用。适体与靶标的结合通常伴随着构象变化，适体的传感通常依赖于这些变化，从外在标记物提供读出信号来检测靶标结合。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响应的方法。为了解决这一挑战，我们开发了一种结构信息丰富的拉曼光谱法，以确定固定在多孔硅表面的 15 -mer 凝血酶结合适体（TBA）的构象。TBA 之所以有趣，是因为它与 α-凝血酶的结合依赖于适体形成反平行 G-四链体，这被认为驱动了允许检测凝血酶结合的信号变化。然而，即使在没有其蛋白质靶标的情况下，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 TBA 的 G-四链体与展开态的影响。然后，我们在 K 或 Li 溶液中检测α-凝血酶与固定化适体的结合，这种结合可以从结合的蛋白质的拉曼散射中观察到。结果表明，在 K 溶液中，α-凝血酶的结合不会导致适体构象发生可检测的变化，在结合其靶标之前和之后，适体都以 G-四链体的形式存在。然而，在 Li 溶液中，TBA 在与α-凝血酶结合之前是展开的，蛋白质结合伴随着适体形成 G-四链体。
