Lehrstuhl für Bioelektronik, Physik Department, Technische Universität München, Garching, Germany.
Biophys J. 2013 Sep 3;105(5):1199-207. doi: 10.1016/j.bpj.2013.07.047.
The stability of aptamer-ligand complexes is probed in nanopore-based dynamic force spectroscopy experiments. Specifically, the ATP-binding aptamer is investigated using a backward translocation technique, in which the molecules are initially pulled through an α-hemolysin nanopore from the cis to the trans side of a lipid bilayer membrane, allowed to refold and interact with their target, and then translocated back in the trans-cis direction. From these experiments, the distribution of bound and unbound complexes is determined, which in turn allows determination of the dissociation constant Kd ≈ 0.1 mM of the aptamer and of voltage-dependent unfolding rates. The experiments also reveal differences in binding of the aptamer to AMP, ADP, or ATP ligands. Investigation of an aptamer variant with a stabilized ATP-binding site indicates fast conformational switching of the original aptamer before ATP binding. Nanopore force spectroscopy is also used to study binding of the thrombin-binding aptamer to its target. To detect aptamer-target interactions in this case, the stability of the ligand-free aptamer-containing G-quadruplexes-is tuned via the potassium content of the buffer. Although the presence of thrombin was detected, limitations of the method for aptamers with strong secondary structures and complexes with nanomolar Kd were identified.
在基于纳米孔的动态力谱实验中研究了适体-配体复合物的稳定性。具体来说，使用反向易位技术研究了 ATP 结合适体，在该技术中，分子最初从顺式侧通过α-溶血素纳米孔被拉到脂质双层膜的反式侧，允许它们重新折叠并与靶标相互作用，然后在反式-顺式方向上再次易位。通过这些实验，确定了结合和未结合复合物的分布，这反过来又允许确定适体的解离常数 Kd ≈ 0.1 mM 和电压依赖性展开速率。实验还揭示了适体与 AMP、ADP 或 ATP 配体结合的差异。对具有稳定 ATP 结合位点的适体变体的研究表明，在 ATP 结合之前，原始适体的构象快速切换。纳米孔力谱也用于研究凝血酶结合适体与其靶标的结合。在这种情况下，为了检测适体-靶标相互作用，通过缓冲液中的钾含量来调整无配体的适体含有的 G-四链体的稳定性。尽管检测到了凝血酶的存在，但该方法对于具有强二级结构的适体和具有纳摩尔 Kd 的复合物存在局限性。
