We have used magnetization transfer NMR experiments to measure the exchange rate constant (kex) of the imino protons in the unbound, cocaine-bound, and quinine-bound forms of the cocaine-binding DNA aptamer. Both long-stem 1 (MN4) and short-stem 1 (MN19) variants were analyzed, corresponding to structures with a prefolded secondary structure and ligand-induced-folding versions of this aptamer, respectively. The kex values were measured as a function of temperature from 5 to 45°C to determine the thermodynamics of the base pair opening for MN4. We find that the base pairs close to the ligand-binding site become stronger upon ligand binding, whereas those located away from the binding site do not strengthen. With the buffer conditions used in this study, we observe imino 1H signals in MN19 not previously seen, which leads us to conclude that in the free form, both stem 2 and parts of stem 3 are formed and that the base pairs in stem 1 become structured or more rigid upon binding. This is consistent with the kex values for MN19 decreasing in both stem 1 and at the ligand-binding site. Based on the temperature dependence of the kex values, we find that MN19 is more dynamic than MN4 in the free and both ligand-bound forms. For MN4, ligand-binding results in the reduction of dynamics that are localized to the binding site. These results demonstrate that an aptamer in which the base pairs are preformed also experiences a reduction in dynamics with ligand binding.

中文翻译：

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可卡因结合适体结合配体时碱基对开放动力学的降低

我们使用磁化转移 NMR 实验来测量未结合、可卡因结合和奎宁结合形式的可卡因结合 DNA 适体中亚氨基质子的交换率常数 (kex)。分析了长茎 1 (MN4) 和短茎 1 (MN19) 变体，分别对应于该适体具有预折叠二级结构和配体诱导折叠版本的结构。测量 kex 值作为 5 至 45°C 温度的函数，以确定 MN4 碱基对开口的热力学。我们发现靠近配体结合位点的碱基对在配体结合时变得更强，而远离结合位点的碱基对不会增强。使用本研究中使用的缓冲条件，我们观察到以前未见的 MN19 中的亚氨基 1H 信号，这使我们得出结论，在自由形式中，茎 2 和茎 3 的部分都形成了，并且茎 1 中的碱基对在结合时变得结构化或更刚性。这与 MN19 的 kex 值在茎 1 和配体结合位点降低一致。基于 kex 值的温度依赖性，我们发现 MN19 在游离形式和两种配体结合形式中都比 MN4 更具动态性。对于 MN4，配体结合导致定位于结合位点的动力学减少。这些结果表明，预先形成碱基对的适体也经历了配体结合动力学的降低。这与 MN19 的 kex 值在茎 1 和配体结合位点降低一致。基于 kex 值的温度依赖性，我们发现 MN19 在游离形式和两种配体结合形式中都比 MN4 更具动态性。对于 MN4，配体结合导致定位于结合位点的动力学减少。这些结果表明，预先形成碱基对的适体也经历了配体结合动力学的降低。这与 MN19 的 kex 值在茎 1 和配体结合位点降低一致。基于 kex 值的温度依赖性，我们发现 MN19 在游离形式和两种配体结合形式中都比 MN4 更具动态性。对于 MN4，配体结合导致定位于结合位点的动力学减少。这些结果表明，预先形成碱基对的适体也经历了配体结合动力学的降低。