Department of Physics, Arizona State University, Tempe, Arizona, USA.
Biophys J. 2012 May 16;102(10):2381-90. doi: 10.1016/j.bpj.2012.04.006. Epub 2012 May 15.
Electron-tunneling data suggest that a noncovalently-bonded complex of three molecules, two recognition molecules that present hydrogen-bond donor and acceptor sites via a carboxamide group, and a DNA base, remains bound for seconds. This is surprising, given that imino-proton exchange rates show that basepairs in a DNA double helix open on millisecond timescales. The long lifetime of the three-molecule complex was confirmed using force spectroscopy, but measurements on DNA basepairs are required to establish a comparison with the proton-exchange data. Here, we report on a dynamic force spectroscopy study of complexes between the bases adenine and thymine (A-T, two-hydrogen bonds) and 2-aminoadenine and thymine (2AA-T, three-hydrogen bonds). Bases were tethered to an AFM probe and mica substrate via long, covalently linked polymer tethers. Data for bond-survival probability versus force and the rupture-force distributions were well fitted by the Bell model. The resulting lifetime of the complexes at zero pulling force was ~2 s for two-hydrogen bonds (A-T) and ~4 s for three-hydrogen bonds (2AA-T). Thus, DNA basepairs in an AFM pulling experiment remain bonded for long times, even without the stabilizing influence of base-stacking in a double helix. This result suggests that the pathways for opening, and perhaps the open states themselves, are very different in the AFM and proton-exchange measurements.
电子隧道数据表明，三个分子的非共价键复合物，两个识别分子通过酰胺基团提供氢键供体和受体位点，以及一个 DNA 碱基，保持结合状态数秒。这令人惊讶，因为亚氨基质子交换率表明 DNA 双螺旋中的碱基对在毫秒时间尺度上打开。使用力谱法证实了三分子复合物的长寿命，但需要对 DNA 碱基对进行测量，以与质子交换数据进行比较。在这里，我们报告了腺嘌呤和胸腺嘧啶（A-T，两个氢键）与 2-氨基腺嘌呤和胸腺嘧啶（2AA-T，三个氢键）之间复合物的动态力谱研究。碱基通过长的、共价连接的聚合物键合到 AFM 探针和云母基底上。键存活概率与力的关系以及断裂力分布的数据通过 Bell 模型得到很好的拟合。在零拉力下，复合物的寿命约为 2 秒（A-T，两个氢键）和约 4 秒（2AA-T，三个氢键）。因此，即使没有双链中碱基堆积的稳定影响，在 AFM 拉伸实验中 DNA 碱基对也能保持长时间的结合。这一结果表明，在 AFM 和质子交换测量中，打开的途径，也许还有开放状态本身，非常不同。
