Long Lifetime of Hydrogen-Bonded DNA Basepairs by Force Spectroscopy
Long Lifetime of Hydrogen-Bonded DNA Basepairs by Force Spectroscopy
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DOI：
10.1016/j.bpj.2012.04.006
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发表时间：
2012-05-16
影响因子：
3.4
通讯作者：
Ros, Robert
中科院分区：
文献类型：
--
作者：
Fuhrmann, Alexander;Getfert, Sebastian;Ros, Robert
电子隧道数据表明，由三个分子、两个通过羧酰胺基呈现氢键供体和受体位点的识别分子以及一个 DNA 碱基组成的非共价键复合物，在数秒内保持结合状态。这是令人惊讶的，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。使用力光谱法证实了三分子复合物的长寿命，但需要对 DNA 碱基对进行测量才能与质子交换数据进行比较。在这里，我们报告了碱基腺嘌呤和胸腺嘧啶（A-T，两个氢键）以及 2-氨基腺嘌呤和胸腺嘧啶（2AA-T，三个氢键）之间的复合物的动态力谱研究。碱基通过长的共价连接的聚合物系链连接到 AFM 探针和云母基底上。贝尔模型很好地拟合了键存留概率与力和断裂力分布的数据。在零拉力下，复合物的最终寿命对于二氢键 (A-T) 来说类似于 2 秒，对于三氢键 (2AA-T) 来说类似于 4 秒。因此，即使没有双螺旋中碱基堆积的稳定影响，AFM 拉伸实验中的 DNA 碱基对仍能长时间保持键合状态。这一结果表明，在原子力显微镜和质子交换测量中，打开的途径，也许还有打开态本身，是非常不同的。
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 similar to 2 s for two-hydrogen bonds (A-T) and similar to 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.