DNA scissors device used to measure MutS binding to DNA mis-pairs.
DNA scissors device used to measure MutS binding to DNA mis-pairs.
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DOI：
10.1021/ja910188p
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发表时间：
2010-03-31
影响因子：
15
通讯作者：
Seeman, Nadrian C.
中科院分区：
文献类型：
--
作者：
Gu, Hongzhou;Yang, Wei;Seeman, Nadrian C.
MutS是一种DNA修复蛋白，可以识别未配对和凸出的碱基。当它与DNA结合时，它会弯曲双螺旋。我们开发了一种新的基于DNA的纳米机械装置，可以测量DNA弯曲蛋白质与双螺旋结合时所能做的工作量。我们在这里报告的设备是一个剪刀状的设备组成的两个双交叉（DX）分子相互连接的灵活霍利迪结。这两个DX组件通过包含MutS结合位点的双螺旋连接;当结合位点双链体弯曲时，剪刀收缩。两个DX分子也通过与结合位点相邻的边缘上的粘性末端连接;如果蛋白质以足够的自由能结合，则粘性末端可以被破坏。这些粘性末端两侧是一对染料;当粘性末端被破坏时，染料分离，荧光共振能量转移信号可以监测破坏。粘性末端的强度很容易变化，因此可以定量蛋白质破坏它们的能力。我们使用该装置与测量蛋白质结合产生的弯曲角度的第二装置一起测量功，以便校准系统。我们的数据与以前的MutS结合的测量结果非常一致，表明该设备能够正确测量结合的强度。
MutS is a DNA repair protein that recognizes unpaired and bulged bases. When it binds to DNA it bends the double helix. We have developed a novel DNA-based nanomechanical device that measures the amount of work that a DNA-bending protein can do when it binds to the double helix. The device we report here is a scissors-like device consisting of two double-crossover (DX) molecules connected to each other by a flexible Holliday junction. The two DX components are connected by a double helix that contains the binding site for MutS; when the binding site duplex is bent, the scissors contracts. The two DX molecules are also joined by sticky ends on an edge adjacent to the binding site; the sticky ends can be disrupted if the protein binds with sufficient free energy. Those sticky ends are flanked by a pair of dyes; when the sticky ends are disrupted, the dyes separate, and the fluorescence resonance energy transfer signal can monitor the disruption. The strength of the sticky ends is readily varied, so that the ability of the protein to disrupt them can be quantitated. We use this device to measure work in conjunction with a second device that measures the bending angle resulting from protein binding, so as to calibrate the system. Our data are in good agreement with previous measurements of MutS binding, indicating that this device is able to measure the strength of binding correctly.
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