Monitoring multiple distances within a single molecule using switchable FRET
Monitoring multiple distances within a single molecule using switchable FRET
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DOI：
10.1038/nmeth.1502
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发表时间：
2010-10-01
期刊：
影响因子：
48
通讯作者：
Kapanidis, Achillefs N.
中科院分区：
文献类型：
--
作者：
Uphoff, Stephan;Holden, Seamus J.;Kapanidis, Achillefs N.
生物分子的结构和动力学分析对于理解其功能具有重要意义。为此，我们介绍了一种方法称为“切换FRET”，它结合了单分子荧光共振能量转移（FRET）与可逆的光开关的荧光团。通常，单分子FRET在单个供体-受体对内测量，并且仅报告一个距离。虽然已经开发了监测多个距离的多对FRET方法，但它们在技术上具有挑战性并且难以扩展，主要是因为它们依赖于光谱上不同的受体。相比之下，可切换FRET顺序探测FRET之间的一个单一的供体和光谱相同的光开关受体，大大降低了实验和分析的复杂性，并能够直接监测多个距离。我们对DNA分子，蛋白质-DNA复合物和动态霍利迪路口的实验证明了可切换FRET研究动态，多组分生物分子的潜力。
The analysis of structure and dynamics of biomolecules is important for understanding their function. Toward this aim, we introduce a method called 'switchable FRET', which combines single-molecule fluorescence resonance energy transfer (FRET) with reversible photoswitching of fluorophores. Typically, single-molecule FRET is measured within a single donor-acceptor pair and reports on only one distance. Although multipair FRET approaches that monitor multiple distances have been developed, they are technically challenging and difficult to extend, mainly because of their reliance on spectrally distinct acceptors. In contrast, switchable FRET sequentially probes FRET between a single donor and spectrally identical photoswitchable acceptors, dramatically reducing the experimental and analytical complexity and enabling direct monitoring of multiple distances. Our experiments on DNA molecules, a protein-DNA complex and dynamic Holliday junctions demonstrate the potential of switchable FRET for studying dynamic, multicomponent biomolecules.