Single-molecule fluorescence resonance energy transfer (smFRET) is one of the powerful techniques for deciphering the dynamics of unsynchronized biomolecules. However, smFRET is limited in its temporal resolution for observing dynamics. Here, we report a novel method for observing real-time dynamics with submillisecond resolution by tethering molecules to freely diffusing 100-nm-sized liposomes. The observation time for a diffusing molecule is extended to 100 ms with a submillisecond resolution, which allows for direct analysis of the transition states from the FRET time trace using hidden Markov modelling. We measure transition rates of up to 1,500 s–1 between two conformers of a Holliday junction. The rapid diffusional migration of Deinococcus radiodurans single-stranded DNA-binding protein (SSB) on single-stranded DNA is resolved by FRET, faster than that of Escherichia coli SSB by an order of magnitude. Our approach is a powerful method for studying the dynamics and movements of biomolecules at submillisecond resolution.
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机译：单分子荧光共振能量转移（smFRET）是用于解密非同步生物分子动力学的强大技术之一。但是，smFRET在观察动力学方面的时间分辨率有限。在这里，我们报告了一种新颖的方法，通过束缚分子以自由扩散100 nm大小的脂质体，以亚毫秒级的分辨率观察实时动态。扩散分子的观察时间以亚毫秒级的分辨率扩展到100µms，从而可以使用隐马尔可夫模型直接分析FRET时间迹线中的跃迁状态。我们测量了霍利迪结的两个构象体之间的最高1,500 s –1 的过渡速率。 FRET解决了放射性杜鹃单链DNA结合蛋白（SSB）在单链DNA上的快速扩散迁移，比大肠杆菌SSB快一个数量级。我们的方法是研究亚分子分辨率下生物分子的动力学和运动的有力方法。
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