Sensing the size of individual molecules in an ensemble has proven to be a powerful tool to investigate biomolecular interactions and association–dissociation processes. In biologically relevant solution environments, molecular size is often sensed by translational or rotational diffusivity. The rotational diffusivity is more sensitive to the size and conformation of the molecules as it is inversely proportional to the cube of the hydrodynamic radius, as opposed to the inverse linear dependence of the translational diffusion coefficient. Single-molecule rotational diffusivity has been measured with time-resolved fluorescence anisotropy decay, but the ability to sense different sizes has been restricted by the limited number of photons available or has required surface attachment to observe each molecule longer, and the attachment may be perturbative. To address these limitations, we show how to measure and monitor single-molecule rotational diffusivity by combining the solution-phase Anti-Brownian ELectrokinetic (ABEL) trap and maximum likelihood analysis of time-resolved fluorescence anisotropy based on the information inherent in each detected photon. We demonstrate this approach by resolving a mixture of single- and double-stranded fluorescently labeled DNA molecules at equilibrium, freely rotating in a native solution environment. The rotational diffusivity, fluorescence brightness and lifetime, and initial and steady-state anisotropy are simultaneously determined for each trapped single DNA molecule. The time resolution and precision of this method are analyzed using statistical signal analysis and simulations. We present key parameters that define the usefulness of a particular fluorescent label for extracting molecular size information from single-molecule rotational diffusivity measurements.

中文翻译：

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通过单分子旋转扩散率解析溶液中的混合物

事实证明，感知整体中单个分子的大小是研究生物分子相互作用和缔合-解离过程的有力工具。在生物学相关的溶液环境中，分子大小通常通过平移或旋转扩散率来感知。旋转扩散率对分子的大小和构象更为敏感，因为它与流体力学半径的立方成反比，与平移扩散系数的线性反比相反。已通过时间分辨的荧光各向异性衰减测量了单分子旋转扩散率，但是感测不同大小的能力受到可用光子数量的限制，或者需要更长的表面附着时间才能观察到每个分子，并且附件可能是微扰的。为了解决这些局限性，我们展示了如何结合每个检测到的光子固有的信息，结合溶液相反布朗电子动能（ABEL）阱和时间分辨荧光各向异性的最大似然分析，来测量和监测单分子旋转扩散率。我们通过解析平衡的单链和双链荧光标记DNA分子的混合物，在天然溶液环境中自由旋转来证明这种方法。同时为每个捕获的单个DNA分子确定旋转扩散率，荧光亮度和寿命以及初始和稳态各向异性。使用统计信号分析和仿真分析了该方法的时间分辨率和精度。