绳系粒子运动实验的标定
Calibration of Tethered Particle Motion Experiments
摘要：
系留粒子运动（TPM）方法已被用于观察和表征各种蛋白质-DNA相互作用，包括DNA成环和转录。TPM实验利用DNA系链珠的布朗运动来探测系链的生物相关构象变化。在这些实验中，珠粒的随机运动的程度的变化被用作潜在的大分子动力学的报告者，并且通常被认为足以用于TPM分析。然而，一个完整的了解如何运动取决于拴系颗粒复合物的物理性质将允许更定量和准确的TPM数据的评估。例如，这种理解可以帮助从TPM数据中提取关于环形复杂几何形状（或多个共存几何形状）的细节。为了更好地表征涉及DNA系链的TPM实验的测量能力，我们已经进行了详细的TPM幅度作为DNA长度和颗粒大小的函数的校准。我们还探讨了如何实验参数，如采集时间和曝光时间影响栓系粒子的表观运动。我们将DNA长度从200bp变化到2.6kbp，并考虑200、490和970nm的颗粒直径。我们还提出了一个系统的比较测量粒子的偏移和理论预期，这有助于澄清实验和模型的DNA构象。
英文摘要：
The Tethered Particle Motion (TPM) method has been used to observe and characterize a variety of protein-DNA interactions including DNA looping and transcription. TPM experiments exploit the Brownian motion of a DNA-tethered bead to probe biologically relevant conformational changes of the tether. In these experiments, a change in the extent of the bead's random motion is used as a reporter of the underlying macromolecular dynamics and is often deemed sufficient for TPM analysis. However, a complete understanding of how the motion depends on the physical properties of the tethered particle complex would permit more quantitative and accurate evaluation of TPM data. For instance, such understanding can help extract details about a looped complex geometry (or multiple coexisting geometries) from TPM data. To better characterize the measurement capabilities of TPM experiments involving DNA tethers, we have carried out a detailed calibration of TPM magnitude as a function of DNA length and particle size. We also explore how experimental parameters such as acquisition time and exposure time affect the apparent motion of the tethered particle. We vary the DNA length from 200bp to 2.6kbp and consider particle diameters of 200, 490 and 970nm. We also present a systematic comparison between measured particle excursions and theoretical expectations, which helps clarify both the experiments and models of DNA conformation.
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