Vanzi F, Sacconi L, Pavone F S
LENS-European Laboratory for Nonlinear Spectroscopy, Sesto Fiorentino, Italy.
Biophys J. 2007 Jul 1;93(1):21-36. doi: 10.1529/biophysj.106.094151. Epub 2007 Apr 13.
In the tethered particle motion method the length of a DNA molecule is monitored by measuring the range of diffusion of a microsphere tethered to the surface of a microscope coverslip through the DNA molecule itself. Looping of DNA (induced by binding of a specific protein) can be detected with this method and the kinetics of the looping/unlooping processes can be measured at the single molecule level. The microsphere's position variance represents the experimental variable reporting on the polymer length. Therefore, data windowing is required to obtain position variance from raw position data. Due to the characteristic diffusion time of the microsphere, the low-pass filtering required to attain a good signal/noise ratio (S/N) in the discrimination of looped versus unlooped state impacts significantly the measurement's time resolution. Here we present a method for measuring lifetimes based on half-amplitude thresholding and then correcting the kinetic measurements, taking into account low S/N (leading to false events) and limited time resolution (leading to missed events). This method allows an accurate and unbiased estimation of the kinetic parameters under investigation, independently of the choice of the window used for variance calculation, with potential applications to other single molecule measurements with low S/N.
在拴系粒子运动方法中，通过测量一个经由DNA分子自身拴系在显微镜盖玻片表面的微球的扩散范围，来监测DNA分子的长度。利用这种方法可以检测到DNA的环化（由特定蛋白质的结合诱导），并且可以在单分子水平上测量环化/解环过程的动力学。微球的位置方差代表了反映聚合物长度的实验变量。因此，需要进行数据开窗处理，以便从原始位置数据中获得位置方差。由于微球具有特征扩散时间，在区分环化状态与非环化状态时，为获得良好的信噪比（S/N）而进行的低通滤波会显著影响测量的时间分辨率。在此，我们提出一种基于半幅度阈值化来测量寿命，然后校正动力学测量的方法，该方法考虑到了低信噪比（导致错误事件）和有限的时间分辨率（导致漏检事件）。这种方法能够准确且无偏地估计所研究的动力学参数，而与用于方差计算的窗口选择无关，具有应用于其他低信噪比单分子测量的潜力。
