A method to track rotational motion for use in single-molecule biophysics
A method to track rotational motion for use in single-molecule biophysics
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DOI：
10.1063/1.3650461
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发表时间：
2011-10-01
影响因子：
1.6
通讯作者：
Dekker, Nynke H.
中科院分区：
文献类型：
--
作者：
Lipfert, Jan;Kerssemakers, Jacob J. W.;Dekker, Nynke H.
DNA的双螺旋性质将DNA复制、转录和修复等许多细胞过程与旋转运动和扭转应变的积累联系在一起。磁性镊子(MTS)是一种单分子技术，能够对核酸锚链施加精确校准的拉伸力，并控制其旋转运动。然而，传统的磁镊子不能直接监测旋转或测量扭矩。在这里，我们描述了一种直接测量MT中粒子旋转运动的方法。该方法依赖于将小的非磁珠附着到磁珠上，以充当旋转跟踪的基准标记。利用专为最小化平移和旋转运动之间的串扰而设计的跟踪算法来分析磁珠的CCD图像：首先，使用基于核的跟踪器确定磁珠的面内中心位置，而随后通过基于相关的算法来确定磁珠的高度和旋转角度。使用模拟图像和表面固定的珠子的记录图像对跟踪算法进行的评估表明，旋转分辨率为0.1度，而平移分辨率保持为1-2 nm。DNA拴系珠在不同硬度的磁势中表现出的旋转波动的示踪实例表明了该方法的健壮性和同时跟踪多个珠的潜力。我们的旋转跟踪算法使MTS能够扩展到磁性扭矩镊子(MTT)，以直接测量单分子中的扭矩。此外，我们展望了该算法在一系列生物物理测量中的应用，包括MT的进一步扩展、拴系粒子运动和光学捕获测量。(C)2011年美国物理研究所。[DOI：10.1063/1.3650461]
The double helical nature of DNA links many cellular processes such as DNA replication, transcription, and repair to rotational motion and the accumulation of torsional strain. Magnetic tweezers (MTs) are a single-molecule technique that enables the application of precisely calibrated stretching forces to nucleic acid tethers and to control their rotational motion. However, conventional magnetic tweezers do not directly monitor rotation or measure torque. Here, we describe a method to directly measure rotational motion of particles in MT. The method relies on attaching small, non-magnetic beads to the magnetic beads to act as fiducial markers for rotational tracking. CCD images of the beads are analyzed with a tracking algorithm specifically designed to minimize crosstalk between translational and rotational motion: first, the in-plane center position of the magnetic bead is determined with a kernel-based tracker, while subsequently the height and rotation angle of the bead are determined via correlation-based algorithms. Evaluation of the tracking algorithm using both simulated images and recorded images of surface-immobilized beads demonstrates a rotational resolution of 0.1 degrees, while maintaining a translational resolution of 1-2 nm. Example traces of the rotational fluctuations exhibited by DNA-tethered beads confined in magnetic potentials of varying stiffness demonstrate the robustness of the method and the potential for simultaneous tracking of multiple beads. Our rotation tracking algorithm enables the extension of MTs to magnetic torque tweezers (MTT) to directly measure the torque in single molecules. In addition, we envision uses of the algorithm in a range of biophysical measurements, including further extensions of MT, tethered particle motion, and optical trapping measurements. (C) 2011 American Institute of Physics. [doi: 10.1063/1.3650461]