Liang Jialun, Li Jiaxi, Zhong Zhensheng, Rujiralai Thitima, Ma Jie
School of Physics, Sun Yat-sen University, Guangzhou 510275, Guangdong, China.
State Key Laboratory of Optoelectronic Materials and Technologies, Sun Yat-sen University, Guangzhou 510275, Guangdong, China.
Nanoscale. 2021 Oct 1;13(37):15916-15927. doi: 10.1039/d1nr04748e.
The flow-cell based single-molecule manipulation technique has found many applications in the study of DNA mechanics and protein-DNA interactions. However, the force in these experiments has not been fully characterized and is usually limited to a moderate force regime (<25 pN). In this work, using the "tethered-bead" assay, the hydrodynamic drag of DNA has been quantitatively evaluated based on a "bead-spring chain" model. The force derived from the Brownian motion of the bead thus contains both contributions from this equivalent hydrodynamic drag of DNA and the pulling force from the tethered bead. Next, using flow-cell based DNA pulling experiments, the linear relationship between the flow rate and total hydrodynamic force on the bead-DNA system has been demonstrated to be valid over a wide force range (0-110 pN). Consequently, the force can be directly converted from the flow rate by a linear factor that can be calibrated either by the bead's Brownian motion at low flow rates or using DNA overstretching transition. Furthermore, the hydrodynamic force and torque due to the shear flow on the bead as well as the equivalent stretching force on DNA are calculated based on theoretical models with the hydrodynamic drag on DNA also considered. The calculated force-extension curves show a good agreement with the measured ones. These results offer important insights into the force in flow-cell based single-molecule stretching experiments and provide a foundation for establishing flow-cells as a simple, low-cost, yet flexible and precise tool for single-molecule force measurements over a wide force range.
基于流动池的单分子操纵技术在DNA力学和蛋白质-DNA相互作用研究中已有诸多应用。然而，这些实验中的力尚未得到充分表征，通常局限于中等力值范围（<25皮牛）。在本研究中，使用“ tethered-bead”测定法，基于“珠-弹簧链”模型对DNA的流体动力学阻力进行了定量评估。因此，由珠子的布朗运动产生的力既包含DNA等效流体动力学阻力的贡献，也包含来自系留珠子的拉力。接下来，通过基于流动池的DNA拉伸实验，已证明流速与珠子-DNA系统上的总流体动力学力之间的线性关系在很宽的力值范围（0-110皮牛）内都是有效的。因此，可以通过一个线性因子将力直接从流速转换而来，该线性因子可以在低流速下通过珠子的布朗运动进行校准，也可以使用DNA过度拉伸转变来校准。此外，基于理论模型计算了由于珠子上的剪切流产生的流体动力学力和扭矩以及DNA上的等效拉伸力，同时也考虑了DNA的流体动力学阻力。计算得到的力-伸长曲线与测量结果吻合良好。这些结果为基于流动池的单分子拉伸实验中的力提供了重要见解，并为将流动池建立成为一种简单、低成本但灵活且精确的工具以在很宽的力值范围内进行单分子力测量奠定了基础。
