School of Physics, Sun Yat-sen University, Guangzhou 510275, P.R. China.
State Key Laboratory of Optoelectronic Materials and Technologies, Sun Yat-sen University, Guangzhou 510275, P.R. China.
Nanoscale. 2023 Nov 9;15(43):17443-17454. doi: 10.1039/d3nr03214k.
A flow-cell offers many advantages for single-molecule studies. But, its merit as a quantitative single-molecule tool has long been underestimated. In this work, we developed a gas-pumped fully calibrated flow-cell system combined with fluorescence imaging for simultaneous single-molecule force measurement and visualization. Such a flow-cell system has considered the hydrodynamic drags on biomolecules and hence can apply and measure force up to more than 100 pN in sub-pN precision with an ultra-high force stability (force drift <0.01 pN in 10 minutes) and tuning accuracy (∼0.04 pN). Meanwhile, it also allows acquiring force signals and fluorescence images at the same time, parallelly tracking hundreds of protein motors in real time as well as monitoring the conformational changes of biomolecules under a well-controlled force, as demonstrated by a series of single-molecule experiments in this work, including the studies of DNA overstretching dynamics, transcription under force and DNA folding/unfolding dynamics. Interesting findings, such as the very tight association of single-stranded binding (SSB) proteins with ssDNA and the reversed transcription, have also been made. These results together lay down an essential foundation for a flow-cell to be used as a versatile, quantitative and high-throughput tool for single-molecule manipulation and visualization.
流池为单分子研究提供了许多优势。但是，其作为定量单分子工具的优点长期以来被低估了。在这项工作中，我们开发了一种结合荧光成像的气压驱动全校准流池系统，用于同时进行单分子力测量和可视化。该流池系统考虑了生物分子的流体动力阻力，因此可以施加和测量高达 100 pN 以上的力，具有超高水平的力稳定性（在 10 分钟内力漂移<0.01 pN）和调谐精度（约 0.04 pN）。同时，它还允许同时获取力信号和荧光图像，实时平行跟踪数百个蛋白质马达，并在受控力下监测生物分子的构象变化，这一系列单分子实验在这项工作中得到了证明，包括 DNA 拉伸动力学、力下转录和 DNA 折叠/展开动力学的研究。有趣的发现，如单链结合（SSB）蛋白与 ssDNA 的紧密结合以及反转录，也已经被发现。这些结果共同为流池作为单分子操作和可视化的通用、定量和高通量工具奠定了重要基础。
