Biomacromolecular Systems and Processes, Institute of Biological Information Processing, IBI-4, Forschungszentrum Jülich, Jülich, Germany.
Faculty of Life and Medical Sciences, Doshisha University, Kyotanabe, 610-0394, Japan.
Eur Biophys J. 2021 Dec;50(8):1139-1155. doi: 10.1007/s00249-021-01570-0. Epub 2021 Sep 9.
Bacteriophages with long DNA genomes are of interest due to their diverse mutations dependent on environmental factors. By lowering the ionic strength of a hydrophobic (PPh4Cl) antagonistic salt (at 1 mM), single long T4 DNA strand fluctuations were clearly observed, while condensed states of T4 DNA globules were formed above 5-10 mM salt. These long DNA strands were treated with fluorescently labeled probes, for which photo bleaching is often unavoidable over a short time of measurement. In addition, long (few tens of [Formula: see text]) length scales are required to have larger fields of view for better sampling, with shorter temporal resolutions. Thus, an optimization between length and time is crucial to obtain useful information. To facilitate the challenge of detecting large biomacromolecules, we here introduce an effective method of live image data analysis for direct visualization and quantification of local thermal fluctuations. The motions of various conformations for the motile long DNA strands were examined for the single- and multi-T4 DNA strands. We find that the unique correlation functions exhibit a relatively high-frequency oscillatory behavior superimposed on the overall slower decay of the correlation function with a splitting of amplitudes deriving from local activities of the long DNA strands. This work shows not only the usefulness of an image-time correlation for analyzing large biomacromolecules, but also provides insight into the effects of a hydrophobic antagonistic salt on active T4 bacteriophage long DNA strands, including thermal translocations in their electrostatic interactions.
由于其依赖于环境因素的多样化突变，具有长 DNA 基因组的噬菌体引起了人们的兴趣。通过降低疏水性（PPh4Cl）拮抗盐（在 1mM 时）的离子强度，可以清楚地观察到单个长 T4 DNA 单链的波动，而在盐浓度高于 5-10mM 时形成 T4 DNA 球体的凝聚态。这些长 DNA 链用荧光标记探针进行处理，由于在短时间的测量过程中经常不可避免地发生光漂白，因此这些探针通常需要在较短的时间内进行光漂白。此外，需要较长的（数十个[公式：见文本]）长度尺度才能获得更大的视野，从而进行更好的采样，同时具有较短的时间分辨率。因此，在获得有用信息方面，长度和时间之间的优化至关重要。为了便于检测大型生物大分子的挑战，我们在这里介绍了一种有效的实时图像数据分析方法，用于直接可视化和量化局部热波动。研究了单链和多链 T4 DNA 的各种构象的运动。我们发现，独特的相关函数表现出相对高频的振荡行为，叠加在相关函数的整体较慢衰减上，幅度的分裂来自长 DNA 链的局部活性。这项工作不仅展示了图像时间相关分析在分析大型生物大分子方面的有用性，还深入了解了疏水性拮抗盐对活性 T4 噬菌体长 DNA 链的影响，包括它们静电相互作用中的热易位。
