Abstract
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 upmu m) 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.
Highlights
The bacteriophage T4 capsid carrying its DNA genome consists of several functional regions including a structural RNA
It should be noted that the unique results of the image–time correlation turn out to be a splitting of the data in the zoomed-in view of the correlation function
The red regions are filled with the modulating lines between upper and lower data points, qualitatively indicating that the amplitude of a locally fluctuating T4 DNA strand is the measure for thermal Brownian motions depicted by the image–time correlation
Summary
The bacteriophage T4 capsid carrying its DNA genome consists of several functional regions including a structural RNA. Similar findings relating to the high speed of the DNA packaging motor of bacteriophage T4 have been observed via optical tweezers (Lin et al 2017). Maintaining such a high speed of the molecular motor is crucial to functions being sustained. We have demonstrated the use of image–time correlation spectroscopy to analyze the real-time movie data of long DNA strands that are fluorescently labeled. The image–time correlation function serves as a useful means of “quantifying” the fluctuating long DNA strands, over mesoscopic time scales (a few seconds), exploiting their elastic deformations directly from images of single- and multi-interacting strands in solution
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