Single-Particle Light Microscopy of Bacteriophages

Bacteriophage capsids have a protein shell with a symmetrical, fullerene-like arrangement of sub-units. In the case of double-stranded DNA bacteriophages, the capsid joins with accessory proteins to form a DNA packaging motor that packages a genome in a cavity of the capsid. The motor cleaves ATP to obtain the needed energy. Light microscopy of single bacteriophages and single bacteriophage DNA packaging intermediates is being developed for the following reasons: (1) A synchronization-independent, fractionation-independent procedure is needed for the in vitro analysis of bacteriophage DNA packaging motors. (2) A non-biological procedure is needed for identifying and characterizing new bacteriophages needed for studies of bacteriophage gene homologies. In a recent study, light microscopy-based nanometry is used to follow the in vitro packaging of DNA in real time. Fluorescence microscopy of stained DNA is similarly used. Towards a more thorough analysis by fluorescence microscopy, single bacteriophage capsids are visualized by the unenhanced fluorescence of covalently bound protein-specific dyes. Dimerization of capsids is observed in real time. The dimerizing capsids had been restricted to a thin planar zone so that single-particle tracking was performed before, during, and after dimerization. Photobleaching is not a major problem. Thermal motion-based procedures are used for distinguishing binding from accidental co-migration. The long-range objective is the simultaneous real time monitoring of multiple state variables during cycling of a single DNA packaging motor. The results of these basic studies are applicable to both nanotechnological drug delivery and biological therapy.