The interaction between four related cyanine
dyes and bacteriophage T5 is investigated with fluorescence and absorption spectroscopy. The
dyes, which differ in size, charge, and mode of
DNA-binding, penetrate the capsid and bind the
DNA inside. The rate of association decreases progressively with increasing
dye size, from a few minutes for YO to more than 50 h for
YOYO (at 37 degrees C). The relative affinity for the phage
DNA is
a factor of about 0.2 lower than for the same T5-DNA when free in
solution. Comparison of groove-bound
BOXTO-PRO and intercalating YO-PRO shows that the reduced affinity is not due to
DNA extension but perhaps influenced by competition with other cationic
DNA-binding agents inside the capsid. Although, the extent of
dye binding to the phages decreases with increasing external ionic strength, the affinity relative to free
DNA increases, which indicates a comparatively weak screening of electrostatic interactions inside the phage. The rate of binding increases with increasing ionic strength, reflecting an increase in effective pore size of the capsid as electrostatic interactions are screened and/or a faster diffusion of the
dye through the
DNA matrix inside the capsid as the
DNA affinity is reduced. A combination of electron microscopy, light scattering, and linear dichroism show that the phages are intact after YO-PRO binding, whereas a small degree of capsid
rupture cannot be excluded with
BOXTO-PRO.