Enhanced DNA dynamics due to cationic reagents, topological states of dsDNA and high mobility group box 1 as probed by PicoGreen

Abstract

We report a study of dynamics with a dsDNA-specific dye called PicoGreen bound to plasmid DNA (3.4 kb), and show that at low dye/DNA phosphate ratios (1 : 100 and below), PicoGreen dynamics reflect the motional dynamics of dsDNA. We further evaluated the usefulness of this probe by measuring the time-resolved fluorescence dynamics of PicoGreen bound to dsDNA in the presence of cationic reagents that affect DNA dynamics [MgCl₂ and polyethyleneimine (PEI)] and also with plasmid DNA in different topological states. Among these conditions, MgCl₂, PEI and the supercoiled form of plasmid resulted in increases in the very short component (0.2–0.4 ns) of the rotational correlation time. Separately, HMGB1 protein enhanced DNA dynamics, as observed from the rotational correlation times of very short (0.2–0.4 ns) and short (2–4 ns) rotational correlation timescale components. By studying the effect of specific deletion mutants HMGB1-ΔA (deletion of 98 N-terminal amino acids) and HMGB1-ΔC (deletion of 30 C-terminal amino acids), we show that the acidic C-terminal tail is required for enhancement of DNA dynamics. We then discuss the possible mechanisms and implications of HMGB1-mediated flexibility of DNA in the context of formation of large nucleoprotein complexes.