Stabilization of naked and condensed plasmid DNA against degradation induced by ultrasounds and high-shear vortices

摘要

Micrometre-sized aggregates of a 6050-bp plasmid obtained by the addition of 1.5-3.0 mM CaCl_2 and 20% (v/v) t-butanol or 0.3-1.0% (v/v) APG (aluminium phosphate gel) were subjected to degradation induced by sonication or vortex flows. Dynamic light scattering revealed that the plasmid hydrodynamic radius increases from 116 nm to >1300 nm and approx. 1000 nm, when formulated with CaCl_2/t-butanol and APG respectively. CD showed that addition of CaCl_2/t_butanol leads to transition in plasmid structure from B-DNA to a ψ-DNA negative form, whereas no detectable transitions were observed for APG formu_lations. The ability of the condensing agents to stabi_lize supercoiled plasmid isoforms subjected to son_ication or turbulent Taylor vortices was assessed by agarose-gel electrophoresis. Although naked plasmid was completely fragmented after 5 s of sonication, condensing agents increased the plasmid stability dramatically [e.g. up to 80% after 30 s with 1.5 mM CaCl_2 + 20% (v/v) t-butanol]. In the case of the vorticular flow system, the extent of degradation correlated well with the shear stress associated with flow of the solutions being processed. Overall, the results from the present study demonstrate that condensing agents such as CaCl_2/t-butanol and APG can effectively stabilize plasmids against shear-induced degradation; the extent of protection, however, depends on both the condensing agents and the shear-inducing system used.