Biotechnol. Appl. Biochem., 53, 237-246 (2009) doi:10.1042/BA20080215

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

Wu, M. L., Freitas, S. S., Monteiro, G. A., Prazeres, D. M. F., Santos, J. A. L.

Abstract

Micron-sized aggregates of a 6050 base pair plasmid obtained by the addition of 1.5-3.0 mM CaCl₂ and 20% v/v t-butanol or 0.3-1.0% v/v aluminium phosphate gel were subjected to degradation induced by sonication or vorticular flows. Dynamic light scattering revealed that the plasmid hydrodynamic radius increases from 116 nm to > 1300 nm and ca. 1000 nm, when formulated with CaCl₂/t-butanol and aluminium phosphate gel, respectively. Circular dichroism showed that addition of CaCl₂/t-butanol leads to a B→ψ (-) transition in the plasmid structure, whereas no detectable transitions were observed for aluminium phosphate gel formulations. The ability of the condensing agents to stabilise supercoiled plasmid isoforms subjected to sonication or turbulent Taylor vortices was assessed by agarose 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 CaCl₂ + 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 this study demonstrate that condensing agents such as CaCl₂/t-butanol and aluminium phosphate gel can effectively stabilize plasmids against shear-induced degradation; the protection degree, however, depends of both the condensing agents and the shear inducing system used.

Keywords: condensation; plasmid DNA; protection; shear stress; sonication; vorticular flow system

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