Stabilization of naked and condensed plasmid DNA against degradation induced by ultrasounds and high-shear vortices.
Micrometre-sized aggregates of a 6050-bp 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) 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 CaCl2/t-butanol and APG respectively. CD showed that addition of CaCl2/t-butanol leads to transition in plasmid structure from B-DNA to a psi-DNA negative form, whereas no detectable transitions were observed for APG formulations. The ability of the condensing agents to stabilize supercoiled plasmid isoforms subjected to sonication 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 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 the present study demonstrate that condensing agents such as CaCl2/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.
Wu
Ming L
ML
IBB (Institute for Biotechnology and Bioengineering), Centre for Biological and Chemical Engineering, Instituto Superior Técnico, 1049-001 Lisbon, Portugal.
Freitas
Sindélia S
SS
Monteiro
Gabriel A
GA
Prazeres
Duarte M F
DM
Santos
José A L
JA
D002942:Circular Dichroism; D004247:DNA / Q000737:chemistry*; D004587:Electrophoresis, Agar Gel; D005453:Fluorescence; D010316:Particle Size; D010957:Plasmids / Q000737:chemistry*; D033081:Shear Strength; D014465:Ultrasonics
D004247:DNA