The effect of lyophilization on plasmid DNA activity

Area of Science:

• Molecular Biology
• Biochemistry
• Biotechnology

Background:

• Plasmid DNA is crucial for protein expression in biotechnology.
• Lyophilization (freeze-drying) is a common storage method, but its effect on plasmid DNA activity is not fully understood.

Purpose of the Study:

• To investigate the impact of lyophilization on plasmid DNA's protein expression capability.
• To determine if carbohydrates can mitigate any negative effects of lyophilization on plasmid DNA.

Main Methods:

• Purified plasmid DNA (pRL-CMV) was lyophilized with and without carbohydrates.
• Transfection efficiency in COS-1 cells was measured.
• Structural integrity was assessed using agarose gel electrophoresis, DMED assay, circular dichroism, and UV spectroscopy.

Main Results:

• Lyophilization caused a significant decrease in plasmid DNA transfection efficiency.
• Mono- and disaccharides completely restored transfection efficiency.
• No significant changes in DNA structure or abasic sites were observed; transient structural changes and a hyperchromic effect occurred but were not the primary cause of activity loss.

Conclusions:

• Lyophilization diminishes plasmid DNA activity for protein expression.
• Carbohydrates effectively protect plasmid DNA activity during and after lyophilization, likely by stabilizing structural changes.