Analytical and Functional Characterization of Plasmid DNA Topological Forms and Multimers

Area of Science:

• Molecular Biology
• Biochemistry
• Analytical Chemistry

Background:

• Plasmid DNA (pDNA) is crucial for genetic engineering and therapies.
• pDNA exists in supercoiled (SC), open circular (OC), and linear forms, with potential for multimerization.
• The impact of these forms and multimers on functional activity, especially for smaller pDNAs, lacks consensus.

Purpose of the Study:

• To develop a workflow for characterizing pDNA topological forms and multimers.
• To evaluate the impact of these forms on functional activity (knock-in efficiency).
• To compare analytical methods for pDNA characterization.

Main Methods:

• Development and optimization of an anion exchange chromatography (AEC) method for pDNA quantification.
• Systematic study of chromatographic parameters (mobile phase pH, salts, temperature, acetonitrile).
• Comparison of AEC with capillary gel electrophoresis (CGE) and assessment of a forced degradation study.

Main Results:

• A superior AEC method was established for quantifying pDNA topological forms and multimers.
• The study revealed unexpectedly high degradation of SC pDNA forms under certain conditions.
• Comparison of knock-in efficiency between SC and OC forms, and multimers, was performed.

Conclusions:

• The developed AEC workflow provides robust analytical characterization of pDNA forms and multimers.
• Topological forms and multimerization significantly influence pDNA stability and functional efficiency.
• Further research is needed to fully elucidate the role of pDNA topology in gene therapy applications.