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Synthesizing unmodified, supercoiled circular DNA molecules in vitro.

Sepideh Rezaei1,2, Monica Moncada-Restrepo1,2, Sophia Leng1,2,3

  • 1Biomolecular Sciences Institute, Florida International University, Miami, FL 33199, United States.

Biorxiv : the Preprint Server for Biology
|February 3, 2025
PubMed
Summary

Novel biochemical methods create pure, supercoiled circular DNA (Sc circular DNA) for therapeutic applications. This approach overcomes limitations of bacterial plasmids, enabling efficient gene delivery with enhanced DNA therapeutics.

Keywords:
EGFP-FLPCRRCASupercoiled circular DNA moleculesplasmids

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Gene Therapy

Background:

  • Supercoiled (Sc) circular DNA shows therapeutic promise but is hindered by bacterial contaminants and modifications.
  • Existing methods for producing Sc circular DNA often result in unwanted sequences and potential immune responses.

Purpose of the Study:

  • To develop scalable, biochemical methods for synthesizing unmodified Sc circular DNA.
  • To overcome the limitations associated with bacterial plasmid production for therapeutic applications.

Main Methods:

  • Linear DNA with loxP sites was amplified using PCR or rolling circle amplification.
  • Cre recombinase facilitated the conversion of linear DNA to relaxed circular DNA.
  • T5 exonuclease and topoisomerases were used to generate purified, Sc circular DNA.

Main Results:

  • Developed two novel scalable biochemical synthesis methods for unmodified Sc circular DNA.
  • Synthesized EGFP-FL, a mini-circular DNA demonstrating higher transfection efficiency in human and mouse cells compared to E. coli plasmids.
  • Methods produce Sc circular DNA ranging from 196 bp to several kilobases, in microgram to milligram quantities.

Conclusions:

  • The new biochemical methods provide a scalable platform for producing high-purity, unmodified Sc circular DNA.
  • This technology offers a promising alternative to bacterial plasmids for diverse applications, including gene therapy.
  • The synthesized mini-circular DNA exhibits superior transfection efficiency, paving the way for advanced DNA therapeutics.