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Synthesizing unmodified, supercoiled circular DNA molecules in vitro. Article

Rezaei, Sepideh, Moncada-Restrepo, Monica, Leng, Sophia et al. (2025). Synthesizing unmodified, supercoiled circular DNA molecules in vitro. . 10.1101/2025.01.24.634800

cited authors

  • Rezaei, Sepideh; Moncada-Restrepo, Monica; Leng, Sophia; Chambers, Jeremy W; Leng, Fenfei

authors

abstract

  • Supercoiled (Sc) circular DNA, such as plasmids, has shown therapeutic potential since the 1990s, but is limited by bacterial modifications, unnecessary DNA sequences, and contaminations that may trigger harmful responses. To overcome these challenges, we have developed two novel scalable biochemical methods to synthesize unmodified Sc circular DNA. Linear DNA with two loxP sites in the same orientation is generated via PCR or rolling circle amplification. Cre recombinase then converts this linear DNA into relaxed circular DNA. After T5 exonuclease removes unwanted linear DNA, topoisomerases are employed to generate Sc circular DNA. We have synthesized EGFP-FL, a 2,002 bp mini-circular DNA carrying essential EGFP expression elements. EGFP-FL transfected human HeLa and mouse C2C12 cells with much higher efficiency than E. coli-derived plasmids. These new biochemical methods can produce unmodified Sc circular DNA, in length from 196 base pairs to several kilobases and in quantities from micrograms to milligrams, providing a promising platform for diverse applications.

publication date

  • January 25, 2025

webpage

keywords

  • EGFP-FL
  • PCR
  • RCA
  • Supercoiled circular DNA molecules
  • plasmids

Location

  • United States

Digital Object Identifier (DOI)