ArticlebioRxiv : the preprint server for biology2025
Synthesizing unmodified, supercoiled circular DNA molecules
Article in bioRxiv : the preprint server for biology, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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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
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