Synthesizing supercoiled circular DNA molecules <i>in vitro</i>

Supercoiled (Sc) circular DNA, such as plasmids, are essential in molecular biology and hold strong therapeutic potential. However, they are typically produced in Escherichia coli, resulting in bacterial methylations, unnecessary sequences, and contaminants that hinder certain applications including clinical uses. These limitations could be avoided by synthesizing plasmids entirely in vitro, but synthesizing high-purity Sc circular DNA biochemically remains a significant technical challenge. To overcome this challenge, we have developed two novel biochemical methods for in vitro synthesis of Sc circular DNA. Linear DNA with two loxP sites in the same orientation is generated by polymerase chain reaction or rolling circle amplification. Cre recombinase efficiently converts the linear DNA into relaxed circular DNA. T5 exonuclease is then used to digest unwanted linear DNA, and topoisomerases are employed to generate Sc circular DNA. Using this approach, we synthesized EGFP-FL, a 2 kb mini-circular DNA encoding essential EGFP expression elements. EGFP-FL transfected HeLa and C2C12 cells with significantly higher efficiency than its E. coli-derived counterpart. These methods enable the efficient production of Sc circular DNA from 196 bp to several kb, and in quantities from micrograms to milligrams, providing a versatile, scalable, and bacteria-free platform for basic research and therapeutic applications.