ArticleNucleic acids research2025
Synthesizing supercoiled circular DNA molecules in vitro.
Article in Nucleic acids research, 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 5 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
The trial behind it
Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
5 citing papers in PubMed.
- Cryo-EM structure of a 95-base-pair double-stranded DNA minicircle at 5.3 Å resolution.Nucleic acids research · 2026Article
- Large serine recombinase-mediated gene insertion for high-throughput screens: advantages, design principles, and applications.Nucleic acids research · 2026Review
- Reply to: Overestimations in the cyclization efficiency of small, restriction enzyme-digested DNA fragments.Nature protocols · 2026Article
- Overestimations in the cyclization efficiency of small, restriction enzyme-digested DNA fragments.Nature protocols · 2026Article
- Minicircle DNA Vaccines: Overcoming Delivery and Expression Barriers in Next-Generation Immunization.Vaccines · 2026Review
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Authors and funding
5 authors.
Funding
Abstract
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.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the Socratic graph.