Rezaei Sepideh, Moncada-Restrepo Monica, Leng Sophia, Chambers Jeremy W, Leng Fenfei
Biomolecular Sciences Institute, Florida International University, Miami, FL 33199, United States.
Department of Chemistry and Biochemistry, Florida International University, Miami, FL 33199, United States.
Nucleic Acids Res. 2025 Sep 5;53(17). doi: 10.1093/nar/gkaf889.
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.
超螺旋（Sc）环状DNA，如质粒，在分子生物学中至关重要且具有强大的治疗潜力。然而，它们通常在大肠杆菌中产生，会导致细菌甲基化、不必要的序列以及污染物，这些会阻碍某些应用，包括临床应用。通过完全在体外合成质粒可以避免这些限制，但生化合成高纯度Sc环状DNA仍然是一项重大的技术挑战。为了克服这一挑战，我们开发了两种用于体外合成Sc环状DNA的新型生化方法。通过聚合酶链反应或滚环扩增产生具有两个同向loxP位点的线性DNA。Cre重组酶可有效地将线性DNA转化为松弛环状DNA。然后使用T5核酸外切酶消化不需要的线性DNA，并使用拓扑异构酶生成Sc环状DNA。使用这种方法，我们合成了EGFP-FL，一种编码基本EGFP表达元件的2 kb微型环状DNA。EGFP-FL转染HeLa和C2C12细胞的效率明显高于其大肠杆菌来源的对应物。这些方法能够高效生产196 bp至数kb的Sc环状DNA，产量从微克到毫克不等，为基础研究和治疗应用提供了一个通用、可扩展且无细菌的平台。
