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.
bioRxiv. 2025 Jan 25:2025.01.24.634800. doi: 10.1101/2025.01.24.634800.
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 -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.
自20世纪90年代以来，超螺旋（Sc）环状DNA，如质粒，已显示出治疗潜力，但受到细菌修饰、不必要的DNA序列以及可能引发有害反应的污染物的限制。为了克服这些挑战，我们开发了两种新型的可扩展生化方法来合成未修饰的Sc环状DNA。通过PCR或滚环扩增产生具有两个同向loxP位点的线性DNA。然后，Cre重组酶将这种线性DNA转化为松弛环状DNA。在T5核酸外切酶去除不需要的线性DNA后，使用拓扑异构酶生成Sc环状DNA。我们已经合成了EGFP-FL，一种携带必需EGFP表达元件的2002 bp微型环状DNA。EGFP-FL转染人HeLa细胞和小鼠C2C12细胞的效率比衍生质粒高得多。这些新的生化方法可以生产长度从196个碱基对到几千碱基、数量从微克到毫克的未修饰Sc环状DNA，为各种应用提供了一个有前景的平台。
