title: Production of DNA minicircles less than 250 base pairs through a novel concentrated DNA circularization assay enabling minicircle design with NF-κB inhibition activity.
abstract: Double-stranded DNA minicircles of less than 1000 bp in length have great interest in both fundamental research and therapeutic applications. Although minicircles have shown promising activity in gene therapy thanks to their good biostability and better intracellular trafficking, minicircles down to 250 bp in size have not yet been investigated from the test tube to the cell for lack of an efficient production method. Herein, we report a novel versatile plasmid-free method for the production of DNA minicircles comprising fewer than 250 bp. We designed a linear nicked DNA double-stranded oligonucleotide blunt-ended substrate for efficient minicircle production in a ligase-mediated and bending protein-assisted circularization reaction at high DNA concentration of 2 μM. This one pot multi-step reaction based-method yields hundreds of micrograms of minicircle with sequences of any base composition and position and containing or not a variety of site-specifically chemical modifications or physiological supercoiling. Biochemical and cellular studies were then conducted to design a 95 bp minicircle capable of binding in vitro two NF-κB transcription factors per minicircle and to efficiently inhibiting NF-κB-dependent transcriptional activity in human cells. Therefore, our production method could pave the way for the design of minicircles as new decoy nucleic acids.
authors.affiliation: Centre de Biophysique Moléculaire, CNRS UPR 4301, affiliated to the University of Orléans and Inserm, 45071 Orléans Cedex 02 France.
authors.forename: Thomas
authors.initials: T
authors.lastname: Thibault
authors.affiliation: Laboratoire de Physique des Solides, Université Paris Sud, CNRS UMR 8502, 91405 Orsay Cedex, France.
authors.forename: Jeril
authors.initials: J
authors.lastname: Degrouard
authors.affiliation: Centre de Biophysique Moléculaire, CNRS UPR 4301, affiliated to the University of Orléans and Inserm, 45071 Orléans Cedex 02 France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Baril
authors.affiliation: Centre de Biophysique Moléculaire, CNRS UPR 4301, affiliated to the University of Orléans and Inserm, 45071 Orléans Cedex 02 France.
authors.forename: Chantal
authors.initials: C
authors.lastname: Pichon
authors.affiliation: Centre de Biophysique Moléculaire, CNRS UPR 4301, affiliated to the University of Orléans and Inserm, 45071 Orléans Cedex 02 France.
authors.forename: Patrick
authors.initials: P
authors.lastname: Midoux
authors.affiliation: Centre de Biophysique Moléculaire, CNRS UPR 4301, affiliated to the University of Orléans and Inserm, 45071 Orléans Cedex 02 France.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Malinge
mesh_terms: D020029:Base Pairing; D001483:Base Sequence; D004270:DNA, Circular / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D008025:Ligases / Q000235:genetics* / Q000378:metabolism; D016328:NF-kappa B / Q000235:genetics* / Q000378:metabolism; D009838:Oligodeoxyribonucleotides / Q000138:chemical synthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014158:Transcription, Genetic
chemical_list: C072843:ABF2 protein, S cerevisiae; D004270:DNA, Circular; D004268:DNA-Binding Proteins; D016328:NF-kappa B; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; D008025:Ligases