Artículos de revistas
Production Of Dna Minicircles Less Than 250 Base Pairs Through A Novel Concentrated Dna Circularization Assay Enabling Minicircle Design With Nf-kappa B Inhibition Activity
Registro en:
Nucleic Acids Research. Oxford Univ Press, v. 45, p. , 2017.
0305-1048
1362-4962
WOS:000397286600001
10.1093/nar/gkw1034
Autor
Thibault
Thomas; Degrouard
Jeril; Baril
Patrick; Pichon
Chantal; Midoux
Patrick; Malinge
Jean-Marc
Institución
Resumen
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 plasmidfree method for the production of DNA minicircles comprising fewer than 250 bp. We designed a linear nicked DNA double-stranded oligonucleotide bluntended substrate for efficient minicircle production in a ligase-mediated and bending protein-assisted circularization reaction at high DNA concentration of 2 mu M. 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-kappa B transcription factors per minicircle and to efficiently inhibiting NF-kappa B-dependent transcriptional activity in human cells. Therefore, our production method could pave the way for the design of minicircles as new decoy nucleic acids. 45 5 Ministere de l'Enseignement Superieur et de la Recherche University of Orleans Ligue contre le cancer Region Centre et Loiret CNRS-CEA `METSA' French network [FR CNRS 3507]