An optimized extended DNA kappa B site that enhances plasmid DNA nuclear import and gene expression

Background The nuclear factor kappa B (NF kappa B) transcription factor, which shuttles between the cytoplasm and the nucleus under specific conditions, is a suitable intracellular target to increase the nuclear import of plasmid DNA. We report the design of an optimized and extended NF kappa B DNA binding sequence that promotes an efficient plasmid nuclear import. Methods On the basis of structural studies, the 5'-CTGGGGACTTTCCAGCT-GGGGACTTTCCAGCTGGGGACTTTCCAGG-3' segment (termed 3NF) comprising three 10-bp kappa B sites (GGGACTTTCC) separated by a 5-bp optimized spacer (AGCTG) was selected for its capacity to ensure the best Structural fit with NF kappa B and to fix simultaneously three proteins. Plasmids encoding-luciferase and bearing this sequence (3NF-plasmids) were constructed and their nuclear import and gene expression efficiencies compared with that of plasmids containing classical kappa B motifs. Results A high luciferase expression was associated with plasmids containing one (p3NF-luc) or two (p3NF-luc-3NF) 3NF sequences. In situ hybridization experiments and quantitative measurement of the number of plasmid copies demonstrated that the nuclear delivery of 3NF-plasmids was more efficient than that of 3NF-free plasmids. Cross-linked immunoprecipitation showed that 3NF-plasmids were recognized by NF kappa B inside Cells upon transfection. The nuclear delivery was inhibited with BAY 11-7085, an inhibitor of NF kappa B activation. Finally, p3NF-luc-3NF the, most efficient construct for in vitro transfection had a long-lived luciferase, expression in vivo. Conclusions The results obtained in the present Study demonstrate the NF kappa B-mediated nuclear delivery of 3NF-plasmids. Due to its high affinity for fixing several NF kappa B, the 3NF sequence is a very promising helper for a nonviral gene delivery system. Copyright (C) 2009 John Wiley & Sons, Ltd.