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Increase in nuclease resistance and incorporation of NF‐κB decoy oligodeoxynucleotides by modification of the 3′‐terminus
Author(s) -
Osako Mariana Kiomy,
Tomita Naruya,
Nakagami Hironori,
Kunugiza Yasuo,
Yoshino Masafumi,
Yuyama Kazuhiko,
Tomita Tetsuya,
Yoshikawa Hideki,
Ogihara Toshio,
Morishita Ryuichi
Publication year - 2007
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.1077
Subject(s) - decoy , nuclease , transfection , microbiology and biotechnology , chemistry , biology , dna , biochemistry , receptor , gene
Background For the development of molecular therapy based on oligodeoxynucleotides (ODN), ODN have to be stable against nucleases and be specific to the target transcription factor. To decrease non‐specific binding and degradation from the 3′‐terminus of ODN, we designed partially annealed ODN by binding the extremities of two single strands, resulting in a ribbon‐shaped ODN, so called ribbon‐type decoy ODN (R‐ODN). Methods We evaluated the efficiency in the process of enzymatic ligation of R‐ODN, the binding activity to nuclear factor‐κB (NF‐κB), and the stability against Exonuclease III and nucleases present in serum. The functional activity of R‐ODN to inhibit NF‐κB in vitro was evaluated in human aortic smooth muscle cells (VSMC): TNF‐α‐induced proliferation rate and MMP‐9 expression were assessed after R‐ODN transfection. Results and conclusions Although R‐ODN have a phosphodiester backbone, their physical conformation was designed to provide nuclease resistance without interfering with their binding activity. As expected, R‐ODN showed more resistance to exonucleases and stability in 100% serum than non‐modified decoy ODN (N‐ODN). Importantly, the R‐ODN construction did not interfere with its binding activity to NF‐κB, similar to N‐ODN. Transfection of R‐ODN significantly inhibited the expression of MMP‐9 induced by TNF‐α in VSMC as assessed by real‐time polymerase chain reaction (PCR), and R‐ODN also inhibited the proliferation of VSMC induced by TNF‐α (10 ng/ml), similar to phosphorothioate decoy ODN. Overall, the development of ribbon NF‐κB decoy ODN could provide a useful tool for basic and clinical research. Copyright © 2007 John Wiley & Sons, Ltd.