Increase in nuclease resistance and incorporation of NF-kappaB decoy oligodeoxynucleotides by modification of the 3'-terminus.
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-kappaB (NF-kappaB), and the stability against Exonuclease III and nucleases present in serum. The functional activity of R-ODN to inhibit NF-kappaB in vitro was evaluated in human aortic smooth muscle cells (VSMC): TNF-alpha-induced proliferation rate and MMP-9 expression were assessed after R-ODN transfection.


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-kappaB, similar to N-ODN. Transfection of R-ODN significantly inhibited the expression of MMP-9 induced by TNF-alpha in VSMC as assessed by real-time polymerase chain reaction (PCR), and R-ODN also inhibited the proliferation of VSMC induced by TNF-alpha (10 ng/ml), similar to phosphorothioate decoy ODN. Overall, the development of ribbon NF-kappaB decoy ODN could provide a useful tool for basic and clinical research.
Osako
Mariana Kiomy
MK
Division of Clinical Gene Therapy, Osaka University Graduate School of Medicine, 2-2 Yamada-Oka, Suita, Osaka, Japan.
Tomita
Naruya
N
Nakagami
Hironori
H
Kunugiza
Yasuo
Y
Yoshino
Masafumi
M
Yuyama
Kazuhiko
K
Tomita
Tetsuya
T
Yoshikawa
Hideki
H
Ogihara
Toshio
T
Morishita
Ryuichi
R
D001483:Base Sequence; D002470:Cell Survival / Q000187:drug effects; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D032389:Myocytes, Smooth Muscle / Q000166:cytology / Q000187:drug effects; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
C468119:NF-kappaB decoy; D009838:Oligodeoxyribonucleotides; D014409:Tumor Necrosis Factor-alpha; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D020780:Matrix Metalloproteinase 9