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.
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-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.