Structural and immunostimulatory properties of Y-shaped DNA consisting of phosphodiester and phosphorothioate oligodeoxynucleotides.
Y-shape formation increased the immunostimulatory activity of phosphodiester (PO) oligodeoxynucleotides (ODNs) containing CpG motif. In this study, PO CpG ODN or CpG ODN containing nuclease-resistant phosphorothioate (PS) linkages, i.e., PS CpG ODN or PO CpG ODN with three PS linkages at the both ends (PS3), was mixed with two PO- or PS ODNs to prepare Y-shaped DNA (Y-DNA) containing a potent CpG motif. The melting temperature of Y-DNA decreased with increasing number of PS linkages. Y(PS/PO/PO), which contained PS CpG ODN, showed the greatest activity to induce tumor necrosis factor-α release from macrophage-like RAW264.7 cells, followed by Y(PS3/PO/PO). However, the high activity of Y(PS/PO/PO) was due to that of PS CpG ODN, and Y-shape formation had no significant effect on the activity. Furthermore, PS CpG ODN of Y(PS/PO/PO) was efficiently taken up by cells, but other PO ODNs in the Y-DNA were not, indicating that PS CpG ODN in Y-DNA behave like single stranded PS CpG ODN. In quite contrast, the immunostimulatory activity of PS3 CpG ODN was significantly increased by Y-shape formation. In conclusion, Y-shape formation and PS substitution can be used simultaneously to increase the immunostimulatory activity of CpG ODN, but extensive substitution should be avoided because it diminishes the benefits of Y-shape formation.
Matsuoka
Nao
N
Department of Biopharmaceutics and Drug Metabolism, Graduate School of Pharmaceutical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8501, Japan.
Nishikawa
Makiya
M
Mohri
Kohta
K
Rattanakiat
Sakulrat
S
Takakura
Yoshinobu
Y
D000276:Adjuvants, Immunologic / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D002463:Cell Membrane Permeability; D004247:DNA / Q000737:chemistry* / Q000276:immunology* / Q000378:metabolism; D008264:Macrophages / Q000276:immunology / Q000378:metabolism; D051379:Mice; D009690:Nucleic Acid Conformation; D009691:Nucleic Acid Denaturation; D009838:Oligodeoxyribonucleotides / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D010755:Organophosphates / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D054735:Phosphorothioate Oligonucleotides / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology
D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D009838:Oligodeoxyribonucleotides; D010755:Organophosphates; D054735:Phosphorothioate Oligonucleotides; D014409:Tumor Necrosis Factor-alpha; D004247:DNA