Immunologic Research

, Volume 39, Issue 1–3, pp 4–14 | Cite as

Structural requirements and applications of inhibitory oligodeoxyribonucleotides



Synthetic oligodeoxyribonucleotides (ODN) bearing certain sequence characteristics mimic bacterial DNA by activating B cells and dendritic cells through Toll-like receptor (TLR) 9, an event that potentiates both humoral and cell-mediated immunity. ODN sharing some of the sequence characteristics of strong stimulatory (ST-) ODN, but substituting GGG for CGTT, competitively inhibit ST-ODN-driven events. An ODN with the same length and base composition as a strong ST-ODN, but lacking both ST- and IN-sequence requirements, has neither ST- nor IN-activity. Whereas, certain sequence changes strongly influence ST-ODN activity in human cells relative to mouse cells and B cells relative to non B cells, the strongest IN-ODN appear to work well in both species and multiple cell types. Converting from the natural phosphodiester backbone to a nuclease-resistant phosphorothioate backbone increases the sensitivity to ST-ODN about 2 logs and to IN-ODN 3 logs, while increasing the impact of critical base changes in ST-ODN and diminishing it in IN-ODN. Examples where IN-ODN have been used in vivo to interrupt autoimmune and other TLR-9-induced inflammatory states are described.


Inhibitory oligonucleotides TLR9 Stimulatory oligonucleotides Autoimmunity Bacterial DNA 



Expert secretarial assistance by Deanna Ollendick, technical assistance by Adam Goeken, and collaboration with Eicke Latz and Douglas Golenbock are gratefully acknowledged. Supported by RO1 AI 47374–04 from the NIH.


  1. 1.
    Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA, Klinman D. CpG motifs in bacterial DNA trigger direct B cell activation. Nature 1995;374:546–9.PubMedCrossRefGoogle Scholar
  2. 2.
    Krieg AM. CpG motifs in bacterial DNA and their immune effects. Ann Rev Immunol 2002;20:709–60.CrossRefGoogle Scholar
  3. 3.
    Klinman DM. Adjuvant activity of CpG oligodeoxynucleotides. Int Rev Immunol 2006;25(3–4):135–54.PubMedCrossRefGoogle Scholar
  4. 4.
    Leadbetter EA, Rifkin IR, Hohlbau AH, Beaudette B, Shlomchik MJ, Marshak-Rothstein A. Immune complexes activate autoreactive B cells by co-engagement of surface IgM and Toll-like receptors. Nature 2002;419:603–7.CrossRefGoogle Scholar
  5. 5.
    Lenert P. Inhibitory oligodeoxynucleotides—therapeutic promise for systemic autoimmune diseases? Clin Exp Immunol 2004;140:1–10.CrossRefGoogle Scholar
  6. 6.
    Latz E, Schoenemeyer A, Visintin A, Fitzgerald KA, Monks BG, Knetter CF, Lien E, Nilsen NJ, Espevic T, Golenbock DT. TLR9 signals after translocating from the ER to CpG DNA in the lysosome. Nat Immunol 2004;5:190–8.PubMedCrossRefGoogle Scholar
  7. 7.
    Ahmad-Nejad P, Hacker H, Rutz M, Bauer S, Vabulas R, Wagner H. Bacterial CpG-DNA and lipopolysaccharides activate Toll-like receptors at distinct cellular compartments. Eur J Immunol 2002;32:1958–68.PubMedCrossRefGoogle Scholar
  8. 8.
    Schnare M, Holt AC, Takeda K, Akira S, Medzhitov R. Recognition of CpG DNA is mediated by signaling pathways dependent on the adaptor protein MyD88. Curr Biol 2000;10:1139–42.PubMedCrossRefGoogle Scholar
  9. 9.
    Hemmi H, Takeuchi O, Kawal T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K, Akira S. A Toll-like receptor recognizes bacterial DNA. Nature 2000;408:740–5.PubMedCrossRefGoogle Scholar
  10. 10.
    Rutz M, Metzger J, Gellert T, Luppa P, Lipford GB, Wagner H, Bauer S. Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur J Immunol 2004;34:2541–50.PubMedCrossRefGoogle Scholar
  11. 11.
    Ishii KJ, Takeshita F, Gursel I, Gursel M, Conover J, Nussenzweig A, et al. Potential role of phosphatidylinositol 3 kinase, rather than DNA-dependent protein kinase, in CpG DNA-induced immune activation. J Exp Med 2002;196:269–74.PubMedCrossRefGoogle Scholar
  12. 12.
    Lenert P, Stunz L, Yi AK, Krieg AM, Ashman RF. CpG stimulation of primary mouse B cells is blocked by inhibitory oligodeoxyribonucleotides at a site proximal to NF-kappaB activation. Antisense Nucleic Acid Drug Dev 2001;11(4):247–56.PubMedCrossRefGoogle Scholar
  13. 13.
    Lenert P, Yi AK, Krieg AM, Stunz LL, Ashman RF. Inhibitory oligonucleotides block the induction of AP-1 transcription factor by stimulatory CpG oligonucleotides in B cells. Antisense Nucleic Acid Drug Dev 2003;13(3):143–50.PubMedCrossRefGoogle Scholar
  14. 14.
    Ballas ZA, Rasmussen WL, Krieg AM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J Immunol 1996;157:1840–5.PubMedGoogle Scholar
  15. 15.
    Klinman DM, Yi AK, Beaucage SL, Conover J, Krieg AM. CpG motifs present in bacteria DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon γ. Proc Natl Acad Sci 1996;93:2879–83.PubMedCrossRefGoogle Scholar
  16. 16.
    Lenert P, Goeken AJ, Ashman RF. Extended sequence preferences for oligodeoxyribonucleotide activity. Immunology 2006;117:474–81.PubMedCrossRefGoogle Scholar
  17. 17.
    Yi AK, Chang M, Peckham DW, Krieg AM, Ashman RF. CpG oligodeoxyribonucleotides rescue mature spleen B cells from spontaneous apoptosis and promote cell cycle entry. J Immunol 1998;160:5898–906.PubMedGoogle Scholar
  18. 18.
    Hartmann G, Krieg AM. Mechanism and function of a newly identified CpG DNA motif in human primary B cells. J Immunol 2000;164:944–53.PubMedGoogle Scholar
  19. 19.
    Hartmann G, Weeratna RD, Ballas ZK, et al. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J Immunol 2000;164:1617–24.PubMedGoogle Scholar
  20. 20.
    Verthelyi D, Ishii KJ, Gursel M, Takeshita F, Klinman D. Human peripheral blood cells differentially recognize and respond to two distinct CpG motifs. J Immunol 2001;166:2372–7.PubMedGoogle Scholar
  21. 21.
    Krug A, Rothenfusser S, Hornung V, Jahrsdorfer B, Blackwell S, Ballas Z, Endres S, Krieg AM, Hartmann G. Identification of CpG oligonucleotide sequences with high induction of INF-α/β in plasmacytoid dendritic cells. Eur J Immunol 2001;31:2154–63.PubMedCrossRefGoogle Scholar
  22. 22.
    Hartmann G, Battiany J, Poeck H, Wagner M, Kerkmann M, Lubenow N, Rothenfusser S, Endres S. Rational design of new CpG oligonucleotides that combine B cell activation with high IFN-α induction in plasmacytoid dendritic cells. Eur J Immunol 2003;33:1633–41.PubMedCrossRefGoogle Scholar
  23. 23.
    Stunz LL, Lenert P, Peckham D, Yi AK, Haxhinasto S, Chang M, et al. Inhibitory oligonucleotides specifically block effects of stimulatory CpG oligonucleotides in B cells. Eur J Immunol 2002;32(5):1212–22.PubMedCrossRefGoogle Scholar
  24. 24.
    Ashman RF, Goeken JA, Drahos J, Lenert P. Sequence requirements for oligodeoxyribonucleotide inhibitory activity. Int Immunol 2005;17(4):411–20.PubMedCrossRefGoogle Scholar
  25. 25.
    Bauer S, Kirschning CJ, Hacker H, Redecke V, Hausmann S, Akira S, et al. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci 2001;98:9237–42.PubMedCrossRefGoogle Scholar
  26. 26.
    Viglianti GA, Lau CM, Hanley TM, Miko BA, Shlomchik MJ, Marshak-Rothstein A. Activaton of autoreactive B cells by CpG dsDNA. Immunity 2003;19:837–47.PubMedCrossRefGoogle Scholar
  27. 27.
    Pawar RD, Patole PS, Ellwart A, Lech M, Segerer S, Schlondorff D, Anders HJ. Ligands to nucleic acid-specific Toll-like receptors and the onset of lupus nephritis. J Am Soc Nephrol 2006;17(12):3365–73.PubMedCrossRefGoogle Scholar
  28. 28.
    Patole PS, Zecher D, Pawar RD, Grone HJ, Schlondorff D, Anders HJ. G-rich DNA suppresses systemic lupus. J Am Soc Nephrol 2005;16(11):3273–80.PubMedCrossRefGoogle Scholar
  29. 29.
    Duramad O, Fearon KL, Chang B, Chan JH, Gregorio J, Coffman RL, Barrat FJ. Inhibitors of TLR-9 act on multiple cell subsets in mouse and man in vitro and prevent death in vivo from systemic inflammation. J Immunol 2005;174(9):5193–200.PubMedGoogle Scholar
  30. 30.
    Yamada H, Gursel I, Takeshita F, Conover J, Ishii KJ, Gursel M, Takeshita S, Klinman DM. Effect of Suppressive DNA on CpG-Induced Immune Activation. J Immunol 2002;169:5590–4.PubMedGoogle Scholar
  31. 31.
    Gursel I, Gursel M, Yamada H, Ishii KJ,Takeshita F, Klinman DM. Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation. J Immunol 2003;117:1393–400.Google Scholar
  32. 32.
    Blasco MA, Gasser SM, Lingner J. Telomeres and telomerase. Genes Dev 1999;13:2353–9.PubMedCrossRefGoogle Scholar
  33. 33.
    Stacey KJ, Young GR, Clark F, Sester DP, Roberts TL, Naik S, Sweet MJ, Hume DA. The molecular basis for the lack of immunostimulatory activity of vertebrate DNA. J Immunol 2003;170:3614–20.PubMedGoogle Scholar
  34. 34.
    Choe J, Kelker MS, Wilson IA. Crystal structure of human Toll-like receptor 3 (TLR3) ectodomain. Science 2005;309:581–5.PubMedCrossRefGoogle Scholar
  35. 35.
    Aderem A, Ulevitch RJ. Toll-like receptors in the induction of the innate immune response. Nature 2000;406:782–7.PubMedCrossRefGoogle Scholar
  36. 36.
    Roberts TL, Sweet MJ, Hume DA, Stacey KJ. Cutting edge: species-specific TLR9-mediated recognition of CpG and non-CpG phosphorothioate-modified oligonucleotides. J Immunol 2005;174:605–8.PubMedGoogle Scholar
  37. 37.
    Pisetsky DS, Reich CF. Inhibition of murine macrophage IL-12 production by natural and synthetic DNA. Clin Immunol 2000;96:198–204.PubMedCrossRefGoogle Scholar
  38. 38.
    Zhu FG, Reich CF, Pisetsky DS. Inhibition of murine dendritic cell activation by synthetic phosphorothioate oligodeoxynucleotides. J Leukoc Biol 2002;72(6):1154–63.PubMedGoogle Scholar
  39. 39.
    Fields ML, Metzgar MH, Hondowicz BD, Kang S, Alexander ST, Hazard KD, Hsu AC, Du Y, Prak EL, Monestier M, Erickson J. Exogenous and endogenous TLR ligands activate anti-chromatin and polyreactive B cells. J Immunol 2006;176:6491–502.PubMedGoogle Scholar
  40. 40.
    Hasegawa K, Hayashi T. Synthetic CpG oligodeoxynucleotides accelerate the development of lupus nephritis during preactive phase in NZB × NZWF1 mice. Lupus 2003;12(11):838–45.PubMedCrossRefGoogle Scholar
  41. 41.
    Dong L, Ito S, Ishii K, Klinman DM. Suppressive oligodeoxynucleotides delay the onset of glomerulonephritis and prolong the survival of lupus-prone NZB × NZW mice. Arthritis Rheum 2005;52(2):651–8.PubMedCrossRefGoogle Scholar
  42. 42.
    Klinman DM, Gursel I, Klaschik S, Dong L, Currie D, Shirota H. Therapeutic potential of oligonucleotides expressing immunosuppressive TTAGGG motifs. Ann NY Acad Sci 2005;1058:87–95.PubMedCrossRefGoogle Scholar
  43. 43.
    Yamada H, Ishii KJ, Klinman DM. Suppressive oligodeoxynucleotides inhibit CpG-induced inflammation of the mouse lung. Crit Care Med 2004;32(10):2045–9.PubMedCrossRefGoogle Scholar
  44. 44.
    Shirota H, Gursel I, Gursel M, Klinman DM. Suppressive oligodeoxynucleotides protect mice from lethal endotoxic shock. J Immunol 2005;174:4579–82.PubMedGoogle Scholar
  45. 45.
    Deng GM, Nilsson IM, Verdrengh M, et al. Intra-articularly localized bacterial DNA containing CpG motifs induces arthritis. Nat Med 1999;5:702–5.PubMedCrossRefGoogle Scholar
  46. 46.
    Zeuner RA, Ishii KJ, Lizak MJ, Gursel I, Yamada H, Klinman DM, Verthelyi D. Reduction of CpG-induced arthritis by suppressive oligodeoxynucleotides. Arthritis Rheum 2002;46(8):2219–24.PubMedCrossRefGoogle Scholar
  47. 47.
    Zeuner RA, Verthelyi D, Gursel M, Ishii KJ, Klinman DM. Influence of stimulatory and suppressive DNA motifs on host susceptibility to inflammatory arthritis. Arthritis Rheum 2003;48(6):1701–7.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  1. 1.Graduate Program in Immunology, Carver College of MedicineUniversity of IowaIowa CityUSA
  2. 2.Department of Internal Medicine, Carver College of MedicineUniversity of IowaIowa CityUSA

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