Chemistry of CpG DNA

Ekambar R. Kandimalla1, Sudhir Agrawal1

1 Hybridon, Inc., Cambridge, Massachusetts
Publication Name:  Current Protocols in Nucleic Acid Chemistry
Unit Number:  Unit 4.16
DOI:  10.1002/0471142700.nc0416s12
Online Posting Date:  May, 2003
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library


The vertebrate immune system can recognize specific pathogen‐associated molecular patterns in invading microorganisms, including the unmethylated CpG dinucleotide. This unit discusses the receptors that recognize CpG motifs and important aspects of the sequence context of CpG motifs to the end of understanding and designing CpG DNA for therapeutic purposes.

PDF or HTML at Wiley Online Library

Table of Contents

  • History
  • Receptors
  • Significance of CpG Dinucleotides and Chemistry of CpG DNA
  • Conclusions
  • Literature Cited
  • Figures
  • Tables
PDF or HTML at Wiley Online Library


PDF or HTML at Wiley Online Library



Literature Cited

Literature Cited
   Agrawal, S. 1999a. Factors affecting the specificity and mechanism of action of antisense oligonucleotides. Antisense Nucleic Drug Dev. 9:371‐375.
   Agrawal, S. 1999b. Importance of nucleotide sequence and chemical modifications of antisense oligonucleotides. Biochim. Biophys. Acta 1489:53‐68.
   Agrawal, S. and Kandimalla, E.R. 2000. Antisense therapeutics: Is it as simple as complementary base recognition? Mol. Med. Today 6:72‐81.
   Agrawal, S. and Kandimalla, E.R. 2001. Antisense and/or immunostimulatory oligonucleotide therapeutics. Current Cancer Drug Targets 1:197‐209.
   Agrawal, S. and Kandimalla, E.R. 2002. Medicinal chemistry and therapeutic potential of CpG DNA. Trends Mol. Med. 8:114‐121.
   Agrawal, S. and Zhao, Q. 1998a. Antisense therapeutics. Curr. Opin. Chem. Biol. 2:519‐528.
   Agrawal, S. and Zhao, Q. 1998b. Mixed backbone oligonucleotides: Improvement oligonucleotide‐induced toxicity in vivo. Antisense Nucleic Acid Drug Dev. 8:135‐139.
   Agrawal, S., Iadarola, P.L., Temsamani, J., Zhao, Q., and Shaw, D. 1996. Effect of G‐rich sequences on the synthesis, purification, binding, cell uptake, and hemolytic activity of oligonucleotides. Bioorg. Med. Chem. Let. 6:2219‐2224.
   Ahmad‐Nejad, P., Hacker, H., Rutz, M., Bauer, S., Vabulas, R.M., and Wagner, H. 2002. Bacterial CpG‐DNA and lipopolysaccharides activate Toll‐like receptors at distinct cellular compartments. Eur. J. Immunol. 32:1958‐1968.
   Alexopoulou, L., Holt, A.C., Medzhitov, R., and Flavell, R.A. 2001. Recognition of double‐stranded RNA and activation of NF‐κB by Toll‐like receptor 3. Nature 413:732‐738.
   Ballas, Z.K., Krieg, A.M., Warren, T., Rasmussen, W., Davis, H.L., Waldschmidt, M., and Weiner, G.J. 2001. Divergent therapeutic and immunologic effects of oligodeoxynucleotides with distinct CpG motifs. J. Immunol. 167:4878‐4886.
   Bauer, S., Kirschning, C.J., Hacker, H., Redecke, V., Hausmann, S., Akira, S., Wagner, H., and Lipford, G.B. 2001a. Human TLR9 confers responsiveness to bacterial DNA via species‐specific CpG motif recognition. Proc. Natl. Acad. Sci. U.S.A. 98:9237‐9242.
   Bauer, M., Redecke, V., Ellwart, J.W., Scherer, B., Kremer, J.P., Wagner, H., and Lipford, G.B. 2001b. Bacterial CpG‐DNA triggers activation and maturation of human CD11c‐, CD123+ dendritic cells. J. Immunol. 166:5000‐5007.
   Bird, A.P. 1986. CpG‐rich islands and the function of DNA methylation. Nature 321:209‐213.
   Branda, R.F., Moore, A.L., Mathews, L., McCormack, J.J., and Zon, G. 1993. Immune stimulation by an antisense oligomer complementary to the rev gene of HIV‐1. Biochem. Pharmacol. 45:2037‐2043.
   Dalpke, A.H., Zimmermann, S., Albrecht, I., and Heeg, K. 2002. Phosphodiester CpG oligonucleotides as adjuvants: Polyguanosine runs enhance cellular uptake and improve immunostimulative activity of phosphodiester CpG oligonucleotides in vitro and in vivo. Immunology 106:102‐112.
   Gursel, M., Verthelyi, D., Gursel, I., Ishii, K.J., and Klinman, D.M. 2002. Differential and competitive activation of human immune cells by distinct classes of CpG oligodeoxynucleotides. J. Leukoc. Biol. 71:813‐820.
   Gurunathan, S., Klinman, D.M., and Seder, R.A. 2000. DNA vaccines: Immunology, application, and optimization. Annu. Rev. Immunol. 18:927‐974.
   Hartmann, G., Weeratna, R.D., Ballas, Z.K., Payette, P., Blackwell, S., Suparto, I., Rasmussen, W.L., Waldschmidt, M., Sajuthi, D., Purcell, R.H., Davis, H.L., and Krieg, A.M. 2000. Delineation of a CpG phosphorothioate oligodeoxynucleotide for activating primate immune responses in vitro and in vivo. J. Immunol. 164:1617‐1624.
   Hemmi, H., Takeuchi, O., Kawai, T., Kaisho, T., Sato, S., Sanjo, H., Matsumoto, M., Hoshino, K., Wagner, H., Takeda, K., and Akira, S. 2000. A Toll‐like receptor recognizes bacterial DNA. Nature 408:740‐745.
   Horner, A.A., Takabaysahi, K., Zubeldia, J.M., and Raz, E. 2002. Immunostimulatory DNA‐based therapeutics for experimental and clinical allergy. Allergy 57:24‐29.
   Iho, S., Yamamoto, T., Takahashi, T., and Yamamoto, S. 1999. Oligodeoxynucleotides containing palindrome sequences with internal 5′‐CpG‐3′ act directly on human NK and activated T cells to induce IFN‐γ production in vitro. J. Immunol. 163:3642‐3652.
   Jahrsdorfer, B., Jox, R., Muhlenhoff, L., Tschoep, K., Krug, A., Rothenfusser, S., Meinhardt, G., Emmerich, B., Endres, S., and Hartmann, G. 2002. Modulation of malignant B cell activation and apoptosis by bcl‐2 antisense ODN and immunostimulatory CpG ODN. J. Leukoc. Biol. 72:83‐92.
   Kadowaki, N., Antonenko, S., and Liu, Y.J. 2001. Distinct CpG DNA and polyinosinic‐polycytidylic acid double‐stranded RNA, respectively, stimulate CD11c− type 2 dendritic cell precursors and CD11c+ dendritic cells to produce type I IFN. J. Immunol. 166:2291‐2295.
   Kandimalla, E.R. and Agrawal, S. 2002. Towards optimal design of second‐generation immunomodulatory oligonucleotides. Curr. Op. Mol. Ther. 4:122‐129.
   Kandimalla, E.R., Yu, D., Zhao, Q., and Agrawal, S. 2001. Effect of chemical modifications of cytosine and guanine in a CpG‐motif of oligonucleotides: Structure‐immunostimulatory activity relationships. Bioorg. Med. Chem. 9:807‐813.
   Kandimalla, E.R., Bhagat, L., Yu, D., Cong, Y., Tang, J., and Agrawal, S. 2002. Conjugation of ligands at the 5′‐end of CpG DNA affects immunostimulatory activity. Bioconj. Chem. 13:966‐974.
   Kimura, Y., Sonehara, K., Kuramoto, E., Makino, T., Yamamoto, S., Yamamoto, T., Kataoka, T., and Tokunaga, T. 1994. Binding of oligoguanylate to scavenger receptors is required for oligonucleotides to augment NK cell activity and induce IFN. J. Biochem. 116:991‐994.
   Klinman, D.M., Yi, A.K., Beaucage, S.L., Conover, J., and Krieg, A.M. 1996. CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon γ. Proc. Natl. Acad. Sci. U.S.A. 93:2879‐2883.
   Krieg, A.M. 2002. CpG motifs in bacterial DNA and their immune effects. Annu. Rev. immunol. 20:709‐760.
   Krieg, A.M., Yi, A.K., Matson, S., Waldschmidt, T.J., Bishop, G.A., Teasdale, R., Koretzky, G.A., and Klinman, D.M. 1995. CpG motifs in bacterial DNA trigger direct B‐cell activation. Nature 374:546‐549.
   Krug, A., Rothenfusser, S., Hornung, V., Jahrsdorfer, B., Blackwell, S., Ballas, Z.K., Endres, S., Krieg, A.M., and Hartmann, G. 2001a. Identification of CpG oligonucleotide sequences with high induction of IFN‐α/β in plasmacytoid dendritic cells. Eur. J. Immunol. 31:2154‐2163.
   Krug, A., Towarowski, A., Britsch, S., Rothenfusser, S., Hornung, V., Bals, R., Giese, T., Engelmann, H., Endres, S., Krieg, A.M., and Hartmann, G. 2001b. Toll‐like receptor expression reveals CpG DNA as a unique microbial stimulus for plasmacytoid dendritic cells which synergizes with CD40 ligand to induce high amounts of IL‐12. Eur. J. Immunol. 31:3026‐3037.
   Lewis, E.J., Agrawal, S., Bishop, J., Chadwick, J., Cristensen, N.D., Cuthill, S., Dunford, P., Field, A.K., Francis, J., Gibson, V., Greenham, A.K., Kelly, F., Kilkuskie, R., Kreider, J.W., Mills, J.S., Mulqueen, M., Roberts, N.A., Roberts, P., and Szymkowski, D.E. 2000. Non‐specific antiviral activity of antisense molecules targeted to the E1 region of human papillomavirus. Antiviral Res. 48:187‐196.
   Lien, E. and Ingalls, R.R. 2002. Toll‐like receptors. Crit. Care Med. 30:S1‐S11.
   Messina, J.P., Gilkeson, G.S., and Pisetsky, D.S. 1991. Stimulation of in vitro murine lymphocyte proliferation by bacterial DNA. J. Immunol. 147:1759‐1764.
   Pearson, A.M., Rich, A., and Krieger, M. 1993. Polynucleotide binding to macrophage scavenger receptors depends on the formation of base‐quartet‐stabilized four‐stranded helices. J. Biol. Chem. 268:3546‐3554.
   Pisetsky, D.S. 1999. The influence of base sequence on the immunostimulatory properties of DNA. Immunol. Res. 19:35‐46.
   Rankin, R., Pontarollo, R., Ioannou, X., Krieg, A.M., Hecker, R., Babiuk, L.A., and van den Hurk, S.v.d.L. 2001. CpG motif identification for veterinary and laboratory species demonstrates that sequence recognition is highly conserved. Antisense Nucleic Acid Drug Dev. 11:333‐340.
   Rothenfusser, S., Hornung, V., Krug, A., Towarowski, A., Krieg, A.M., Endres, S., and Hartmann, G. 2001. Distinct CpG oligonucleotide sequences activate human gamma delta T cells via interferon‐α/‐β. Eur. J. Immunol. 31:3525‐3534.
   Sato, Y., Roman, M., Tighe, H., Lee, D., Corr, M., Nguyen, M.D., Silverman, G.J., Lotz, M., Carson, D.A., and Raz, E. 1996. Immunostimulatory DNA sequences necessary for effective intradermal gene immunization. Science 273:352‐354.
   Shirota, H., Sano, K., Kikuchi, T., Tamura, G., and Shirato, K. 2000. Regulation of murine airway eosinophilia and Th2 cells by antigen‐conjugated CpG oligodeoxynucleotides as a novel antigen‐specific immunomodulator. J. Immunol. 164:5575‐5582.
   Shirota, H., Sano, K., Hirasawa, N., Terui, T., Ohuchi, K., Hattori, T., Shirato, K., and Tamura, G. 2001. Novel roles of CpG oligodeoxynucleotides as a leader for the sampling and presentation of CpG‐tagged antigen by dendritic cells. J. Immunol. 167:66‐74.
   Sonehara, K., Saito, H., Kuramoto, E., Yamamoto, S., Yamamoto, T., and Tokunaga, T. 1996. Hexamer palindromic oligonucleotides with 5′‐CG‐3′ motif(s) induce production of interferon. J. Interferon Cytokine Res. 16:799‐803.
   Stacey, K.J., Sester, D.P., Sweet, M.J., and Hume, D.A. 2000. Macrophage activation by immunostimulatory DNA. Curr. Top. Microbiol. Immunol. 247:41‐58.
   Takeshita, F., Leifer, C.A., Gursel, I., Ishii, K.J., Takeshita, S., Gursel, M., and Klinman, D.M. 2001. Role of Toll‐like receptor 9 in CpG DNA‐induced activation of human cells. J. Immunol. 167:3555‐3558.
   Tang, J.Y., Roskey, A.R., Li, Y., and Agrawal, S. 1995. Enzymatic synthesis of stereoregular (all Rp) oligonucleotide phosphorothioate and its properties. Nucleosides Nucleotides 14:985‐989.
   Tighe, H., Takabayashi, K., Schwartz, D., Van Nest, G., Tuck, S., Eiden, J.J., Kagey‐Sobotka, A., Creticos, P.S., Lichtenstein, L.M., Spiegelberg, H.L., and Raz, E. 2000a. Conjugation of immunostimulatory DNA to the short ragweed allergen amb a 1 enhances its immunogenicity and reduces its allergenicity. J. Allergy Clin. Immunol. 106:124‐134.
   Tighe, H., Takabayashi, K., Schwartz, D., Marsden, R., Beck, L., Corbeil, J., Richman, D.D., Eiden, J.J. Jr., Spiegelberg, H.L., and Raz, E. 2000b. Conjugation of protein to immunostimulatory DNA results in a rapid,long‐lasting and potent induction of cell‐mediated and humoral immunity. Eur. J. Immunol. 30:1939‐1947.
   Tokunaga, T., Yamamoto, H., Shimada, S., Abe, H., Fukuda, T., Fujisawa, Y., Furutani, Y., Yano, O., Kataoka, T., Sudo, T., Makiguchi, N., and Suganuma, T. 1984. Antitumor activity of deoxyribonucleic acid fraction from Mycobacterium bovis BCG. I. Isolation, physicochemical characterization, and antitumor activity. J. Natl. Cancer Inst. 72:955‐962.
   Van Uden, J., and Raz, E. 2000. Introduction to immunostimulatory DNA sequences. Springer Semin. Immunopathol. 22:1‐9.
   Verthelyi, D., Ishii, K., Gursel, M., Takeshita, F., and Klinman, D. 2001. Human peripheral blood cells differentially recognize and respond to two distinct CpG motifs. J. Immunol. 166:2372‐2377.
   Verthelyi, D., Kenney, R.T., Seder, R.A., Gam, A.A., Friedag, B., and Klinman, D.M. 2002. CpG oligodeoxynucleotides as vaccine adjuvants in primates. J. Immunol. 168:1659‐1663.
   Wernette, C.M., Smith, B.F., Barksdale, Z.L., Hecker, R., and Baker, H.J. 2002. CpG oligodeoxynucleotides stimulate canine and feline immune cell proliferation. Vet. Immunol. Immunopathol. 84:223‐236.
   Yamamoto, S., Yamamoto, T., Kataoka, T., Kuramoto, E., Yano, O., and Tokunaga, T. 1992a. Unique palindromic sequences in synthetic oligonucleotides are required to induce INF and augment INF‐mediated natural killer activity. J. Immunol. 148:4072‐4076.
   Yamamoto, S., Yamamoto, T., Shimada, S., Kuramoto, E., Yano, O., Kataoka, T., and Tokunaga, T. 1992b. DNA from bacteria, but not from vertebrates, induces interferons, activates natural killer cells and inhibits tumor growth. Microbiol. Immunol. 36:983‐997.
   Yamamoto, S., Yamamoto, T., Iho, S., and Tokunaga, T. 2000. Activation of NK cell (human and mouse) by immunostimulatory DNA sequence. Springer Semin. Immunopathol. 22:35‐43.
   Yu, D., Kandimalla, E.R., Roskey, A., Zhao, Q., Chen, L., Chen, J., and Agrawal, S. 2000a. Stereo‐enriched phosphorothioate oligodeoxynucleotides: Synthesis, biophysical and biological properties. Bioorg. Med. Chem. 8:275‐284.
   Yu, D., Zhao, Q., Kandimalla, E.R., and Agrawal, S. 2000b. Accessible 5′‐end of CpG‐containing phosphorothioate oligodeoxynucleotides is essential for immunostimulatory activity. Bioorg. Med. Chem. Lett. 10:2585‐2588.
   Yu, D., Kandimalla, E.R., Zhao, Q., Cong, Y., and Agrawal, S. 2001a. Modulation of immunostimulatory activity of CpG oligonucleotides by site‐specific deletion of nucleobases. Bioorg. Med. Chem. Lett. 11:2263‐2267.
   Yu, D., Kandimalla, E.R., Zhao, Q., Cong, Y., and Agrawal, S. 2001b. Immunostimulatory activity of CpG oligonucleotides containing non‐ionic methylphosphonate linkages. Bioorg. Med. Chem. 9:2803‐2808.
   Yu, D., Zhu, F.G., Bhagat, L., Wang, H., Kandimalla, E.R., Zhang, R., and Agrawal, S. 2002a. Potent CpG oligonucleotides containing phosphodiester linkages: In vitro and in vivo immunostimulatory properties. Biochem. Biophys. Res. Commun. 297:83‐90.
   Yu, D., Kandimalla, E.R., Zhao, Q., Cong, Y., and Agrawal, S. 2002b. Immunostimulatory properties of phosphorothioate CpG DNA containing both 3′‐5′‐ and 2′‐5′‐internucleotide linkages. Nucl. Acids Res. 30:1613‐1619.
   Yu, D., Kandimalla, E.R., Cong, Y., Tang, J., Tang, J.Y., Zhao, Q., and Agrawal, S. 2002c. Design, synthesis, and immunostimulatory properties of CpG DNAs containing alkyl‐linker substitutions: Role of nucleosides in the flanking sequences. J. Med. Chem. 45:4540‐4548.
   Zhang, Y., Shoda, L.K., Brayton, K.A., Estes, D.M., Palmer, G.H., and Brown, W.C. 2001. Induction of interleukin‐6 and interleukin‐12 in bovine B lymphocytes, monocytes, and macrophages by a CpG oligodeoxynucleotide (ODN 2059) containing the GTCGTT motif. J. Interferon Cytokine Res. 21:871‐881.
   Zhao, Q., Temsamani, J., Iadarola, P.L., Jiang, Z., and Agrawal, S. 1996. Effect of different chemically modified oligodeoxynucleotides on immune stimulation. Biochem. Pharmacol. 51:173‐182.
   Zhao, Q., Temsamani, J., Zhou, R.Z., and Agrawal, S. 1997. Pattern and kinetics of cytokine production following administration of phosphorothioate oligonucleotides in mice. Antisense Nucleic Acid Drug. Dev. 7:495‐502.
   Zhao, Q., Yu, D., and Agrawal, S. 1999. Site of chemical modifications in CpG containing phosphorothioate oligodeoxynucleotide modulates its immunostimulatory activity. Bioorg. Med. Chem. Lett. 9:3453‐3458.
   Zhao, Q., Yu, D., and Agrawal, S. 2000. Immunostimulatory activity of CpG containing phosphorothioate oligodeoxynucleotide is modulated by modification of a single deoxynucleoside. Bioorg. Med. Chem. Lett. 10:1051‐1054.
PDF or HTML at Wiley Online Library