Single Stranded DNA Immune Modulators with Unmethylated CpG Motifs: Structure and Molecular Recognition by Toll-Like Receptor 9

Author(s): Krisztina Fehér*.

Journal Name: Current Protein & Peptide Science

Volume 20 , Issue 11 , 2019

Become EABM
Become Reviewer

Graphical Abstract:


Single stranded microbial DNA fragments with unmethylated deoxycytidylyldeoxyguanosine dinucleotide (CpG) motifs are interpreted as danger signals by the innate immune system via recognition by the Toll-like Receptor 9 (TLR9). Their synthetic analogues, Oligodeoxynucleotides (ODN) comprise a promising class of immune modulators with potential applications in the treatment of multiple diseases, such as cancer, autoimmune diseases or allergy. ODN molecules contain a core hexamer sequence, which is species specific consisting of GACGTT and AACGT for mouse and GTCGTT in humans. Assessment of structural features of different type of ODNs is highly challenging. NMR spectroscopic insights were gained for a short, single CpG motif containing ODN 1668. The structural basis of ODN recognition by TLR9 recently started to unravel as crystal structures of TLR9 orthologues in complex with ODN 1668 were solved. Systematic investigations of ODN sequences revealed that ODNs with a single CpG motif are capable of activating mouse TLR9, but two closely positioned CpG motifs are necessary for activation of human TLR9. Furthermore, longer ODNs with TCC and TCG sequences at the 5’ end were shown to activate TLR9 with higher efficiency. It was revealed that 5’-xCx motif containing short ODNs (sODN) are able to augment the immune response of short, single CpG containing ODNs, which are incapable of activating of TLR9 alone. All these observations pointed to the existence of a second binding site on TLR9, which was characterized in crystal structures that delivered further insights of the nucleic acid recognition of the innate immune system by TLR9.

Keywords: Innate immunity, molecular recognition, immune modulation, single stranded DNA, CpG motif, Pattern Recognition Receptors, Pathogen Associated Molecular Patterns, Toll-like receptor 9.

Bartneck, M. Immunomodulatory nanomedicine. Macromol. Biosci., 2017, 17(10), 13.
Medzhitov, R.; Janeway, C.A. An ancient system of host defense. Curr. Opin. Immunol., 1998, 10(1), 12-15.
Krieg, A.M.; Yi, A.K.; Matson, S.; Waldschmidt, T.J.; Bishop, G.A.; Teasdale, R.; Koretzky, G.A.; Klinman, D.M. CPG motifs in bacterial-DNA trigger direct B-cell activation. Nature, 1995, 374(6522), 546-549.
Medzhitov, R. Toll-like receptors and innate immunity. Nat. Rev. Immunol., 2001, 1(2), 135-145.
Kumagai, Y.; Takeuchi, O.; Akira, S. TLR9 as a key receptor for the recognition of DNA. Adv. Drug Deliv. Rev., 2008, 60(7), 795-804.
Hornung, V.; Rothenfusser, S.; Britsch, S.; Krug, A.; Jahrsdorfer, B.; Giese, T.; Endres, S.; Hartmann, G. Quantitative expression of Toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides. J. Immunol., 2002, 168(9), 4531-4537.
Hemmi, H.; Takeuchi, O.; Kawai, 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(6813), 740-745.
Klinman, D.M. Immunotherapeutic uses of CpG oligodeoxynucleotides. Nat. Rev. Immunol., 2004, 4(4), 248-257.
Chan, M.P.; Onji, M.; Fukui, R.; Kawane, K.; Shibata, T.; Saitoh, S.; Ohto, U.; Shimizu, T.; Barber, G.N.; Miyake, K. DNase II-dependent DNA digestion is required for DNA sensing by TLR9. Nat. Commun., 2015, 6, 10.
Hara, T.; Tanegashima, K.; Takahashi, R.; Nuriya, H.; Naruse, N.; Tsuji, K.; Shigenaga, A.; Otaka, A. A novel function of a CXC-type chemokine CXCL14 as a specific carrier of CpG DNA into dendritic cells for activating toll-like receptor 9-mediated adaptive immunity. Blood, 2016, 128(22), 5.
Tanegashima, K.; Takahashi, R.; Nuriya, H.; Iwase, R.; Naruse, N.; Tsuji, K.; Shigenaga, A.; Otaka, A.; Hara, T. CXCL14 acts as a specific carrier of CpG DNA into dendritic cells and activates toll-like receptor 9-mediated adaptive immunity. EBioMed., 2017, 24, 247-256.
Montomoli, E.; Piccirella, S.; Khadang, B.; Mennitto, E.; Camerini, R.; De Rosa, A. Current adjuvants and new perspectives in vaccine formulation. Expert Rev. Vaccines, 2011, 10(7), 1053-1061.
Shirota, H.; Tross, D.; Klinman, D.M. CpG oligonucleotides as cancer vaccine adjuvants. Vaccines, 2015, 3(2), 390-407.
Scheiermann, J.; Klinman, D.M. Clinical evaluation of CpG oligonucleotides as adjuvants for vaccines targeting infectious diseases and cancer. Vaccine, 2014, 32(48), 6377-6389.
Pohar, J.; Krajnik, A.K.; Jerala, R.; Bencina, M. Minimal sequence requirements for oligodeoxyribonucleotides activating human TLR9. J. Immunol., 2015, 194(8), 3901-3908.
Heeg, K.; Dalpke, A.; Peter, M.; Zimmermann, S. Structural requirements for uptake and recognition of CpG oligonucleotides. Int. J. Med. Microbiol., 2008, 298(1-2), 33-38.
Klinman, D.M. Use of CpG oligodeoxynucleotides as immunoprotective agents. Expert Opin. Biol. Ther., 2004, 4(6), 937-946.
Krug, A.; Rothenfusser, S.; Hornung, V.; Jahrsdorfer, B.; Blackwell, S.; Ballas, Z.K.; Endres, S.; Krieg, A.M.; Hartmann, G. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur. J. Immunol., 2001, 31(7), 2154-2163.
Hartmann, G.; Krieg, A.M. Mechanism and function of a newly identified CpG DNA moth in human primary B cells. J. Immunol., 2000, 164(2), 944-952.
Vollmer, J.; Jurk, M.; Samulowitz, U.; Lipford, G.; Forsbach, A.; Wullner, M.; Tluk, S.; Hartmann, H.; Kritzler, A.; Muller, C.; Schetter, C.; Krieg, A.M. CpG oligodeoxynucleotides stimulate IFN-gamma-inducible protein-10 production in human B cells. J. Endotoxin Res., 2004, 10(6), 431-438.
Guiducci, C.; Ott, G.; Chan, J.H.; Damon, E.; Calacsan, C.; Matray, T.; Lee, K.D.; Man, R.L.C.; Barrat, F.J. Properties regulating the nature of the plasmacytoid dendritic cell response to Toll-like receptor 9 activation. J. Exp. Med., 2006, 203(8), 1999-2008.
Verthelyi, D.; Ishii, K.J.; Gursel, M.; Takeshita, F.; Klinman, D.M. Human peripheral blood cells differentially recognize and respond to two distinct CpG motifs. J. Immunol., 2001, 166(4), 2372-2377.
Gursel, M.; Verthelyi, D.; Gursel, I.; Ishii, K.J.; Klinman, D.M. Differential and competitive activation of human immune cells by distinct classes of CpG oligodeoxynucleotide. J. Leukoc. Biol., 2002, 71(5), 813-820.
Pohar, J.; Lainscek, D.; Kunsek, A.; Cajnko, M.M.; Jerala, R.; Bencina, M. Phosphodiester backbone of the CpG motif within immunostimulatory oligodeoxynucleotides augments activation of Toll-like receptor 9. Sci. Rep., 2017, 7, 11.
Klinman, D.M.; Yi, A.K.; Beaucage, S.L.; Conover, J.; Krieg, A.M. CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon gamma. Proc. Natl. Acad. Sci. USA, 1996, 93(7), 2879-2883.
Vollmer, J.; Weeratna, R.; Payette, P.; Jurk, M.; Schetter, C.; Laucht, M.; Wader, T.; Tluk, S.; Liu, M.; Davis, H.L.; Krieg, A.M. Characterization of three CpG oligodeoxynucleotide classes with distinct immunostimulatory activities. Eur. J. Immunol., 2004, 34(1), 251-262.
Abel, K.; Wang, Y.C.; Fritts, L.; Sanchez, E.; Chung, E.; Fitzgerald-Bocarsly, P.; Krieg, A.M.; Miller, C.J. Deoxycytidyl-deoxyguanosine oligonucleotide classes A, B, and C induce distinct ctokine gene expression patterns in rhesus monkey peripheral blood mononuclear cells and distinct alpha interferon responses in TLR9-expressing rhesus monkey plasmacytoid dendritic cells. Clin. Diagn. Lab. Immunol., 2005, 12(5), 606-621.
Marshall, J.D.; Fearon, K.L.; Higgins, D.; Hessel, E.M.; Kanzler, H.; Abbate, C.; Yee, P.; Gregorio, J.; Dela Cruz, T.; Lizcano, J.O.; Zolotorev, A.; McClure, H.M.; Brasky, K.M.; Murthy, K.K.; Coffman, R.L.; Van Nest, G. Superior activity of the type C class of ISS in vitro and in vivo across multiple species. DNA Cell Biol., 2005, 24(2), 63-72.
Samulowitz, U.; Weber, M.; Weeratna, R.; Uhlmann, E.; Noll, B.; Krieg, A.M.; Vollmer, J. A novel class of immune-stimulatory CpG oligodeoxynucleotides unifies high potency in type I interferon induction with preferred structural properties. Oligonucleotides, 2010, 20(2), 93-101.
Pohar, J.; Lainscek, D.; Fukui, R.; Yamamoto, C.; Miyake, K.; Jerala, R.; Bencina, M. Species-specific minimal sequence motif for oligodeoxyribonucleotides activating mouse TLR9. J. Immunol., 2015, 195(9), 4396-4405.
Pohar, J.; Lainscek, D.; Ivicak-Kocjan, K.; Cajnko, M.M.; Jerala, R.; Bencina, M. Short single-stranded DNA degradation products augment the activation of toll-like receptor 9. Nat. Commun., 2017, 8, 13.
Ohto, U.; Ishida, H.; Shibata, T.; Sato, R.; Miyake, K.; Shimizu, T. Toll-like receptor 9 contains two DNA binding sites that function cooperatively to promote receptor dimerization and activation. Immunity, 2018, 48(4), 649-658.e4.
Martinez, J.M.; Elmroth, S.K.C.; Kloo, L. Influence of sodium ions on the dynamics and structure of single-stranded DNA oligomers: A molecular dynamics study. J. Am. Chem. Soc., 2001, 123(49), 12279-12289.
Zhang, Y.; Zhou, H.J.; Ou-Yang, Z.C. Stretching single-stranded DNA: Interplay of electrostatic, base-pairing, and base-pair stacking interactions. Biophys. J., 2001, 81(2), 1133-1143.
Zuker, M. Mfold web server for nucleic acid folding and hybridization prediction. Nucleic Acids Res., 2003, 31(13), 3406-3415.
Kerkmann, M.; Costa, L.T.; Richter, C.; Rothenfusser, S.; Battiany, J.; Hornung, V.; Johnson, J.; Englert, S.; Ketterer, T.; Heckl, W.; Thalhammer, S.; Endres, S.; Hartmann, G. Spontaneous formation of nucleic acid-based nanoparticles is responsible for high interferon-alpha induction by CpG-A in plasmacytoid dendritic cells. J. Biol. Chem., 2005, 280(9), 8086-8093.
Narayanan, S.; Dalpke, A.H.; Siegmund, K.; Heeg, K.; Richert, C. CpG Oligonucleotides with modified termini and nicked dumbbell structure show enhanced immunostimulatory activity. J. Med. Chem., 2003, 46(23), 5031-5044.
He, G.Y.; Patra, A.; Siegmund, K.; Peter, M.; Heeg, K.; Dalpke, A.; Richert, C. Immunostimulatory CpG oligonucleotides form defined three-dimensional structures: Results from an NMR study. ChemMedChem, 2007, 2(4), 549-560.
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-alpha induction in plasmacytoid dendritic cells. Eur. J. Immunol., 2003, 33(6), 1633-1641.
Marshall, J.D.; Fearon, K.; Abbate, C.; Subramanian, S.; Yee, P.; Gregorio, J.; Coffman, R.L.; Van Nest, G. Identification of a novel CpG DNA class and motif that optimally stimulate B cell and plasmacytoid dendritic cell functions. J. Leukoc. Biol., 2003, 73(6), 781-792.
Ohto, U.; Shibata, T.; Tanji, H.; Ishida, H.; Krayukhina, E.; Uchiyama, S.; Miyake, K.; Shimizu, T. Structural basis of CpG and inhibitory DNA recognition by toll-like receptor 9. Nature, 2015, 520(7549), 702-U303.
Ishida, H.; Ohto, U.; Shibata, T.; Miyake, K.; Shimizu, T. Structural basis for species-specific activation of mouse toll-like receptor 9. FEBS Lett., 2018, 592(15), 2636-2646.
Collins, B.; Wilson, I.A. Crystal structure of the C-terminal domain of mouse TLR9. Proteins, 2014, 82(10), 2874-2878.
Pohar, J.; Yamamoto, C.; Fukui, R.; Cajnko, M.M.; Miyake, K.; Jerala, R.; Bencina, M. Selectivity of human TLR9 for double CpG motifs and implications for the recognition of genomic DNA. J. Immunol., 2017, 198(5), 2093-2104.
Stacey, K.J.; Young, G.R.; Clark, F.; Sester, D.P.; Roberts, T.L.; Naik, S.; Sweet, M.J.; Hume, D.A. The molecular basis for the lack of immunostimulatory activity of vertebrate DNA. J. Immunol., 2003, 170(7), 3614-3620.
Barton, G.M.; Kagan, J.C. A cell biological view of Toll-like receptor function: Regulation through compartmentalization. Nat. Rev. Immunol., 2009, 9(8), 535-542.
Feher, K. Atomistic simulations of immune stimulatory single stranded bacterial DNA. In ISQBP Presindet's Meeting 2018, Barcelona, Spain 2018.

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2019
Page: [1060 - 1068]
Pages: 9
DOI: 10.2174/1389203720666190830162149
Price: $65

Article Metrics

PDF: 34
PRC: 1