ODN 2006 (ODN 7909)

Class B CpG oligonucleotide - Human TLR9-preferred ligand

TLR9 activation with ODN 2006

ODN 2006 (ODN 7909) is a Class B CpG oligonucleotide (ODN) with a preference for the human Toll-like receptor 9 (TLR9). It is a short synthetic single-stranded DNA molecule containing unmethylated CpG dinucleotides (CpG motifs). These unmethylated CpG motifs mimic microbial DNA and act as immunostimulants via TLR9. 

 More details

Mode of action:

Stimulatory CpG ODNs are internalized and activate the endosomal receptor TLR9. Activation of TLR9 triggers NF-κB- and interferon regulatory factor (IRF)-mediated pro-inflammatory responses upon the recognition of unmethylated cytosine-phosphorothioate-guanosine (CpG) forms of DNA [1-3]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA and mitochondrial self-DNA [3, 4]. Class B (Type K) CpG ODNs, such as ODN 2006, contain a full phosphorothioate backbone with one or more CpG dinucleotides. They strongly activate B cells but weakly stimulate IFN-α secretion in plasmacytoid dendritic cells [5]. 

InvivoGen's ODN 2006 is a strong TLR9 agonist as verified using our HEK-Blue™ reporter cell lines expressing human or mouse TLR9 (see figure). Moreover, ODN 2006 activates the hTLR9-dependent NF-κB and IRF pathways, as assessed using our THP1-Dual™ hTLR9 reporter cell line expressing two reporter genes, for the NF-κB-inducible SEAP and IRF-inducible Lucia luciferase, as well as hTLR9 (see figure). 

Key features of ODN 2006:

• Potent activator of human TLR9
• Synthetic ODN with unmethylated CpG motifs
• Each lot is functionally tested
• High-quality, pre-clinical ODN 2006 VacciGrade™ is also available for in vivo studies

Get more information about CpG ODNs Classes.

Read our review on TLR9 agonists: double-edged sword for immune therapies.

References

1. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
2. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
3. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
4. Kumar V., 2021. The trinity of cGAS, TLR9, and ALRs: guardians of the cellular galaxy against host-derived self-DNA. Front. Immunol. 11:624597.
5. Krieg A.M. et al., 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 374(6522):546-9.

Figures

Species-driven TLR9 differential responses. HEK-Blue™ mTLR9 and HEK-Blue™ human (h)TLR9 cells were cultured in HEK-Blue™ Detection reagent and stimulated for 24 hours with 100 nM of the following TLR9 agonists: ODN 1826, ODN 2006, and ODN 2007. Human TNF-α (1 ng/ml) served as an NF-κ-positive control. After 24h incubation, the NF-κB-induced SEAP activity was assessed by measuring the SEAP level in the supernatant. Data are shown as optical density (OD) at 650 nm (mean ± SEM).

THP1-Dual™ hTLR9 cells were incubated with increasing concentrations of ODN 2006. After overnight incubation, the NF-κB response was assessed by measuring the SEAP activity using QUANTI-Blue™ Solution. Data are shown as a fold increase (mean ± SEM).

THP1-Dual™ hTLR9 cells were incubated with increasing concentrations of ODN 2006. After overnight incubation, the interferon regulatory receptor (IRF) activity was assessed by measuring the Lucia luciferase activity in the supernatant using QUANTI‑Luc™ 4 Lucia/Gaussia. Data are shown as a fold increase (mean ± SEM).

Specifications

Synonyms: ODN 7909, PF_3512676

Specificity: Human TLR9 agonist

Working concentration: 1-5 µM

Solubility:  5 mg/ml in water

ODN 2006 sequence:
5’-tcgtcgttttgtcgttttgtcgtt-3’ (24 mer)
Note: Bases are phosphorothioate (nuclease resistant).

Quality control:

• TLR9 activity has been tested using HEK-Blue™ TLR9 cells.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ TLR2 and HEK-Blue™ TLR4 cells.

Contents

ODN2006 is provided lyophilized and is available in three quantities:

tlrl-2006 (formerly tlrl-hodnb):

• 200 μg (25.96 nmol) lyophilized ODN2006
• 1.5 ml sterile endotoxin-free water

tlrl-2006-1 (formerly tlrl-hodnb-1):

• 1 mg (129.8 nmol) lyophilized ODN2006
• 1.5 ml sterile endotoxin-free water

tlrl-2006-5 (formerly tlrl-hodnb-5):

• 5 mg (649 nmol) lyophilized ODN2006
• 10 ml sterile endotoxin-free water

ODN 2006 (ODN 7909) is shipped at room temperature.

Upon receipt, store at -20 °C.

Details

CpG ODNs

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs (CpG ODNs), such as ODN 1018, have been extensively studied as adjuvants [1]. These CpG motifs are present at a 20-fold greater frequency in bacterial DNA compared to mammalian DNA [2]. CpG ODNs are recognized by the Toll-like receptor 9 (TLR9), which is expressed on human B cells and plasmacytoid dendritic cells (pDCs), thereby inducing Th1-dominated immune responses [3]. Pre-clinical studies, conducted in rodents and non-human primates, as well as human clinical trials, have demonstrated that CpG ODNs can significantly improve vaccine-specific antibody responses [1]. Three types of stimulatory CpG ODNs have been identified, types A, B, and C, which differ in their immune-stimulatory activities [4-5]. 

Toll-like receptor 9

The Toll-like Receptor 9 (TLR9) is an endosomal receptor that triggers NF-κB- and interferon regulatory factor (IRF)-mediated pro-inflammatory responses upon the recognition of unmethylated cytosine-phosphorothioate-guanosine (CpG) forms of DNA [6-8]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA, as well as mitochondrial self-DNA [8,9]. These TLR9 agonists can be mimicked by synthetic oligonucleotides containing CpG motifs (CpG ODNs), which have been extensively studied to improve adaptive immune responses in the context of vaccination [6,8].

TLR9 is mainly expressed in subsets of Dendritic Cells and B cells of all mammals. In rodents, but not in humans, TLR9 is also expressed in monocytes and macrophages [8]. The structure of the receptor varies by 24% between human TLR9 (hTLR9) and mouse TLR9 (mTLR9) [8]. They recognize different CpG motifs, the optimal sequences being GTCGTT and GACGTT for hTLR9 and mTLR9, respectively [10].
 

References:

1. Steinhagen F. et al., 2011. TLR-based immune adjuvants. Vaccine 29(17):3341-55.
2. Hemmi H. et al., 2000. A Toll-like receptor recognizes bacterial DNA. Nature 408:740-5.
3. Coffman RL. et al., 2010. Vaccine adjuvants: Putting innate immunity to work. Immunity 33(4):492-503.
4. Krug A. et al., 2001. Identification of CpG oligonucleotide sequences with high induction of IFN-alpha/beta in plasmacytoid dendritic cells. Eur J Immunol, 31(7): 2154-63.
5. Marshall JD. et al., 2005. Superior activity of the type C class of ISS in vitro and in vivo across multiple species. DNA Cell Biol. 24(2):63-72.
6. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
7. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
8. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
9. Kumar V., 2021. The trinity of cGAS, TLR9, and ALRs: guardians of the cellular galaxy against host-derived self-DNA. Front. Immunol. 11:624597.
10. Bauer S. et al., 2001. Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. Proc Natl Acad Sci USA, 98(16):9237-42.