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ODN 1585 - TLR9 ligand

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ODN 1585

CpG ODN, Class A (mouse) - TLR9 agonist

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200 µg

1 mg

5 mg

Bulk

tlrl-1585
+-
$214

Class A CpG oligonucleotide - Mouse TLR9-preferred ligand

TLR9 activation with ODN 1585
TLR9 activation with ODN 1585

ODN 1585 is a Class A CpG oligonucleotide (ODN), specifically designed for mouse Toll-like receptor 9 (TLR9) [1]. CpG ODNs are short synthetic single-stranded DNA molecules containing unmethylated CpG dinucleotides (CpG motifs). These unmethylated CpG motifs mimic microbial DNA and act as immunostimulants via TLR9. 

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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 [2-4]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA and mitochondrial self-DNA [4,5].

CpG Class A ODNs, such as ODN 1585, are characterized by a phosphodiester (PO) central CpG-containing palindromic motif and a phosphorothioate (PS)-modified 3’ poly-G string.

In HEK-Blue™ mTLR9 reporter cells, ODN 1585 efficiently activates mTLR9. Interestingly, ODN 1585 is able to activate the human (h)TLR9-mediated NF-κB and IRF pathways as assessed using InvivoGen's THP1-Dual™ hTLR9 reporter cell line. This monocytic cell line overexpresses the human TLR9 gene as well as features two reporter genes for the NF-κB-inducible SEAP and IRF-inducible Lucia luciferase (see figures).

 

Key features of ODN 1585

  • Potent activator of mouse TLR9
  • Synthetic ODN with unmethylated CpG motifs
  • Each lot is functionally tested
  • High-quality, pre-clinical ODN 1585 VacciGrade™ is also available for in vivo studies

 

Get more information about CpG ODNs Classes.

 

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

 

 

References

1. Ballas ZK. et al., 2001. Divergent therapeutic and immunologic effects of oligodeoxynucleotides with distinct CpG motifs. J Immunol. 167(9):4878-86.
2. Kumagai Y. et al., 2008. TLR9 as a key receptor of the recognition of DNA. Adv. Drug. Deliv. Rev. 60(7):795-804.
3. Heinz L.X. et al., 2021. TASL is the SLC15A4-associated adaptor for IRF5 activation by TLR7-9. Nature. 581(7808):316-322.
4. Kayraklioglu N. et al., 2021. CpG oligonucleotides as vaccine adjuvants. DNA Vaccines: Methods and Protocols. Methods in Molecular Biology. Vol. 2197. p51-77.
5. Kumar V., 2021. The trinity of cGAS, TLR9, and ALRs: guardians of the cellular galaxy against host-derived self-DNA. Front. Immunol. 11:624597.
6. Krieg A.M. et al., 1995. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature. 374(6522):546-9.

Figures

Dose-dependent NF-κB response of ODN 1585 in HEK-Blue™ mTLR9 cells
Dose-dependent NF-κB response of ODN 1585 in HEK-Blue™ mTLR9 cells

Dose-dependent NF-κB response in HEK-Blue™ mTLR9 cells. HEK-Blue™ mTLR9 cells were incubated with increasing concentrations of ODN 1585 VacciGrade™. After 24h, the mTLR9-induced NF‑κB response was assessed by measuring the SEAP activity using QUANTI-Blue™. Data are shown as optical density (OD) at 650 nm (mean + SEM).

NF-κB and IRF responses of ODN 1585 in THP1-Dual™ hTLR9 cells
NF-κB and IRF responses of ODN 1585 in THP1-Dual™ hTLR9 cells

Dose-dependent NF-κB and IRF responses in THP1-Dual™ hTLR9 cells. Cells were incubated with increasing concentrations of ODN 1585 VacciGrade™. After 24h, the hTLR9 induced (A) NF‑κB and (B) IRF responses were assessed by measuring SEAP and Lucia activity using QUANTI-Blue™ and QUANTI-Luc™, respectively. Data are shown as optical density (OD) at 650 nm or in fold increase over non-induced cells.

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Specifications

Specificity: Mouse TLR9 agonist.

Working concentration: 1-5 µM.

Solubility:  5 mg/ml in water.

ODN 1585 sequence: 5’- ggGGTCAACGTTGAgggggg -3’ (20 mer).
Note: Bases in capital letters are phosphodiester and those in lowercase are phosphorothioate.

Quality control:

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Contents

ODN 1585 is provided lyophilized and is available in three quantities.

tlrl-1585 (formerly tlrl-modna):

  • 200 μg (31.1 nmol) lyophilized ODN 1585
  • 1.5 ml sterile endotoxin-free water

tlrl-1585-1 (formerly tlrl-modna-1):

  • 1 mg (155.5 nmol) lyophilized ODN 1585
  • 1.5 ml sterile endotoxin-free water

tlrl-1585-5 (formerly tlrl-modna-1):

  • 5 mg (777.5 nmol) lyophilized ODN 1585
  • 10 ml sterile endotoxin-free water

room temperature ODN 1585 is shipped at room temperature.

store Upon receipt, store at -20 °C.

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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.

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Citations

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