ODN TTAGGG (A151)

Suppressive oligonucleotide - TLR9, AIM2, and cGAS antagonist

ODN TTAGGG (A151), also known simply as A151, is a synthetic oligonucleotide (ODN) containing 4 consecutive repeats of the immunosuppressive TTAGGG motif commonly found in mammalian telomeric DNA [1].

Initially, this ODN was identified as a TLR9 antagonist that inhibits immune activation by CpG-containing ODNs [2].

Subsequently, the cytosolic DNA sensors (CDSs) AIM2 and IFI16 were identified as additional targets for ODN TTAGGG (A151). By binding to these CDSs, ODN TTAGGG (A151) prevents AIM2 inflammasome activation [3,4].

This ODN is also described as a cGAS inhibitor, acting through competition with DNA [1].

Overall, these findings show that ODN TTAGGG (A151) is a multiple pattern recognition receptor (PRR) suppressor that can prove useful for immunomodulatory studies.

Key features of ODN TTAGGG (A151):

• ODN containing 4 repeats of TTAGGG
• TLR9, AIM2, and cGAS antagonist 
• Each lot is functionally tested

Read our review on cGAS.

References:

1. Steinhagen F. et al., 2017. Suppressive oligodeoxynucleotides containing TTAGGG motifs inhibit cGAS activation in human monocytes. Eur J Immunol. DOI: 10.1002/ eji.201747338.
2. Gursel I. et al., 2003. Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation. J Immunol. 171(3):1393-400.
3. Eichholz K. et al., 2016. Immune-Complexed Adenovirus Induce AIM2-Mediated Pyroptosis in Human Dendritic Cells. PLoS Pathog. 12(9):e1005871.
4. Kaminski J. et al., 2013. Synthetic oligodeoxynucleotides containing suppressive TTAGGG motifs inhibit AIM2 inflammasome activation. J Immunol. 191(7):3876-83.

Figures

Dose-dependent inhibition of TLR9 activity in HEK-Blue™ hTLR9 cells. Cells were treated with the human CpG-ODN 2006 (1 μg/ml) in the presence of increasing concentrations of ODN TTAGGG (A151). After overnight incubation, the NF-κB response was determined using QUANTI-Blue™ Solution, a SEAP detection reagent. Data represent % inhibition of the signal.

Effect of inflammasome inhibitors on IL-1β release by THP1-HMGB1-Lucia™ cells. Cells were primed with LPS (1 μg/ml) for 3 hours prior to treatment with 200 μg/ml MSU (NLRP3 inflammasome inducer) or 0.5 μg/ml Poly(dA:dT) complexes (AIM2 inflammasome inducer) in the presence of 0.5 μM Z-VAD (pan-capase inhibitor), Ac-YVAD-cmk (caspase-1 inhibitor), MCC950 (NLRP3 inflammasome inhibitor), VX-765 (caspase-1 inhibitor) and ODN TTAGGG (A151; AIM2 inhibitor). After overnight incubation, cell supernatants were added to HEK-Blue™ IL-1β cells for 16 hours. IL-1β levels were determined by assessing SEAP activity in the supernatant using QUANTI-Blue™ Solution.

Inhibition of CDS activity in THP1-Dual™ cells. Cells were pre-treated with increasing concentrations of ODN TTAGGG (A151) for 6 hours followed by stimulation with cytosolic double-stranded DNA (1 μg/ml). Prior to experimentation, the DNA was complexed with a transfection reagent to facilitate intracellular delivery. After overnight incubation, the IRF and NF-κB responses were determined using QUANTI-Luc™ 4 Lucia/Gaussia and QUANTI-Blue™ Solution, respectively.

Specifications

Working concentration: 100 nM - 10 µM

Solubility:  5 mg/ml in water

Molecular weight: 7944 g/mol

ODN TTAGGG sequence
5’- tt agg gtt agg gtt agg gtt agg g -3’ (24 mer)
Note: Bases are phosphorothioate-linked (nuclease resistant).

Quality control:
- Biological 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

ODN TTAGGG (A151) is provided lyophilized and is available in two quantities.

tlrl-ttag151:

• 200 μg (25.2 nmol) lyophilized ODN TTAGGG (A151)
• 1.5 ml endotoxin-free water

tlrl-ttag151-1:

• 1 mg (126 nmol) lyophilized ODN TTAGGG (A151)
• 1.5 ml endotoxin-free water

ODN TTAGGG (A151) is shipped at room temperature.

Upon receipt, store at -20 °C.

Details

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 [1-3]. Unmethylated CpG dinucleotides are a hallmark of microbial (bacterial, viral, fungal, and parasite) DNA, as well as mitochondrial self-DNA [3,4]. 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 [1,3].

AIM2 Inflammasome

AIM2 (absent in melanoma 2), a receptor for cytoplasmic DNA, forms an inflammasome with its ligand and ASC to activate caspase-1 [5-7]. AIM2 is an interferon-inducible HIN-200 family member that contains an amino-terminal pyrin domain and a carboxy-terminal oligonucleotide/oligosaccharide-binding domain. AIM2 senses cytoplasmic double-stranded DNA through its oligonucleotide/oligosaccharide-binding domain and interacts with ASC via its pyrin domain to activate caspase-1. The interaction of AIM2 with ASC also leads to the formation of the ASC pyroptosome, which induces pyroptotic cell death in cells containing caspase-1. AIM2 is necessary and sufficient for inflammasome activation in response to cytoplasmic DNA. 

cGAS

Cyclic GMP-AMP synthase (cGAS, cGAMP synthase) is a critical cytosolic DNA sensor that triggers innate immune responses through the production of type I interferons (IFNs) [8]. In response to cytosolic double‑stranded DNA (dsDNA), cGAS produces the cyclic dinucleotide (CDN) 2’3’-cGAMP.
CDNs bind directly to STING, leading to TBK1‑IRF3-mediated activation of IFN-stimulated response elements (ISRE) in the promoters of IFN-stimulated genes (ISG). The most potent agonist of human STING is 2’3’-cGAMP [9,10].

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. Hornung V. et al., 2009. AIM2 recognizes cytosolic dsDNA and forms a caspase-1-activating inflammasome with ASC. Nature. 458(7237):514-8.
6. Fernandes-Alnemri T. et al., 2009. AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature.458(7237):509-13.
7. Bürckstümmer T. et al., 2009. An orthogonal proteomic-genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat Immunol. 10(3):266-72.
8. Sun L. et al., 2013. Cyclic GMP-AMP synthase is a cytosolic DNA sensor that activates the type I interferon pathway. Science 339(6121):786-91.
9. Gao P. et al., 2013. Cyclic [G(2’,5’)pA(3’,5’)p] is the metazoan second messenger produced by DNA-activated cyclic GMP-AMP synthase. Cell. 153(5):1094-107.
10. Ablasser A. et al., 2013. cGAS produces a 2’-5’-linked cyclic dinucleotide second messenger that activates STING. Nature. 498(7454):380-4.