Suppressive oligonucleotide - TLR9, AIM2 and cGAS antagonist

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

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

Subsequently, additional targets for this inhibitor were found: by binding to the cytosolic DNA sensors (CDSs) AIM2 and IFI16, 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 receptors (PRR) suppressor that can prove useful for immunomodulatory studies.

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™, 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™.

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™ and QUANTI-Blue™, respectively.

Specifications

Working concentration: 100 nM - 10 µM

Solubility:  5 mg/ml in water

Molecular weight: 7944 g/mol

ODN TTAGGG sequence5’- 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.