TLR9 Ligands

Three types of stimulatory CpG ODNs have been identified, types A, B and C, which differ in their immune-stimulatory activities. They induce differentially the stimulation of human and murine immune cells in vitro, a species-specificity that is also observed with nonresponsive cells such as HEK293 cells transfected with human or mouse TLR9.
- Type A CpG ODNs are characterized by a phosphodiester central CpG-containing palindromic motif and a phosphorothioate 3ʼ poly-G string. They induce high IFN-α production from plasmacytoid dendritic cells (pDC) but are weak stimulators of TLR9-dependent NF-κB signaling.
- Type B CpG ODNs contain a full phosphorothioate backbone with one or more CpG dinucleotides. They strongly activate B cells but stimulate weakly IFN-α secretion.
- Type C CpG ODNs combine features of both types A and B. They contain a complete phosphorothioate backbone and a CpG containing palindromic motif. Type C CpG ODNs induce strong IFN-α production from pDC and B cell stimulation.

Control CpG ODNs that do not stimulate TLR9 have been designed for each stimulatory CpG ODN. They feature the same sequence as their stimulatory counterparts but contain GpC dinucleotides in place of CpG dinucleotides.
Note: ODN 2006 control was shown to activate PBMC due to the presence of a non-CpG immunostimulatory sequence (Bartz H. et al., Vaccine 2004, 23:148-155).

Stimulatory CpG ODNs are available labeled with FITC at their 3ʼ terminus. FITC-labeled CpG ODNs are u seful to study their cellular uptake and localization by confocal laser-scanning microscopy or flow cytometry.

Stimulatory CpG ODNs and their control ODNs are provided as lyophilized ultrapure DNA and are available in three sizes: 200 μg, 1 mg and 5 mg. They are supplied with 2 ml (for ODN sizes 200 μg and 1 mg) or 10 ml (for ODN size 5 mg) sterile endotoxin-free water.
Labeled CpG ODNs are provided as 50 μg lyophilized ultrapure DNA with 2 ml sterile endotoxin-free water.
ODNs are shipped at room temperature.
Store at -20°C.
Lyophilized and resuspended ODNs are stable 6 months at -20°C. Avoid repeated freeze-thaw cycles.

E. coli DNA ef is an ultrapure, endotoxin-free (ef) preparation of E. coli K12 double-stranded DNA devoid of TLR2 and TLR4 activities.

E. coli ssDNA is an ultrapure, endotoxin-free preparation of bacterial single-stranded DNA (ssDNA). It is a better TLR9 ligand than E. coli DNA ef (dsDNA endotoxin-free) as TLR9 binds directly and sequence-specifically to single-stranded unmethylated CpG-DNA. E. coli ssDNA is complexed with the cationic lipid LyoVec™ to allow a better internalization of the immunostimulatory DNA to the acidic compartment where TLR9 is expressed.

E. coli DNA ef and E. coli ssDNA are provided lyophilized and shipped at room temperature.
Store at -20°C. Lyophilized E. coli DNAs are stable 6 months at -20°C.

Inhibitory ODNs are characterized by three consecutive Gs downstream of a C or A, addition of a fourth G (G-tetrads) increases the inhibitory capability. They act by disrupting the colocalization of CpG ODNs with TLR9 in endosomal vesicles without affecting cellular binding and uptake.
ODN 2088 derives from a stimulatory ODN by replacement of 3 bases [1].
ODN TTAGGG, which contains 4 TTAGGG motifs, is found in mammalian telomeres [2].
G-ODN, a guanosine rich-ODN, is suppressive in murine DC and macrophages as well as in human plasmacytoid DC [3].
ODN 2088 control and ODN TTAGGG control are negative controls for ODN 2088 [1] and ODN TTAGGG [2], respectively.

1. Stunz LL. et al., 2002. Inhibitory oligodinucleotides specifically block effects of stimulatory CpG oligonucleotides in B cells. Eur J Immunol. 32(5): 1212-22.
2.Gursel I. et al., 2003. Repetitive elements in mammalian telomeres suppress bacterial DNA-induced immune activation. J Immunol. 171(3):1393-400.
3. Peter M. et al., 2008. Characterization of suppressive oligodeoxynucleotides that inhibit Toll-like receptor-9-mediated activation of innate immunity. Immunology. 123(1):118-28.

Inhibitory ODNs and their controls are provided lyophilized with sterile endotoxin-free water and shipped at room temperature.
Store at -20°C. Lyophilized ODNs are stable 6 months at -20°C.

pCpG Giant is a high molecular weight plasmid entirely devoid of CpG dinucleotides. This DNA also features no detectable amounts of endotoxin, as determined by the LAL assay and the HEK-Blue™-4 cell line-based assay and no detectable TLR2 activity. In addition, this plasmid DNA displays no Dcm methylation and a reduced level of Dam methylation. pCpG Giant can be used as a control in studies on CpG methylations.
Salmon sperm DNA does not activate any TLR and can be used as a negative control for TLR induction experiments in particular in TLR9 studies. Salmon sperm DNA is endotoxin-free (<0.125EU/mg). Non-induction of TLR-expressing cells has been confirmed at concentrations up to 1 mg/ml.

pCpG Giant and salmon sperm DNA are provided lyophilized and shipped at room temperature.
Store at -20°C. Products are stable 6 months at -20°C.