E. coli ssDNA / LyoVec™

TLR9 activation with E. coli ssDNA/LyoVec™

TLR9 Agonist - E. coli single-stranded DNA/LyoVec complexes

E. coli ssDNA/LyoVec™ is a Toll-like receptor 9 (TLR9) ligand. It comprises E. coli ssDNA an ultrapure, endotoxin-free preparation of bacterial single‑stranded DNA (ssDNA) which is complexed with the cationic lipid-based transfection reagent LyoVec™. This allows for a better internalization of the immunostimulatory DNA to the acidic endosomal compartment where TLR9 is expressed.

TLR9 specifically recognizes CpG DNA that is unmethylated and single-stranded (ss). Upon recognition, it initiates a signaling cascade leading to the activation of the transcription factors NF-κB, AP-1, and interferon regulatory factor 7 (IRF7) with the subsequent production of pro‑inflammatory cytokines. Methylation of the cytosine within the CpG motif strongly reduces the affinity of TLR9 [1, 2]. In addition, double‑stranded (ds) CpG DNA is a much weaker stimulator of TLR9 compared to its ss counterpart [1]. Unlike mammalian DNA, bacterial DNA is rich in unmethylated CpG motifs and thus activates TLR9.

InvivoGen provides bacterial ssDNA as sheared single‑stranded DNA fragments produced by treating genomic E. coli DNA with ultrasound followed by heat denaturation. These fragments are complexed with LyoVec™ to allow penetration of the DNA in the cells. E. coli ssDNA/LyoVec™ activates TLR9 similarly to CpG-ODNs in a species‑independent manner. Of note, the ability of E. coli ssDNA/LyoVec™ to induce an inflammatory response has been described in the literature [3].

Key Features of E. coli ssDNA/LyoVec™:

• E. coli ssDNA / LyoVec™ is a TLR9 agonist
• E. coli ssDNA / LyoVec™ is complexed with a cationic-lipid for better internalization
• E. coli ssDNA / LyoVec™ is free of microbial contaminants
• Each lot of E. coli ssDNA / LyoVec™ is functionally tested

Read our review on TLR9

References:

1. Rutz M. et al., 2004. Toll-like receptor 9 binds single-stranded CpG-DNA in a sequence- and pH-dependent manner. Eur J Immunol. 16(1):17-22.
2. Cornelie S. et al., 2004. Direct evidence that toll-like receptor 9 (TLR9) functionally binds plasmid DNA by specific cytosine-phosphate-guanine motif recognition. J Biol Chem. 279(15):15124-9.
3. Yu S. et al.., 2014. . TLR sorting by Rab11 endosomes maintains intestinal epithelial-microbial homeostasis. EMBO J. 33:1882-95.

Specifications

Specificity: TLR9 agonist

Working Concentration: 500 ng/ml- 5 µg/ml

Solubility: 0.05 mg/ml in water

Quality control:

• Activation of TLR9 by E. coli ssDNA/LyoVec™ has been confirmed using cellular assays.
• The absence of bacterial contamination (e.g. lipoproteins and endotoxins) has been confirmed using HEK-Blue™ hTLR2 and HEK-Blue™ hTLR4 cells.

Contents

E. coli ssDNA/LyoVec™ is provided lyophilized.

• 4 x 50 μg lyophilized E. coli ssDNA/LyoVec™ 1:2 ratio (w/w)

Note: Each vial contains 50 μg of E. coli K12 ssDNA complexed with 100 μg LyoVec™.

• 10 ml endotoxin-free water

E. coli ssDNA/LyoVec™ is shipped at room temperature.

Store at -20°C. Upon resuspension, store product at 4°C.

Lyophilized product is stable for 1 year at -20°C. Resuspended product is stable for 1 week at 4°C.