ORN06/LyoVec™

TLR7/8 Agonist - minimal ssRNA motif of ssRNA40 with 6 UUGU repeats - complexed with LyoVec™

Activation of TLR7/8 by ORN06/LyoVec™

The oligoribonucleotide ORN06 contains six repeats of the UUGU sequence motif, identified as the minimal motif responsible for ssRNA40 immunoreactivity. ssRNA40, also known as R-1075, is a U-rich single-stranded (ss)RNA derived from the long terminal repeat of HIV-1, and a strong agonist of the human (h) Toll-like receptor 8 (TLR8) [1]. 

ssRNA derived from HIV-1 or the influenza virus were shown to induce the production of proinflammatory cytokines by activation of TLR7 and/or TLR8 [2].
 More details

Description

InvivoGen's ORN06 is complexed with the cationic lipid LyoVec™ to protect it from degradation and facilitate its uptake. Moreover, phosphorothioate linkages were incorporated in order to extend the effective molecular lifetime by minimizing extra and intracellular nuclease degradation. ORN06/LyoVec™ is a strong agonist of human TLR8 and mouse TLR7, as verified by using our HEK-Blue™ reporter cell lines. Moreover, it slightly activates human TLR7 in our HEK-derived cells (see figure). 

Key features of ORN06/LyoVec™

• Strong activator of human TLR8 and mouse TLR7
• ssRNA40 and its negative control ssRNA41 are available
• Complexed with LyoVec™ 
• Each lot of ORN06/LyoVec™ is functionally tested

Read our review about TLR7 and TLR8.

References:

1. Forsbach A, et al., 2008. Identification of RNA sequence motifs stimulating sequence-specific TLR8-dependent immune responses. J Immunol. 2008 Mar 15;180(6):3729-38.
2. Heil F. et al., 2004. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science. 5;303(5663):1526-9.

Figures

NF-κB response of HEK-Blue™-derived cells to ORN06/LyoVec™. HEK-Blue™ cells expressing hTLR7, mTLR7, or hTLR8 were cultured in HEK-Blue™ Detection reagent and stimulated with increasing concentrations of ORN06/LyoVec™. After 24h incubation, the NF-κB-induced SEAP activity was assessed by measuring the SEAP level in the supernatant. Data are shown as optical density (OD) at 650 nm (mean ± SEM). Of note, HEK-Blue™ Null* comprises data from parental cell lines HEK-Blue Null1, HEK-Blue Null1-v, HEK-Blue Null2-k.

Specifications

Specificity: TLR8 and TLR7 agonist

Working Concentration: 0.25 - 5 μg/ml

Sequence: ORN 06 5’-U*U*G*U*U*G*U*U*G*U*U*G*U*U*G*U*U*G*U*U-3’ (20 mer)

(“*” depicts the phosphorothioate linkage)

Solubility: 0.05 mg/ml in water

Quality control:

• The activation of ORN06/LyoVec™ has been validated 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

• 4 x 25 mg lyophilized ORN 06/LyoVec™
• 10 ml endotoxin-free water

ORN 06/LyoVec™ is provided lyophilized and shipped at room temperature.

Store at -20°C.

Lyophilized product is stable 1 year at -20 ̊C.

Upon resuspension, store product at 4°C. Resuspended product is stable 1 week at 4°C.

Details

Single-stranded RNA (ssRNA) has been identified as the natural ligand of TLR7 and TLR8 [1, 2]. ssRNA derived from HIV-1 or the influenza virus were shown to induce the production of proinflammatory cytokines in pDC.

This induction was reproduced using U-rich single-stranded RNAs, such as polyU or GU-rich (ssRNA40) ODNs complexed with cationic lipids to protect them from degradation.

According to Diebold et al., this activation is sequence-independent as long as the ssRNAs contain several uridines in close proximity [1].

However, recent studies suggest sequence-dependent recognition of U-rich ssRNAs by TLR7 and TLR8.

They demonstrate that AU-rich and GU-rich oligoribonucleotides (ORNs) are capable of activating TLR8, whereas only GU-rich ORNs are able to stimulate TLR7 [3].

ORN 06 contains 6 repeats of the UUGU sequence motif, identified as the minimal motif responsible for ssRNA40 immunoactivity [3].

1. Diebold S. et al., 2006. Nucleic acid agonists for Toll-like receptor 7 are defined by the presence of uridine ribonucleotides. Eur. J. Immunol., 36:3256-67.
2. Heil F. et al., 2004. Species-specific recognition of single-stranded RNA via toll-like receptor 7 and 8. Science. 5;303(5663):1526-9.
3. Forsbach A. et al., 2008. Identification of RNA Sequence Motifs Stimulating Sequence-Specific TLR8-Dependent Immune Responses. J. Immunol., 180: 3729-38.