Nucleic acid agonists for Toll‐like receptor 7 are defined by the presence of uridine ribonucleotides

Abstract

Toll-like receptor 7 (TLR7) mediates innate responses by responding to viral RNA in endocytic compartments. However, the molecular pattern recognised by TLR7 and whether it differs between RNA of viral and self origin remains unclear. Here, we identify nucleic acids that act as TLR7 agonists for mouse and human cells. We show that uridine and ribose, the two defining features of RNA, are both necessary and sufficient for TLR7 stimulation, and that short single-stranded RNA (ssRNA) act as TLR7 agonists in a sequence-independent manner as long as they contain several uridines in close proximity. Consistent with the notion that TLR7 lacks specificity for sequence motifs, we show that it is triggered equally efficiently by viral or self RNA delivered to endosomes. Our results support the notion that TLR7 recognises uracil repeats in RNA and that it discriminates between viral and self ligands on the basis of endosomal accessibility rather than sequence.

Antitumor Activity and Immune Response Induction of a Dual Agonist of Toll-Like Receptors 7 and 8

“…Imidazoquinolines also interfere with the adenosine and opioid growth factor receptor signaling pathways independent of TLR7 and TLR8 (29,30) Recent studies have shown that synthetic RNAs formulated with lipid carriers also activate TLR7 and TLR8 in a sequence-dependent manner (2,23). However, singlestranded RNA is highly susceptible to nuclease degradation, and the use of lipid carriers has been required to stabilize RNAs against nucleases (12,(31)(32)(33)(34)(35)(36). We have designed RNAs linked through their 3′-ends to prevent 3′-exonuclease degradation, referred to as SIMRA compounds, which act as agonists of TLR7, TLR8, or both TLR7 and TLR8, depending on the nucleotide sequence and chemical modifications incorporated (14)(15)(16)(17).…”

The Use of TLR7 and TLR8 Ligands for the Enhancement of Cancer Immunotherapy

“…Although the content of U or GU in the RNA molecule seems to be important for both TLR7 and TLR8 activation [21,23,27,28], the specific RNA motif recognized by TLR7 and/or TLR8 has not yet been identified. It was suggested by Diebold et al [29] that the uracil content and motifs in ORNs define the ability of the ORN to act as a TLR7 ligand, whereas the G content is important for TLR8 ligands. The finding of Peng et al [28] that poly-G3 oligonucleotides are recognized by TLR8 fits into this hypothesis.…”

“…Other immunostimulatory ligands of TLR7/TLR8 that have been described are self-RNA [29,30] and short interfering RNA [31][32][33][34]. Moreover, 2Ј-O-methyl modification of these RNA molecules inhibits TLR7/8-mediated immune activation, thereby paving the way to the development of TLR7/8 antagonists [35][36][37][38].…”

Evading innate immunity in nonviral mRNA delivery: don’t shoot the messenger

“…Accordingly, the TLRs involved in the recognition of foreign mRNA -TLR3, TLR7 and TLR8 -are located in the endosomal compartment. As such, especially uridine-rich ssRNA was identified as a strong immune inducer, mainly via stimulation of TLR7 [26,27], whereas dsRNA rather activates TLR3 [28,29]. Generally, mRNA is considered ssRNA, causing the foreign IVT mRNA to be mostly recognized by the structurally homologous TLR7 and TLR8 receptors [26,30].…”