RNA therapeutics all rely on chemical modifications, which help to stabilise these molecules against nucleases and are paramount to their drug-like properties. Critically, such modifications are also essential to blunt activation of innate immune nucleic acid sensors by such RNA therapeutics, but a detailed mechanistic understanding of how this operates remains poorly defined.
We have now made the ground-breaking discovery that degradation fragments as short as three bases (3-mers) from 2’O-methyl-modified phosphorothioate gapmer antisense oligonucleotides can directly bind Toll-Like Receptor (TLR) 7 and TLR8 to impair their RNA sensing activity. Systematic analyses have identified the optimal 3-mer sequences and chemistries impacting TLR7/8 signalling. Functionally, select 3-mer oligonucleotides have divergent effects on TLR8 activity, allowing for both inhibition or potentiation in a sequence-dependent manner, while TLR7 sensing is strictly suppressed by immunoregulatory 3-mers. Mechanistically, molecular dynamics analyses indicate that antagonistic 3-mers do not bind as well as agonistic 3-mers to site 2 of TLR7, potentially underlying their inhibitory function. Critically, TLR7-inhibiting 3-mer oligonucleotides showed significant protection against systemic and topical TLR7-driven inflammation in vivo.
Collectively, our findings add to the understanding of TLR7/8 sensing of RNA, indicating a complex interplay between activating and inhibiting fragments according to base and sugar modification of their nucleotides. Our studies suggest that the distinction between self and non-self RNA by TLR7/8 relies on the competitive activities of 3-base long “immune codons”, acting as agonists or antagonists. Finally, our work defines a novel class of ultra-short immunomodulatory oligonucleotides with a broad range of potential therapeutic applications.