Modulation of the Innate Immune System by Catalytically Inactive Single Stranded Oligonucleotides

Young, Tom and Unterholzner, Leonie (2025) Modulation of the Innate Immune System by Catalytically Inactive Single Stranded Oligonucleotides. PhD thesis, Lancaster University.

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Abstract

The innate immune system is the first line of defence against pathogens, as such it must be tightly controlled to prevent excessive damage to the host. Therefore, there is an interest in developing inflammatory inhibitors which can be used to reduce harmful inflammation. Here we report the development of a series of oligonucleotides which we show are able to specifically inhibit nucleic acid sensing pathways. The cGAS-STING pathway controls the response to DNA in the cytoplasm through the pattern recognition receptor cGAS which produces the second messenger cGAMP in response to DNA in the cytoplasm. The adaptor protein STING then facilitates a signalling cascade which triggers a pro-inflammatory interferon-based response. The Rig-I like receptor pathway is able to detect the presence of dsRNA in the cytoplasm through RIG-I and MDA5 which signal through MAVS to also drive a pro-inflammatory response. Through RT-PCR and ELISA, we show that both of these responses can be inhibited by the co-transfection of short single stranded oligonucleotides in THP1 cells and that this inhibition is specific to nucleic acid sensing pathways, having no effect on autophagy or the LPS stimulated response. This behaviour is consistent over time and not driven through any toxic effects. Through the use of computer modelling, we show that the presence of a predicted stem-loop structure in these oligonucleotides is essential for their behaviour, as variants without a predicted stem-loop are unable to modulate the immune response to DNA or dsRNA. Additionally, through confocal microscopy we show that these oligonucleotides act either at the level or on a regulator of STING. AlphaFold predicted ligand binding shows that these oligonucleotides are predicted to bind to and interact with a number of important residues in STING, RIG-I and MDA5. Overall, these oligonucleotides have unique previously unidentified activity against nucleic acid sensing pathways and could be useful in the development of new therapeutics against pro-inflammatory conditions.