McAllan, A. L. | bioRxiv Science

bioRxiv2026

Defining the Immunomodulatory Determinants of 3-mer Oligonucleotides on TLR7 and TLR8 sensing

Chemical modifications such as 2'-O-methyl (2'-OMe) are central to the efficacy and tolerability of RNA therapeutics. We recently identified that 2'-OMe RNA fragments as short as three nucleotides can exert opposing effects on Toll-like receptor 8 (TLR8) sensing in a motif-dependent manner. This discovery raises important considerations for degradation products of chemically modified RNA therapeutics, which may generate such immunologically active fragments. Here, leveraging their short length, we systematically map how base and sugar modifications within 3-mer oligonucleotides regulate TLR7 and TLR8 responses, and we resolve the structural basis for both TLR8 potentiation and TLR7/8 antagonism by RNA fragments. Building on these insights, we report the development of a dual TLR7/8 inhibitory oligonucleotide with therapeutic potential in autoimmune disease. Together, these findings provide unprecedented resolution of the immunomodulatory properties of oligonucleotide modifications on TLR7/8 and establish 3-mer oligonucleotides as the shortest functional class of RNA therapeutics described to date.

immunology↗

bioRxiv2025

Annexin A2 mediates RIG-I-like receptor responses to viral infection

RIG-I and MDA5 are critical sensors of virus infection and detect many important human pathogens including SARS-CoV-2, influenza A virus (IAV), Zika virus and West Nile virus (WNV). MDA5 is activated by binding to double-stranded RNA generated during infection and induces strong pro-inflammatory and antiviral responses mediated largely via type I interferons. Activation of MDA5 is also implicated in monogenic (e.g. Aicardi-Goutieres syndrome) and complex polygenic (e.g. systemic lupus erythematosus) autoinflammatory diseases, demonstrating the importance of appropriate regulation of this pathway. Yet, how MDA5 is regulated is poorly defined. We employed SILAC-mass spectrometry to discover MDA5 binding partners in virus-infected cells. Our screen revealed Annexin A2 (ANXA2) as an interactor of MDA5 specifically during infection. Direct binding of ANXA2 and MDA5 was recapitulated in vitro and required Ca2+, and ANXA2 promoted MDA5 oligomerisation in cells. Loss of ANXA2 in human cells significantly blunted interferon responses to EMCV, WNV and IAV, demonstrating a role for ANXA2 in activation of both MDA5 and RIG-I. Using human induced pluripotent stem cells, we found that ANXA2 was required for potent neuronal cell responses to WNV infection and macrophage responses to multiple viral pathogens. Thus, we find that ANXA2 is a critical mediator of RIG-I-like receptor pathways.

immunology↗