Eliminating RNA Immunogenicity via Chemical Modifications: Good to Know

In this essay, group leader Mouldy Sioud goes through recent advances in this field, and how their work is relevant for the development of covid-19 vaccines.


Besides being the physical link between DNA and proteins, RNAs play several other key roles, including RNA catalysis and gene regulation. Recent advances in the delivery and chemical modifications of RNAs have enabled the development of RNA-based therapeutics, including antisense oligonucleotides, ribozymes, small interfering RNAs, and messenger RNAs (1). In addition to their intended targets/functions, RNA therapeutics can exert off-target effects and activation of the immune system (1). Since the activation of the immune system is undesirable for certain therapeutic applications, it is crucial to develop ways to block the sensing of exogenous RNAs by immune cells, such as monocytes, macrophages and dendritic cells. RNA sensing by the immune system can be blocked by chemical modifications of uridines only, a finding that has facilitated the development of therapeutic RNAs, such as mRNA vaccines against COVID-19.   


We started investigating the mechanisms by which small interfering RNAs (sense and antisense strands) activate innate immune cells. In this regard, we reported that the activation of innate immunity by small RNAs is sequence- and cell-dependent (2). In a follow-up study, we demonstrated that lipid-formulated RNAs activate the expression of pro-inflammatory cytokines and type I interferons in blood monocytes/macrophages through endosomal Toll-like receptors (TLR) 7 and 8 (3, 4). Notably, the protein kinase R and TLR3 are not involved as claimed by Karikó et al. (5). In accordance with our data, Judge et al. also identified TLR7/8 to be the receptor for siRNAs (6). Next, we reasoned that RNA modifications may alter the recognition of RNAs by TLRs (4). We have demonstrated that the activation of endosomal TLR7/8 by self or non-self RNAs (sense or antisense strand) can be abrogated by chemical modifications (e.g. 2’-O-methylation) of uridines only (7-9). Moreover, our work showed that uridine is the most critical nucleoside for TLR activation and that RNAs having few uridines would have immunostimulatory effect (Fig. 1). Recent crystallographic studies confirmed that uridine is essential for RNA binding to TLR7/8 (10, 11).  

Early chemical modifications were used to improve the stability and function of chemically synthesized and in vitro transcribed RNAs (12, 13). Modified nucleotides not only increase RNA stability but also block the activation of innate immunity. So, what is the mechanism by which modified RNAs suppress immune responses? Mechanistically, we have shown that 2’-O-methylated RNAs competed with unmodified RNAs for TLR7/8 binding and therefore they function as TLR7/8 antagonists (14). Similarly, Robbins and et al. identified 2’-O-methylated RNA as TLR7 antagonist (15). The binding of 2’-modified RNAs to TLR7/8 seems to block the conformational changes that are required for activation (10, 11). Of note, 2′-O-methylation, where a methyl group (–CH3) is added to the 2′ hydroxyl (–OH) of the ribose moiety, is the most common naturally occurring modification in the RNA world (16). Overall; our work offers the possibility of choosing the chemical modification strategy to evade immune recognition of exogenously delivered RNAs without compromising their intended functions, such as gene silencing (reviewed in 8). Given these findings, one should think that the presence of such modifications in naturally occurring RNAs would decrease their immunogenicity (17). In this respect, Karikó et al. reported that endogenous RNA modifications can suppress RNA recognition by TLRs (17). RNA modifications play an essential role in gene expression regulation by altering and fine tuning messenger RNA, ribosomal RNA, transfer RNA and non-coding RNA functions (16). 

To summarize, the data described above indicate that modified nucleotides not only increase RNA stability but also modulate the activation of innate immunity (8, 18). Of note, the strategy of using chemical modifications to block the sensing of exogenously delivered RNAs by innate immune cells and the subsequent induction of undesirable immune responses was initially suggested by us (4) and further validated in the European Journal of Immunology’s paper (7). What is important is not the nature of the used modifications, but the demonstration that modified RNA does not trigger the signaling of TLR7/8, a significant finding that has facilitated the application of therapeutic RNAs, such as mRNA vaccines against COVID-19. In vitro transcribed mRNA is expected to activate innate immunity when injected to humans similar to what would happen during viral infections. Without RNA modifications to prevent the induction of unwanted immune responses and increase mRNA stability/function, the success of mRNA vaccines would not have been possible.    

Schematic illustration of TLR7/8 activation by RNAs (7, 18)


Mouldy Sioud
Immunomodulation and targeted therapy's group.
Department of Cancer Immunology, ICR, Division of Cancer Medicine, Oslo University Hospital.


  1. Sioud M. RNA therapeutics, Methods In Mol. Biol, 2010 (Sioud Ed.), 629: 1-521, DOI: 10.1007/978-1-60761-657-3  
  2. Sioud M, Sørensen DR. Cationic liposome-mediated delivery of siRNAs in adult mice. Biochem Biophys Res Commun. 2003 Dec 26;312(4):1220-5. 378 citations (GS)
  3. Sioud M. Induction of inflammatory cytokines and interferon responses by double-stranded and single-stranded siRNAs is sequence dependent and requires endosomal localization. J Mol Biol. 2005 May 20; 348(5):1079-90 (received December 2004). 515 citations.
  4. Sioud M. On the delivery of small interfering RNAs into mammalian cells. Expert Opin Drug Deliv. 2005 Jul; 2(4):639-51.  (received April 2005) 101 citations 
  5. Karikó K, Bhuyan P, Capodici J, Weissman D. Small interfering RNAs mediate sequence-independent gene suppression and induce immune activation by signaling through toll-like receptor 3. J Immunol. 2004 Jun 1;172(11):6545-9. 
  6. Judge AD, Sood V, Shaw JR, Fang D, McClintock K, MacLachlan I. Sequence-dependent stimulation of the mammalian innate immune response by synthetic siRNA. Nat Biotechnol. 2005 Apr; 23(4):457-62.
  7. Sioud M. Single-stranded small interfering RNAs are more immunostimulatory than their double-stranded counterparts: a central role for 2'-hydroxyl uridines in immune responses. Eur J Immunol. 2006 May; 36(5):1222-30. (received 18 November 2005). 226 citations.
  8. Sioud M. Innate sensing of self and non-self RNAs by Toll-like receptors. Trends Mol Med. 2006 Apr; 12(4):167-76.  210  citations.
  9. Cekaite L, Furset G, Hovig E, Sioud M. Gene expression analysis in blood cells in response to unmodified and 2'-modified siRNAs reveals TLR-dependent and independent effects. J Mol Biol. 2007 Jan 5; 365(1):90-108. 139 citations.
  10. Tanji H, Ohto U, Shibata T, Taoka M, Yamauchi Y, Isobe T, Miyake K, Shimizu T. Toll-like receptor 8 senses degradation products of single-stranded RNA. Nat Struct Mol Biol. 2015 Feb;22(2):109-15. 
  11. Zhang Z, Ohto U, Shibata T, Krayukhina E, Taoka M, Yamauchi Y, Tanji H, Isobe T, Uchiyama S, Miyake K, Shimizu T. Structural Analysis Reveals that Toll-like Receptor 7 Is a Dual Receptor for Guanosine and Single-Stranded RNA. Immunity. 2016 Oct 18;45(4):737-748. 
  12. Sioud M, Sørensen DR. A nuclease-resistant protein kinase C alpha ribozyme blocks glioma cell growth. Nat Biotechnol. 1998 Jun;16(6):556-61. 112 citations.
  13. Corey DR. Chemical modification: the key to clinical application of RNA interference? J Clin Invest. 2007 Dec;117(12):3615-22.
  14. Sioud M, Furset G, Cekaite L. Suppression of immunostimulatory siRNA-driven innate immune activation by 2'-modified RNAs. Biochem Biophys Res Commun. 2007 Sep 14;361(1):122-6. 152 citations.
  15. Robbins M, Judge A, Liang L, McClintock K, Yaworski E, MacLachlan I. 2'-O-methyl-modified RNAs act as TLR7 antagonists. Mol Ther. 2007 Sep;15(9):1663-9.
  16. Roundtree IA, Evans ME, Pan T, He C. Dynamic RNA modifications in gene expression regulation. Cell. 2017 Jun 15;169(7): 
  17. Karikó K, Buckstein M, Ni H, Weissman D. Suppression of RNA recognition by Toll-like receptors: the impact of nucleoside modification and the evolutionary origin of RNA. Immunity. 2005 Aug; 23(2):165-75. 
  18. Sioud M. RNA interference and innate immunity. Adv Drug Deliv Rev. 2007 Mar 30;59(2-3):153-63.  126 citations.
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