title: Base modification strategies to modulate immune stimulation by an siRNA.
abstract: Immune stimulation triggered by siRNAs is one of the major challenges in the development of safe RNAi-based therapeutics. Within an immunostimulatory siRNA sequence, this hurdle is commonly addressed by using ribose modifications (e.g., 2'-OMe or 2'-F), which results in decreased cytokine production. However, as immune stimulation by siRNAs is a sequence-dependent phenomenon, recognition of the nucleobases by the trigger receptor(s) is also likely. Here, we use the recently published crystal structures of Toll-like receptor 8 (TLR8) bound to small-molecule agonists to generate computational models for ribonucleotide binding by this immune receptor. Our modeling suggested that modification of either the Watson-Crick or Hoogsteen face of adenosine would disrupt nucleotide/TLR8 interactions. We employed chemical synthesis to alter either the Watson-Crick or Hoogsteen face of adenosine and evaluated the effect of these modifications in an siRNA guide strand by measuring the immunostimulatory and RNA interference properties. For the siRNA guide strand tested, we found that modifying the Watson-Crick face is generally more effective at blocking TNFα production in human peripheral blood mononuclear cells (PBMCs) than modification at the Hoogsteen edge. We also observed that modifications near the 5'-end were more effective at blocking cytokine production than those placed at the 3'-end. This work advances our understanding of how chemical modifications can be used to optimize siRNA performance.
authors.affiliation: Department of Chemistry, University of California, One Shields Avenue, Davis, CA 95616 (USA).
authors.forename: Rachel Anne P
authors.initials: RA
authors.lastname: Valenzuela
authors.forename: Scott R
authors.initials: SR
authors.lastname: Suter
authors.forename: Alexi A
authors.initials: AA
authors.lastname: Ball-Jones
authors.forename: José M
authors.initials: JM
authors.lastname: Ibarra-Soza
authors.forename: Yuxuan
authors.initials: Y
authors.lastname: Zheng
authors.forename: Peter A
authors.initials: PA
authors.lastname: Beal
mesh_terms: D000241:Adenosine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D058534:Class I Phosphatidylinositol 3-Kinases; D016207:Cytokines / Q000378:metabolism; D055785:Gene Knockdown Techniques; D006801:Humans; D007114:Immunization / Q000379:methods; D007963:Leukocytes, Mononuclear / Q000187:drug effects; D008958:Models, Molecular; D062105:Molecular Docking Simulation; D019869:Phosphatidylinositol 3-Kinases / Q000235:genetics; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D051200:Toll-Like Receptor 8 / Q000819:agonists / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
chemical_list: D016207:Cytokines; D034741:RNA, Small Interfering; C495349:TLR8 protein, human; D051200:Toll-Like Receptor 8; D014409:Tumor Necrosis Factor-alpha; D019869:Phosphatidylinositol 3-Kinases; D058534:Class I Phosphatidylinositol 3-Kinases; C497132:PIK3CB protein, human; D000241:Adenosine
keywords: TLR8; immune stimulation; nucleobase modification; siRNA; toll-like receptors