title: Sequence-dependent off-target inhibition of TLR7/8 sensing by synthetic microRNA inhibitors.
abstract: Anti-microRNA (miRNA) oligonucleotides (AMOs) with 2'-O-Methyl (2'OMe) residues are commonly used to study miRNA function and can achieve high potency, with low cytotoxicity. Not withstanding this, we demonstrate the sequence-dependent capacity of 2'OMe AMOs to inhibit Toll-like receptor (TLR) 7 and 8 sensing of immunostimulatory RNA, independent of their miRNA-targeting function. Through a screen of 29 AMOs targeting common miRNAs, we found a subset of sequences highly inhibitory to TLR7 sensing in mouse macrophages. Interspecies conservation of this inhibitory activity was confirmed on TLR7/8 activity in human peripheral blood mononuclear cells. Significantly, we identified a core motif governing the inhibitory activity of these AMOs, which is present in more than 50 AMOs targeted to human miRNAs in miRBaseV20. DNA/locked nucleic acids (LNA) AMOs synthesized with a phosphorothioate backbone also inhibited TLR7 sensing in a sequence-dependent manner, demonstrating that the off-target effects of AMOs are not restricted to 2'OMe modification. Taken together, our work establishes the potential for off-target effects of AMOs on TLR7/8 function, which should be taken into account in their therapeutic development and in vivo application.
authors.affiliation: Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Clayton, Victoria 3168, Australia Department of Molecular and Translational Science, Monash University, Clayton, Victoria 3168, Australia.
authors.forename: Soroush T
authors.initials: ST
authors.lastname: Sarvestani
authors.affiliation: Institute of Innate Immunity, Biomedical Center, University Hospitals Bonn, Bonn 53127, Germany.
authors.forename: H James
authors.initials: HJ
authors.lastname: Stunden
authors.affiliation: Integrated DNA Technologies Inc., Coralville, IA 52241, USA.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Behlke
authors.affiliation: Host-Microbiota Interactions Laboratory, Wellcome Trust Sanger Institute, Hinxton, CB10 1SA, UK.
authors.forename: Samuel C
authors.initials: SC
authors.lastname: Forster
authors.affiliation: Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Clayton, Victoria 3168, Australia Department of Molecular and Translational Science, Monash University, Clayton, Victoria 3168, Australia.
authors.forename: Claire E
authors.initials: CE
authors.lastname: McCoy
authors.affiliation: Department of Molecular and Translational Science, Monash University, Clayton, Victoria 3168, Australia Centre for Innate Immunity and Infectious Diseases, MIMR-PHI Institute of Medical Research, Clayton, Victoria 3168, Australia.
authors.forename: Michelle D
authors.initials: MD
authors.lastname: Tate
authors.affiliation: Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Clayton, Victoria 3168, Australia Department of Molecular and Translational Science, Monash University, Clayton, Victoria 3168, Australia.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Ferrand
authors.affiliation: Integrated DNA Technologies Inc., Coralville, IA 52241, USA.
authors.forename: Kim A
authors.initials: KA
authors.lastname: Lennox
authors.affiliation: Institute of Innate Immunity, Biomedical Center, University Hospitals Bonn, Bonn 53127, Germany Division of Infectious Diseases & Immunology, University of Massachusetts Medical School, Worcester, MA 01605, USA Deutsches Zentrum für Neurodegenerative Erkrankungen, Bonn 53127, Germany.
authors.forename: Eicke
authors.initials: E
authors.lastname: Latz
authors.affiliation: Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Clayton, Victoria 3168, Australia Department of Molecular and Translational Science, Monash University, Clayton, Victoria 3168, Australia michael.gantier@mimr-phi.org.
authors.forename: Bryan R G
authors.initials: BR
authors.lastname: Williams
authors.affiliation: Centre for Cancer Research, MIMR-PHI Institute of Medical Research, Clayton, Victoria 3168, Australia Department of Molecular and Translational Science, Monash University, Clayton, Victoria 3168, Australia michael.gantier@mimr-phi.org.
authors.forename: Michael P
authors.initials: MP
authors.lastname: Gantier
mesh_terms: D000276:Adjuvants, Immunologic / Q000494:pharmacology; D000818:Animals; D001483:Base Sequence; D057809:HEK293 Cells; D006801:Humans; D051379:Mice; D035683:MicroRNAs / Q000037:antagonists & inhibitors*; D059372:Nucleotide Motifs; D009841:Oligonucleotides / Q000737:chemistry*; D012313:RNA / Q000494:pharmacology; D051199:Toll-Like Receptor 7 / Q000037:antagonists & inhibitors*; D051200:Toll-Like Receptor 8 / Q000037:antagonists & inhibitors*
chemical_list: D000276:Adjuvants, Immunologic; D035683:MicroRNAs; D009841:Oligonucleotides; C495348:TLR7 protein, human; C495349:TLR8 protein, human; D051199:Toll-Like Receptor 7; D051200:Toll-Like Receptor 8; D012313:RNA