title: Modifications in small interfering RNA that separate immunostimulation from RNA interference.
abstract: Synthetic small interfering RNA (siRNA) can suppress the expression of endogenous mRNA through RNA interference. It has been reported that siRNA can induce type I IFN production from plasmacytoid dendritic cells, leading to off-target effects. To separate immunostimulation from the desired gene-specific inhibitory activity, we designed RNA strands with chemical modifications at strategic positions of the ribose or nucleobase residues. Substitution of uridine residues by 2'-deoxyuridine or thymidine residues was found to decrease type I IFN production upon in vitro stimulation of human PBMC. Thymidine residues in both strands of a siRNA duplex further decreased immunostimulation. Fortunately, the thymidine residues did not affect gene-silencing activity. In contrast, 2'-O-methyl groups at adenosine and uridine residues reduced both IFN-alpha secretion and gene-silencing activity. Oligoribonucleotides with 2'-O-methyladenosine residues actively inhibited IFN-alpha secretion induced by other immunostimulatory RNAs, an effect not observed for strands with 2'-deoxynucleosides. Furthermore, neither 5-methylcytidine nor 7-deazaguanosine residues in the stimulatory strands affected IFN-alpha secretion, suggesting that recognition does not involve sites in the major groove of duplex regions. The activity data, together with structure prediction and exploratory UV-melting analyses, suggest that immunostimulatory sequences adopt folded structures. The results show that immunostimulation can be suppressed by suitable chemical modifications without losing siRNA potency by introducing seemingly minor structural changes.
authors.affiliation: Department of Medical Microbiology and Hygiene, University of Heidelberg, Heidelberg, Germany.
authors.forename: Florian
authors.initials: F
authors.lastname: Eberle
authors.forename: Kerstin
authors.initials: K
authors.lastname: Giessler
authors.forename: Christopher
authors.initials: C
authors.lastname: Deck
authors.forename: Klaus
authors.initials: K
authors.lastname: Heeg
authors.forename: Mirjam
authors.initials: M
authors.lastname: Peter
authors.forename: Clemens
authors.initials: C
authors.lastname: Richert
authors.forename: Alexander H
authors.initials: AH
authors.lastname: Dalpke
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000737:chemistry / Q000235:genetics; D002460:Cell Line; D005229:Fatty Acids, Monounsaturated / Q000494:pharmacology; D020868:Gene Silencing / Q000276:immunology; D006801:Humans; D016898:Interferon-alpha / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism / Q000528:radiation effects; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000276:immunology / Q000378:metabolism; D009690:Nucleic Acid Conformation / Q000187:drug effects / Q000528:radiation effects; D009692:Nucleic Acid Heteroduplexes / Q000235:genetics / Q000276:immunology; D009843:Oligoribonucleotides / Q000737:chemistry* / Q000235:genetics; D011089:Polydeoxyribonucleotides / Q000737:chemistry / Q000235:genetics; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D034622:RNA Interference / Q000276:immunology*; D012330:RNA, Double-Stranded / Q000737:chemistry; D034741:RNA, Small Interfering / Q000737:chemistry* / Q000235:genetics; D017423:Sequence Analysis, RNA; D013936:Thymidine / Q000737:chemistry* / Q000235:genetics; D014466:Ultraviolet Rays; D014529:Uridine / Q000737:chemistry / Q000235:genetics
chemical_list: D005229:Fatty Acids, Monounsaturated; D016898:Interferon-alpha; D009692:Nucleic Acid Heteroduplexes; D009843:Oligoribonucleotides; D011089:Polydeoxyribonucleotides; D000644:Quaternary Ammonium Compounds; D012330:RNA, Double-Stranded; D034741:RNA, Small Interfering; C090949:N(6)-cyclohexyl-2-O-methyladenosine; D000241:Adenosine; C070046:1,2-dioleoyloxy-3-(trimethylammonium)propane; D013936:Thymidine; D014529:Uridine