title: Defining the factors that contribute to on-target specificity of antisense oligonucleotides.
abstract: To better understand the factors that influence the activity and specificity of antisense oligonucleotides (ASOs), we designed a minigene encoding superoxide dismutase 1 (SOD-1) and cloned the minigene into vectors for T7 transcription of pre-mRNA and splicing in a nuclear extract or for stable integration in cells. We designed a series of ASOs that covered the entire mRNA and determined the binding affinities and activities of the ASOs in a cell-free system and in cells. The mRNA bound known RNA-binding proteins on predicted binding sites in the mRNA. The higher order structure of the mRNA had a significantly greater effect than the RNA-binding proteins on ASO binding affinities as the ASO activities in cells and in the cell-free systems were consistent. We identified several ASOs that exhibited off-target hybridization to the SOD-1 minigene mRNA in the cell-free system. Off-target hybridization occurred only at highly accessible unstructured sites in the mRNA and these interactions were inhibited by both the higher order structure of the mRNA and by RNA-binding proteins. The same off-target hybridization interactions were identified in cells that overexpress E. coli RNase H1. No off-target activity was observed for cells expressing only endogenous human RNase H1. Neither were these off-target heteroduplexes substrates for recombinant human RNase H1 under multiple-turnover kinetics suggesting that the endogenous enzyme functions under similar kinetic parameters in cells and in the cell-free system. These results provide a blueprint for design of more potent and more specific ASOs.
authors: Isis Pharmaceuticals Inc., Carlsbad, California, United States of America.
authors: Walt F
authors: WF
authors: Lima
authors: Isis Pharmaceuticals Inc., Carlsbad, California, United States of America.
authors: Timothy A
authors: TA
authors: Vickers
authors: Isis Pharmaceuticals Inc., Carlsbad, California, United States of America.
authors: Josh
authors: J
authors: Nichols
authors: Isis Pharmaceuticals Inc., Carlsbad, California, United States of America.
authors: Cheryl
authors: C
authors: Li
authors: Isis Pharmaceuticals Inc., Carlsbad, California, United States of America.
authors: Stanley T
authors: ST
authors: Crooke
mesh_terms: D020029:Base Pairing; D001665:Binding Sites; D002460:Cell Line; D002474:Cell-Free System; D015870:Gene Expression; D023061:Gene Order; D006801:Humans; D016376:Oligonucleotides, Antisense / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012326:RNA Splicing; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000378:metabolism; D016914:Ribonuclease H / Q000378:metabolism; D013482:Superoxide Dismutase / Q000235:genetics / Q000378:metabolism; D000072105:Superoxide Dismutase-1; D014158:Transcription, Genetic
chemical_list: D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D016601:RNA-Binding Proteins; C000606290:SOD1 protein, human; D013482:Superoxide Dismutase; D000072105:Superoxide Dismutase-1; D016914:Ribonuclease H; C078856:ribonuclease HI