title: Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis.
abstract: RNA interference can be considered as an antisense mechanism of action that utilizes a double-stranded RNase to promote hydrolysis of the target RNA. We have performed a comparative study of optimized antisense oligonucleotides designed to work by an RNA interference mechanism to oligonucleotides designed to work by an RNase H-dependent mechanism in human cells. The potency, maximal effectiveness, duration of action, and sequence specificity of optimized RNase H-dependent oligonucleotides and small interfering RNA (siRNA) oligonucleotide duplexes were evaluated and found to be comparable. Effects of base mismatches on activity were determined to be position-dependent for both siRNA oligonucleotides and RNase H-dependent oligonucleotides. In addition, we determined that the activity of both siRNA oligonucleotides and RNase H-dependent oligonucleotides is affected by the secondary structure of the target mRNA. To determine whether positions on target RNA identified as being susceptible for RNase H-mediated degradation would be coincident with siRNA target sites, we evaluated the effectiveness of siRNAs designed to bind the same position on the target mRNA as RNase H-dependent oligonucleotides. Examination of 80 siRNA oligonucleotide duplexes designed to bind to RNA from four distinct human genes revealed that, in general, activity correlated with the activity to RNase H-dependent oligonucleotides designed to the same site, although some exceptions were noted. The one major difference between the two strategies is that RNase H-dependent oligonucleotides were determined to be active when directed against targets in the pre-mRNA, whereas siRNAs were not. These results demonstrate that siRNA oligonucleotide- and RNase H-dependent antisense strategies are both valid strategies for evaluating function of genes in cell-based assays.
authors.affiliation: GeneTrove Division and Antisense Core Research Department, Isis Pharmaceuticals, Inc., Carlsbad, California 92008, USA. tvickers@isiph.com
authors.forename: Timothy A
authors.initials: TA
authors.lastname: Vickers
authors.forename: Seongjoon
authors.initials: S
authors.lastname: Koo
authors.forename: C Frank
authors.initials: CF
authors.lastname: Bennett
authors.forename: Stanley T
authors.initials: ST
authors.lastname: Crooke
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Dean
authors.forename: Brenda F
authors.initials: BF
authors.lastname: Baker
mesh_terms: D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000096:biosynthesis; D007438:Introns; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D051059:PTEN Phosphohydrolase; D010744:Phosphoric Monoester Hydrolases / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D016914:Ribonuclease H / Q000378:metabolism*; D013997:Time Factors; D014407:Tumor Cells, Cultured; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D009841:Oligonucleotides; D016376:Oligonucleotides, Antisense; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D018799:Intercellular Adhesion Molecule-1; D012313:RNA; D008156:Luciferases; D016914:Ribonuclease H; D010744:Phosphoric Monoester Hydrolases; D051059:PTEN Phosphohydrolase; C494929:PTEN protein, human