Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis

• Published in: The Journal of biological chemistry 2003-02, Vol.278 (9), p.7108-7118
• Main Authors: Vickers, Timothy A, Koo, Seongjoon, Bennett, C Frank, Crooke, Stanley T, Dean, Nicholas M, Baker, Brenda F
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; COS Cells; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Intercellular Adhesion Molecule-1 - biosynthesis; Introns; Luciferases - metabolism; Molecular Sequence Data; Nucleic Acid Conformation; Oligonucleotides - pharmacology; Oligonucleotides, Antisense - pharmacology; Phosphoric Monoester Hydrolases - genetics; Phosphoric Monoester Hydrolases - metabolism; Protein Structure, Tertiary; PTEN Phosphohydrolase; Ribonuclease H - metabolism; RNA - metabolism; RNA, Messenger - metabolism; RNA, Small Interfering - metabolism; Time Factors; Tumor Cells, Cultured; Tumor Suppressor Proteins - genetics; Tumor Suppressor Proteins - metabolism
• ISSN: 0021-9258
• DOI: 10.1074/jbc.M210326200

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.