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.
Highlights
RNA interference can be considered as an antisense mechanism of action that utilizes a double-stranded RNase to promote hydrolysis of the target RNA
To determine whether positions on target RNA identified as being susceptible for RNase H-mediated degradation would be coincident with small interfering RNA (siRNA) target sites, we evaluated the effectiveness of siRNAs designed to bind the same position on the target mRNA as RNase H-dependent oligonucleotides
These results demonstrate that siRNA oligonucleotide- and RNase Hdependent antisense strategies are both valid strategies for evaluating function of genes in cell-based assays
Summary
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 Hdependent antisense strategies are both valid strategies for evaluating function of genes in cell-based assays. It has been shown that transfection of synthetic 21-nucleotide siRNA duplexes into mammalian cells does not elicit the RNA-activated protein kinase response, allowing effective inhibition of endogenous genes in a sequence-specific manner [9, 10] These siRNAs are too short to trigger the nonspecific double-stranded RNA responses, but they still promote degradation of complementary RNA sequences [9, 11]. Active siRNA oligonucleotides and homologous RNase H-dependent oligonucleotides were evaluated for relative potency, efficacy, duration of action, sequence specificity, and site of action within the cell to determine whether significant advantages could be found for the different antisense strategies in cellbased assays. SiRNAs and RNase H oligonucleotides appear to work upon the target mRNA at different stages of its processing/metabolism
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