Antisense Mechanism Of Action

Antisense oligonucleotides are small synthetic oligonucleo-tides that are designed to bind to mRNA through Watson-Crick hybridization. Upon binding to the RNA, the oligo-

nucleotide may inhibit expression of the encoded gene product through either inducing cleavage of the RNA by RNases such as RNase H or by occupancy of critical regulatory sites on the RNA (Fig. 2). Several studies have documented that phos-phorothioate oligodeoxynucleotides promote cleavage of the targeted RNA by a mechanism consistent with RNase H cleavage (2-6). RNase H is a ubiquitously expressed enzyme that cleaves the RNA strand of an RNA-DNA heteroduplex (6,7). If the antisense oligonucleotide use DNA chemistry, it will direct RNase H to specifically cleave the target RNA upon binding.

Another RNase-dependent antisense mechanism that has recently received much attention is interference RNA or RNAi (8-13). Introduction of long double-stranded RNA (dsRNA) into eukaryotic cells leads to the sequence-specific degradation of homologous gene transcripts. The long dsRNA molecules are metabolized to small 21 to 23 nucleotide interfering RNAs (siRNAs) by the action of an endogenous ribonuclease, Dicer (14-16). The siRNA molecules bind to a protein complex, termed RNA-induced silencing complex (RISC), which contains a helicase activity that unwinds the 2 strands of RNA molecules, allowing the antisense strand to bind to the targeted RNA molecule (12,17). The RISC is also believed to contain an endonuclease activity, which hydrolyzes the target RNA at the site where the antisense strand is bound. It is unknown whether the antisense RNA molecule is also hydrolyzed or recycles and binds to another RNA molecule. Therefore, RNA interference is an antisense mechanism of action, as ultimately a single- strand RNA molecule binds to the target RNA molecule by Watson-Crick base pairing rules and recruits a ribonuclease that degrades the target RNA.

Figure 1 Phosphorothioate antisense oligodeoxynucleotide targeting an RNA receptor. Watson-Crick base pairing rules are indicated: nucleobase adenosine hydrogen bonds to nucleobase uracil, nucleobase cytosine hydrogen bonds to nucleobase guanine.

In mammalian cells, long double-stranded RNA molecules were found to promote a global change in gene expression, obscuring any gene-specific silencing (18,19). This reduction in global gene expression is believed to be mediated in part, through activation of double-stranded RNA-activated protein kinase (PKR), which phosphorylates and inactivates the translation factor eIF2a (20). Recently, it has been shown that transfection of synthetic 21-nucleotide siRNA duplexes into mammalian cells does not elicit the PKR response, allowing effective inhibition of endogenous genes in a sequence-specific manner (21,22). These siRNAs are too short to trigger the nonspecific dsRNA responses, but they still promote degradation of complementary RNA sequences (21-23). We have directly compared the activity of optimized oligonucleotides that work by RNase H mechanism with those that work by an RNAi mechanism in human cells (24). The potency, maximal efficacy, duration of action, selectivity and efficiency for identification of leads was similar for both mechanisms in cell-based assays. The one noted difference between the 2 mechanisms, is that RNase H oligonucleotides are able to cleave pre-mRNA in the nucleus, whereas siRNA oligonucleotides appear to only be able to interact with mature mRNA in the cytoplasm. These results suggest that both mechanisms are equally valid for inhibition of gene expression in mammalian cells.

There are other RNases present in cells that may be exploited in a manner similar to RNase H or the dsRNase associ ated with RNAi. As an example, Wu et al., reported that single-stranded, phosphorothioate-modified oligoribonucleo-tides can promote selective loss of ha-ras mRNA in human cells (25). The RNA oligonucleotides could be partially modified with 2'-O-methyl nucleosides and still support enzyme activity. The enzyme activity is consistent with a RNase III type enzyme. RNase III activity is present in both cytosolic and nuclear extracts (25). It is unclear if this enzyme activity is the same RNase III used for siRNA oligonucleotides. Recent work has demonstrated that single-stranded RNA oligonucle-otides can interact with the RISC and promote selective degradation of targeted RNA, consistent with RNAi activity, albeit not as efficiently as double-stranded RNA (26,27). Another RNase enzyme that has been exploited for antisense applications is RNase L (28). RNase L is ribonuclease activated by 2'-5'-linked oligoadenylates generated in response to interferon activation. Selectively, linkage of 2'-5' oligoadenylate to an antisense oligonucleotide has been reported to promote selective cleavage of the targeted mRNA (28-30).

It should be noted that not all oligonucleotide designed to hybridize to a target RNA effectively inhibit target gene expression (2,31-33). This is believed to be due to inaccessibility of some regions of the RNA to the oligonucleotide due to secondary or tertiary structure or to protein interactions with the RNA. At this time, there are no good predictive algorithms for predicting antisense oligonucleotide-binding sites

Table 1 Antisense Oligonucleotides Approved or Currently in Clinical Development


Molecular target

Disease indication


Route of administration




Human CMV IE-2 gene

CMV retinitis

Phosphorothioate oligodeoxynucleotide



Novartis Ophthalmic/ISIS


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