Oligonucleotide‐Based Knockdown Technologies: Antisense Versus RNA Interference

Abstract

The postgenomic era is characterized by an almost intimidating amount of information regarding the sequences and expression of previously unknown genes. In response, researchers have developed an increasing interest in functional studies. At the start of such a study, one may have little more than sequence information and bioinformatic annotation. The next step is to hypothesize a potential role in the context of a cell. Testing of the hypothesis needs to be fast, cheap, and applicable to a large number of genes. Knockdown methods that rely on binding of antisense oligonucleotides to mRNA combined with a subsequent functional assay in cell culture fulfil these requirements: sequence information is sufficient for synthesis of active inhibitors. Depending on the in vitro model chosen, knockdown of gene expression can be achieved with medium or even high throughput. The two most popular methods of knockdown in cell culture are the use of antisense oligonucleotides that rely on ribonuclease H (RNAse H)-dependent cleavage of mRNA, and RNA interference triggered by small double-stranded RNA molecules. Both methods act in a sequence-specific manner and can give efficient knockdown. In both cases, researchers struggle with nonspecific "off-target" effects and the difficulty of site selection. Studies that compare the methods differ in their judgment as to which method is superior.

Intrathecal siRNA against Toll-like receptor 4 reduces nociception in a rat model of neuropathic pain

“…siRNA is a more promising and advantageous strategy. Several studies have indicated that RNAi is more potent than antisense oligonucleotides even in cases where site selection was optimized for antisense effectiveness 29. One of the potential advantages of RNAi technologies is the ability to design precisely targeted therapeutics for almost any gene, regardless of the function of the gene product, whether that function is clearly defined, and in the absence of protein structure information 30.…”

Dissecting role of regulatory factors in NF-kappaB pathway with siRNA1

“…An additional obstacle in exploring siRNA as a therapeutic tool is toxicity. siRNA have the potential to induce a concentration-and cell-type-dependent cell death [105] . In mammalian cells, the utility of RNAi has been limited by the innate immune response triggered by dsRNA.…”

“…Long dsRNA induce an interferon response usually resulting in a generalized inhibition of gene expression. However, this response can usually be avoided in mammalian cell cultures by using synthetic siRNA with a length of 21 nt [105] . Inhibition of viral replication by RNAi has been demonstrated in vitro for a variety of viruses, including RNA viruses such as HIV, respiratory syncytial virus, influenza virus, poliovirus, West Nile virus, dengue virus, and foot and mouth disease virus.…”

Conditionally Replicating Adenoviruses Expressing Short Hairpin RNAs Silence the Expression of a Target Gene in Cancer Cells

“…activity is considered as successful knockdown (25). Moreover, the level of silencing depends to a great extent on which region of the mRNA is targeted (26).…”