RNA interference (RNAi) is a relatively new technology that is revolutionizing the way that researchers study mammalian gene expression. RNAi has had significant impact on the ease, speed, and specificity with which the loss of gene function analysis can be done in mammalian cells and animal models. This is becoming the method of choice for researchers doing loss of function studies.
A brief history of RNA interference (RNAi)
Figure 1. Evolution of RNAi
RNA Interference (RNAi) is one of the most important technological breakthroughs in modern biology, allowing us to directly observe the effects of the loss of function of specific genes in mammalian systems.
In the early 1990s, a number of scientists observed independently that RNA inhibited protein expression in plants and fungi (Figure 1). This phenomenon, identified but not understood, was then known as “posttranscriptional gene silencing” and “quelling”. In 1998 Fire and Mello observed in Caenorthabditis elegans that double-stranded RNA (dsRNA) was the source of sequence-specific inhibition of protein expression, which they called “RNA interference”. While the studies in C. elegans were encouraging at that time the use of RNAi as a tool was limited to lower organisms because delivering long dsRNA for RNAi was nonspecifically inhibitory in mammalian cells. Fire and Mello won the 2006 Nobel Prize in Physiology or Medicine for their discovery of RNA interference.
Further studies in plants and invertebrates demonstrated that the actual molecules that led to RNAi were short dsRNA oligonucleotides, 21 nucleotides in length, processed internally by an enzyme called “Dicer”. The Dicer cleavage products were referred to as “short interfering RNA” now popularly known as “siRNA”. Subsequently in 2001, it was demonstrated that siRNA could directly trigger RNAi in mammalian cells without evoking nonspecific effects.
Today we have a greater understanding of the components that are part of the RNAi pathway, the efficiency with which these components function, the specificity of sequence recognition and cleavage of cellular mRNA and many of the key requirements for designing and generating extremely effective RNAi reagents. Our analysis of novel chemistries will further improve this approach and lead the way to in vivo analysis and potentially make the use of RNAi in therapeutics possible.
More about RNAi
Applications of RNAi
RNAi technology can be used to identify and functionally assess the thousands of genes within the genome that potentially participate in disease phenotypes. In addition, RNAi technology provides an efficient means for blocking expression of a specific gene and evaluating its response to chemical compounds or changes in signaling pathways.
How RNA interference (RNAi) works
RNAi technology takes advantage of the cell’s natural machinery, facilitated by short interfering RNA molecules, to effectively knock down expression of a gene of interest. There are several ways to induce RNAi, synthetic molecules, RNAi vectors, and in vitro dicing (Figure 2). In mammalian cells, short pieces of dsRNA, short interfering RNA (siRNA), initiate the specific degradation of a targeted cellular mRNA.
In this process the antisense strand of the siRNA duplex becomes part of a multi-protein complex, or RNA-induced silencing complex (RISC), which then identifies the corresponding mRNA and cleaves it at a specific site. Next, this cleaved message is targeted for degradation, which ultimately results in the loss of protein expression.
Figure 2. Methods of RNAi knockdown in mammalian cells.