If there is one thing that researchers agree on, it’s that now is a good time to be working with RNA interference (RNAi). RNAi is a natural cellular mechanism that regulates gene expression at the stage of translation by degrading the mRNA or blocking translation. It can also alter the level of transcription of specific genes.
Double-stranded RNA (dsRNA) triggers a series of biochemical events that culminates in sequence-specific suppression of gene expression. “Long dsRNAs have been employed for many years as a means to modulate gene expression in plants, yeast, and C. elegans,” noted Mark Behlke, M.D., Ph.D., svp of molecular genetics and CSO at Integrated DNA Technologies (IDT; www.idtdna.com).
“Similar attempts in higher organisms failed due to interferon activation, however we now know that short RNA duplexes can be safely used in mammalian systems both in vitro and in vivo. The technology has rapidly matured, thanks in large part to all that was learned over the past 20 years using antisense oligonucleotides. RNAi is now routinely employed in vivo as an experimental tool and numerous groups are vigorously pursing the use of RNAi compounds as therapeutics. Several siRNA drugs are already in clinical trials and more are in preclinical development.”
The RNAi pathway is initiated by the enzyme dicer, which cleaves long dsRNA molecules into short fragments of 20–25 base pairs. One of the two strands of each fragment, known as the guide strand, is then incorporated into the RNA-induced silencing complex (RISC) and paired with complementary sequences.
The most well-studied outcome of this recognition event is post-transcriptional gene silencing. This occurs when the guide strand specifically pairs with an mRNA molecule and induces the degradation by argonaute, the catalytic component of the RISC complex. Another outcome is epigenetic changes to a gene—histone modification and DNA methylation—affecting the degree to which the gene is transcribed.
RNAi targets include RNA from viruses. These targets also play a role in regulating development and genome maintenance. Breakthroughs in this technology are making it possible to access biological information that was not possible until recently.
The selective and robust effect of RNAi on gene expression makes it a valuable research tool, both in cell culture and in living organisms, because synthetic dsRNA introduced into cells can induce suppression of specific genes of interest. RNAi may also be used for large-scale screens that systematically shut down each gene in the cell, which can help identify the components necessary for a particular cellular process or an event such as cell division. Exploitation of the pathway is also a promising tool in biotechnology and medicine.
Discussion of these new technologies took center stage at the “Second Annual RNAi World Congress” in Boston last month, where presenters described some of the new opportunities that lay ahead for research and drug discovery. “This is a really exciting time to be in the field,” said Dr. Behlke.