In the fall of 2006, the RNA world arrived on the big stage when it was announced that Andrew Z. Fire, Ph.D., Craig Mello, Ph.D., and Roger Kornberg, Ph.D., were selected to receive the Nobel Prizes in Physiology or Medicine (Drs. Fire and Mellow) and Chemistry (Dr. Kornberg) for their work on RNA interference (RNAi) and RNA polymerase II, respectively.
RNA therapeutics have since emerged as the next big frontier in drug development. Not since the development of gene cloning, antibodies, and PCR has a specific biotechnology had so much impact on molecular biology, biomedicine, and beyond.
RNAi, the sequence-specific gene silencing at the messenger RNA level induced by double-stranded RNA (dsRNA), is a prime example of the value of serendipity as well as research involving model organisms.
Two decades ago, plant scientists first noted the “co-suppression” phenomenon when they created transgenic petunias and found that the transgene not only failed to express properly but was silencing sequence-homologous genes in the genomes of the same plants. Similar phenomena were soon recognized in other lower eukaryotes such as the bread mold Neurospora crassa and the worm Caenorhabditis elegans.
It was in C. elegans that Drs. Fire and Mello made the seminal discovery that it is double-stranded RNA, and not single-stranded sense or antisense RNA, that is the actual trigger for the gene silencing, which the scientists subsequently labeled RNAi. The importance of that discovery was that it made RNAi a reverse genetics tool of sufficient robustness for adoption by the wider scientific community.
Molecular genetics and biochemistry in plants, worms, flies, and molds quickly converged to unravel important principles of RNAi molecular biology, but one nagging question remained: would it also work in human cells?
The reason for some skepticism about the existence of the RNAi pathway in mammals was because, with a few exceptions in early development, long dsRNAs that were used to trigger RNAi gene silencing in lower eukaryotes were known to elicit the interferon response, a potent innate immune reaction that causes the nonspecific shutdown of protein translation. Obviously, the medical and economic value of RNAi would increase dramatically if it also functioned in human cells.
There were, however, hints from plant RNAi studies by the Baulcombe group and the parallel discovery of genetically related endogenous small silencing RNAs, called microRNAs, that small RNAs may mediate RNAi activity downstream of long dsRNAs. This was confirmed by elegant biochemical studies (using the fly) on the processing of long dsRNAs into 19- to 23-nucleotide small RNAs in the test tube.
Based on these results and the speculation that a type III ribonuclease might be involved in this processing, Tuschl and colleagues fully opened up RNAi to human medicine by demonstrating that synthetic 19–20 base-pair dsRNA with two nucleotide 3' overhangs, the classical siRNA structure, mediates efficient RNAi gene silencing in mammalian cells.
From then on, the use of RNAi in basic and applied biomedical research exploded. A number of additional RNAi trigger structures and strategies soon emerged, including DNA-directed RNAi whereby the RNAi trigger is transcribed from a DNA template.
Consequently, RNAi became widely used, from single-gene reverse genetic studies to genome-wide RNAi screens for the discovery of genes involved in biological pathways of interest. RNAi was also employed in the rapid generation of transgenic animal models of human disease and the development of crops, and as a therapeutic modality in its own right, that is, RNAi therapeutics.