Researchers have produced vast libraries of short segments of ribonucleic acid (RNA) that can be used to turn off individual human and mouse genes to study their function.
The libraries will be made widely available to laboratories studying human biology and disease. The researchers are optimistic that the libraries will become a powerful research tool for gene analysis and discovery.
Two independent research groups reported on their respective RNA interference (RNAi) libraries in the March 25, 2004, issue of the journal Nature. Gregory Hannon of the Cold Spring Harbor Laboratory and Howard Hughes Medical Institute investigator <<>> at Harvard Medical School and Brigham and Women's Hospital led the first group. The joint lead authors were Patrick Paddison, Jose Silva and Douglas Conklin in Hannon's laboratory. René Bernards of The Netherlands Cancer Institute led a second group.
Commenting on the significance of the studies in the journal Nature, Andrew Fraser at the Wellcome Trust Sanger Institute wrote: “As no single laboratory can specialize in every aspect of gene function, the general availability of these [short hairpin RNA] libraries as a communal resource is a major step forward, harnessing the screening expertise of the entire mammalian-cell research community.”
RNA interference is a technique used with much success by researchers to switch off genes in lower organisms, including the fruit fly Drosophila and the roundworm C. elegans. Researchers stumbled upon this powerful tool for gene analysis when they discovered that introduced sequences of double-stranded RNA identical to a target messenger RNA actually triggered degradation of the messenger RNA.
Messenger RNA molecules are the genetic templates for proteins. In constructing proteins, the mRNA template is transcribed from DNA genes and transported to the ribosomes — the cell's protein “factories” that are large complexes of protein and RNA. RNA interference is a technique that essentially shuts down the activity of the gene under study.
“But RNAi didn't work in the vast majority of human or mouse cells because there are additional antiviral responses that recognize double-stranded RNA,” said Elledge. “While the machinery to do RNAi is in mammalian cells, the antiviral machinery makes the introduced RNA toxic, and the cells die.”
Researchers subsequently discovered that short segments of interfering RNA could be introduced into mammalian cells and remain unnoticed by the antiviral machinery, said Elledge. Furthermore, they discovered that the cell itself could be engineered to make interfering RNAs by introducing the gene for short hairpin RNA molecules that fold back on themselves to create a small RNA.
To construct a library of mammalian genes for short hairpin RNA molecules, Hannon and his colleagues first had to settle on an optimal design for a short-hairpin-RNA molecule. “We tested a lot of different things — for example, the length of the hairpin, the loop structure, the structure of the transcript and what promoters to use,” said Hannon. “And we arrived at an optimal structure for this phase of the science.”
Hannon emphasized, however, “that set of parameters is something that is going to evolve continuously. There have been many advances over the last year in understanding of the biochemistry of RNAi. So, we are now constructing even more effective structures and even more effective delivery vehicles which will be built into future generations of this library.”