siRNA as a Tool
Several recent discoveries have begun to overcome the difficulties of unspecific responses and the cell death of mammalian cells in RNAi experiments.
Elbashir et al., analyzed the rate of 21-23 nt fragment formation after successfully triggering RNAi by several dsRNA in their described Drosophila lysate in vitro system (28). The authors then triggered RNAi efficiently using chemically synthesized siRNA duplexes of the same structure with 3'-overhang ends (11).
In a following study Tuschl et al., demonstrated that chemically synthesized 21 nt siRNA duplexes specifically suppress expression of endogenous and heterologeous genes in different mammalian cell lines, including human kidney (293) and HeLa cells (29). A key discovery of these studies was that no unspecific effects occurred in mammalian cells by transfection of short sequences (<30nt). The authors suggested that 21 nt siRNA duplexes provide a new tool for studying gene function in mammalian cells and may eventually be used as gene-specific therapeutics.
Caplen et al., supported these discoveries on siRNAs mediating RNAi in cell extracts and presented data that synthetic siRNAs can induce gene-specific inhibition of expression in Caenorhabditis elegans and in cell lines from humans and mice (30). This study also presents evidence that siRNAs can have direct effects on gene expression in C. elegans and mammalian cell culture in vivo.
siRNA an Outlook
Today many researchers are excited about the huge potential of siRNA. In Nature Structural Biology Zamore wrote (10): "The ability to initiate RNAi in cultured mammalian cells using siRNA duplexes should dramatically accelerate the pace of reverse genetic analysis of the human genome. It will not be surprising if in five years the loss of function phenotype of virtually every human gene will have been examined in cultured cells using siRNA-mediated RNAi. In fact, new technologies may soon make it possible to fabricate RNAi chips - arrays of siRNAs on which cultured cells of many types can be grown and scored for the effects of suppressing expression of every gene in the genome, one-by-one."
We are commited to focusing our resources on this fast growing field of research. We strongly believe that siRNA technology is the most powerful tool to unravel the function of genes. In combination with our TOM-Chemistry and our expertise in RNA synthesis we hope to contribute significantly to the growth of this exciting technology. In our eyes siRNA will soon be used in a variety of applications such as high throughput target validation and gene therapy.
Literature and further reading
(1) Jorgensen RA., Cluster PD, English J., Que Q., Napoli CA., "Chalcone synthase cosuppression phenotypes in petunia flowers: comparison of sense vs. antisense constructs and single-copy vs. complex T-DNA sequences", Plant Mol. Biol., 31 (5), 957-73, (1996)
(2) Baulcombe DC., "Fast forward genetics based on virus-induced gene silencing", Curr. Opin. Plant. Biol., 2, 109-113, (1999)
(3) Cogoni C, Irelan JT., Schumacher M., Schmidhauser TJ., Selker EU., Macino G., "Transgene silencing of the al-1 gene in vegetative cells of Neurospora is mediated by a cytoplasmic effector and does not depend on DNA-DNA interactions or DNA methylation", EMBO J., 15 (12), 3153-63, (1996)
(4) Cogoni C., Macino G.,Proc. Natl Acad. Sci. USA 94, 10233-10238, (1997)
(5) Guo S. & Kemphues K., "par-1, a gene required for establishing polarity in embryos, encodes a putative Ser/Thr kinase that is symmetrically disrupted", Cell, 81, 611-620 (1995)
Susan Parrish, Jamie Fleenor, SiQun Xu, Craig Mello and Andrew Fire, "Functional Anatomy of a dsRNA Trigger: Differential Requirement for the Two Trigger Strands in RNA Interference", Molecular Cell, 6, 1077-87, (2000)
(7) Fire A., Xu S., Montgomery M.K., Kostas S.A., Driver S.E., Mello C.C., "Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans", Nature, Vol 391, (1998)
Hamilton AJ. and Baulcombe DC., A species of small antisense RNA in posttranscriptional gene silencing in plants, Science, 286, 950-952, (1999)
(9) Hammond SM., Bernstein E., Beach D., Hannon GJ., An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cells., Nature 404, 293-296, (2000)
(10) Zamore P.D., "RNA interference: listening to the sound of silence", Nature Structural Biology, 8, 9, 746-750, (2001)
(11) Zamore PD., Tuschl T., Sharp PA.& Bartel DP., "RNAi: Double stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals", Cell, 101, 25-33, (2000)
(12) Elbashir SM., Lendeckel W. & Tuschl T., "RNA interference is mediated by 21- and 22-nucleotide RNAs", Genes & Development, 15, 188-200, (2001)
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