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TL;DR: The results suggest that the thermodynamic properties of siRNA play a critical role in determining the molecule's function and longevity, possibly biasing the steps involved in duplex unwinding and strand retention by RISC.
2,677 citations
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TL;DR: Application of an algorithm incorporating all eight characteristics associated with siRNA functionality significantly improves potent siRNA selection and highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.
Abstract: Short-interfering RNAs suppress gene expression through a highly regulated enzyme-mediated process called RNA interference (RNAi). RNAi involves multiple RNA-protein interactions characterized by four major steps: assembly of siRNA with the RNA-induced silencing complex (RISC), activation of the RISC, target recognition and target cleavage. These interactions may bias strand selection during siRNA-RISC assembly and activation, and contribute to the overall efficiency of RNAi. To identify siRNA-specific features likely to contribute to efficient processing at each step, we performed a systematic analysis of 180 siRNAs targeting the mRNA of two genes. Eight characteristics associated with siRNA functionality were identified: low G/C content, a bias towards low internal stability at the sense strand 3'-terminus, lack of inverted repeats, and sense strand base preferences (positions 3, 10, 13 and 19). Further analyses revealed that application of an algorithm incorporating all eight criteria significantly improves potent siRNA selection. This highlights the utility of rational design for selecting potent siRNAs and facilitating functional gene knockdown studies.
2,403 citations
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TL;DR: In this article, the authors used short interfering RNA (siRNA) pools against individual genes to test their ability to inhibit VKOR activity in human cells and confirmed that MGC11276 messenger RNA encodes VKOR through its expression in insect cells and sensitivity to warfarin.
Abstract: Vitamin K epoxide reductase (VKOR) is the target of warfarin, the most widely prescribed anticoagulant for thromboembolic disorders. Although estimated to prevent twenty strokes per induced bleeding episode, warfarin is under-used because of the difficulty of controlling dosage and the fear of inducing bleeding. Although identified in 1974 (ref. 2), the enzyme has yet to be purified or its gene identified. A positional cloning approach has become possible after the mapping of warfarin resistance to rat chromosome 1 (ref. 3) and of vitamin K-dependent protein deficiencies to the syntenic region of human chromosome 16 (ref. 4). Localization of VKOR to 190 genes within human chromosome 16p12-q21 narrowed the search to 13 genes encoding candidate transmembrane proteins, and we used short interfering RNA (siRNA) pools against individual genes to test their ability to inhibit VKOR activity in human cells. Here, we report the identification of the gene for VKOR based on specific inhibition of VKOR activity by a single siRNA pool. We confirmed that MGC11276 messenger RNA encodes VKOR through its expression in insect cells and sensitivity to warfarin. The expressed enzyme is 163 amino acids long, with at least one transmembrane domain. Identification of the VKOR gene extends our understanding of blood clotting, and should facilitate development of new anticoagulant drugs.
715 citations
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TL;DR: This study demonstrates that the efficient gene silencing by rationally designed small interfering RNA can be used as an approach to functionally analyze the entire cellular machineries, such as the clathrin-coated pits and vesicles.
449 citations
Authors
Showing all 52 results
Name | H-index | Papers | Citations |
---|---|---|---|
Anastasia Khvorova | 52 | 299 | 20822 |
Devin Leake | 39 | 128 | 9851 |
William Marshall | 30 | 194 | 9104 |
Angela Reynolds | 28 | 152 | 10993 |
Robert J. Kaiser | 26 | 68 | 7231 |
Emily M. Anderson | 23 | 43 | 3202 |
Stephen Scaringe | 23 | 118 | 4314 |
Jeffrey A. Pleiss | 23 | 38 | 2071 |
Jon Karpilow | 20 | 37 | 3972 |
Annaleen Vermeulen | 19 | 29 | 1781 |
Scott Baskerville | 17 | 27 | 5376 |
Yuriy Fedorov | 15 | 36 | 2817 |
Anja van Brabant Smith | 12 | 22 | 932 |
Steven Read | 11 | 39 | 390 |
Barbara Robertson | 10 | 17 | 438 |