MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells (original) (raw)
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- Published: 12 August 2007
Nature Methods volume 4, pages 721–726 (2007)Cite this article
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Abstract
MicroRNAs are predicted to regulate thousands of mammalian genes, but relatively few targets have been experimentally validated and few microRNA loss-of-function phenotypes have been assigned. As an alternative to chemically modified antisense oligonucleotides, we developed microRNA inhibitors that can be expressed in cells, as RNAs produced from transgenes. Termed 'microRNA sponges', these competitive inhibitors are transcripts expressed from strong promoters, containing multiple, tandem binding sites to a microRNA of interest. When vectors encoding these sponges are transiently transfected into cultured cells, sponges derepress microRNA targets at least as strongly as chemically modified antisense oligonucleotides. They specifically inhibit microRNAs with a complementary heptameric seed, such that a single sponge can be used to block an entire microRNA seed family. RNA polymerase II promoter (Pol II)-driven sponges contain a fluorescence reporter gene for identification and sorting of sponge-treated cells. We envision the use of stably expressed sponges in animal models of disease and development.
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Acknowledgements
This work was funded by US Public Health Service grants U19-AI056900 from the National Cancer Institute, by an Integrative Cancer Biology Program Grant U54 CA112967 from the National Institutes of Health to P.A.S. and partially by Cancer Center Support (core) P30-CA14051 from the National Cancer Institute. M.S.E. is supported by a Howard Hughes Medical Institute Predoctoral Fellowship and a Paul and Cleo Schimmel Scholarship. J.R.N. is supported by the Cancer Research Institute. We thank A. Garfinkel and M. Kumar for luciferase reporter preparations, A. Leung for assistance with fluorescence in situ hybridization, D. Engelke (University of Michigan) for the U6 vector and members of the Sharp laboratory for helpful discussions.
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Authors and Affiliations
- Center for Cancer Research, E17-529B Massachusetts Institute of Technology, Cambridge, 02139, Massachusetts, USA
Margaret S Ebert, Joel R Neilson & Phillip A Sharp
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- Margaret S Ebert
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Contributions
M.S.E. and J.R.N. conceived the experimental design and made the sponge constructs. M.S.E. performed the experiments and wrote the manuscript. P.A.S. supervised the work.
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Correspondence toPhillip A Sharp.
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Ebert, M., Neilson, J. & Sharp, P. MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells.Nat Methods 4, 721–726 (2007). https://doi.org/10.1038/nmeth1079
- Received: 24 April 2007
- Accepted: 20 July 2007
- Published: 12 August 2007
- Issue Date: September 2007
- DOI: https://doi.org/10.1038/nmeth1079