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Design of small molecule-responsive microRNAs based on structural requirements for Drosha processing

Beisel, Chase L. and Chen, Yvonne Y. and Culler, Stephanie J. and Hoff, Kevin G. and Smolke, Christina D. (2011) Design of small molecule-responsive microRNAs based on structural requirements for Drosha processing. Nucleic Acids Research, 39 (7). pp. 2981-2994. ISSN 0305-1048.

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MicroRNAs (miRNAs) are prevalent regulatory RNAs that mediate gene silencing and play key roles in diverse cellular processes. While synthetic RNA-based regulatory systems that integrate regulatory and sensing functions have been demonstrated, the lack of detail on miRNA structure–function relationships has limited the development of integrated control systems based on miRNA silencing. Using an elucidated relationship between Drosha processing and the single-stranded nature of the miRNA basal segments, we developed a strategy for designing ligand-responsive miRNAs. We demonstrate that ligand binding to an aptamer integrated into the miRNA basal segments inhibits Drosha processing, resulting in titratable control over gene silencing. The generality of this control strategy was shown for three aptamer–small molecule ligand pairs. The platform can be extended to the design of synthetic miRNAs clusters, cis-acting miRNAs and self-targeting miRNAs that act both in cis and trans, enabling fine-tuning of the regulatory strength and dynamics. The ability of our ligand-responsive miRNA platform to respond to user-defined inputs, undergo regulatory performance tuning and display scalable combinatorial control schemes will help advance applications in biological research and applied medicine.

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Smolke, Christina D.0000-0002-5449-8495
Additional Information:© 2010 The Author(s). Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( by-nc/2.5), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received August 2, 2010; Revised September 28, 2010; Accepted September 30, 2010. First published online: December 11, 2010. The authors thank A. Brown, M. Greenwood-Goodwin and J. Vowles for constructive comments on the article and V.N. Kim for providing the pCK-Drosha-FLAG and pCK-DGCR8-FLAG plasmids. C.L.B. and C.D.S. designed the experiments, Y.Y.C. performed the inducible promoter experiments, S.J.C. prepared the stable cell lines, K.G.H. helped perform the Drosha cleavage assays, C.L.B. performed all other experiments and C.L.B. and C.D.S. wrote the article. Funding: Caltech Joseph Jacobs Institute for Molecular Engineering for Medicine (to C.D.S.); Alfred P. Sloan Foundation, fellowship (to C.D.S.); Bill and Melinda Gates Foundation (to C.D.S.); Department of Defense (W81XWH-06-1-0250 to C.D.S.); National Institutes of Health (fellowship to K.G.H, GM091298 to C.D.S.); National Science Foundation (fellowship to C.L.B.). Funding for open access charge: National Institutes of Health (GM091298 to C.D.S.).
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Caltech Joseph Jacobs Institute for Molecular Engineering for MedicineUNSPECIFIED
Alfred P. Sloan FoundationUNSPECIFIED
Bill and Melinda Gates FoundationUNSPECIFIED
Department of DefenseW81XWH-06-1-0250
Issue or Number:7
Record Number:CaltechAUTHORS:20110506-141659804
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Official Citation: Chase L. Beisel, Yvonne Y. Chen, Stephanie J. Culler, Kevin G. Hoff, and Christina D. Smolke Design of small molecule-responsive microRNAs based on structural requirements for Drosha processing Nucl. Acids Res. (2011) 39(7): 2981-2994 doi:10.1093/nar/gkq954
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:23584
Deposited By: Ruth Sustaita
Deposited On:09 May 2011 20:16
Last Modified:03 Oct 2019 02:47

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