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Published November 2012 | Supplemental Material + Published
Journal Article Open

A high-throughput, quantitative cell-based screen for efficient tailoring of RNA device activity


Recent advances have demonstrated the use of RNA-based control devices to program sophisticated cellular functions; however, the efficiency with which these devices can be quantitatively tailored has limited their broader implementation in cellular networks. Here, we developed a high-efficiency, high-throughput and quantitative two-color fluorescence-activated cell sorting-based screening strategy to support the rapid generation of ribozyme-based control devices with user-specified regulatory activities. The high-efficiency of this screening strategy enabled the isolation of a single functional sequence from a library of over 106 variants within two sorting cycles. We demonstrated the versatility of our approach by screening large libraries generated from randomizing individual components within the ribozyme device platform to efficiently isolate new device sequences that exhibit increased in vitro cleavage rates up to 10.5-fold and increased in vivo activation ratios up to 2-fold. We also identified a titratable window within which in vitro cleavage rates and in vivo gene-regulatory activities are correlated, supporting the importance of optimizing RNA device activity directly in the cellular environment. Our two-color fluorescence-activated cell sorting-based screen provides a generalizable strategy for quantitatively tailoring genetic control elements for broader integration within biological networks.

Additional Information

© 2012 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 (http://creativecommons.org/licenses/by-nc/3.0), which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received February 22, 2012; Revised June 1, 2012; Accepted June 5, 2012. First published online: July 18, 2012. The authors thank C. Crumpton and M. Bigos of the Stanford Shared FACS facility. J.C.L. designed and performed the research and wrote the article; A.L.C. designed and performed the research associated with the two-color FACS-based screen and wrote the article; A.B.K. designed and performed the research associated with the in vitro characterization of ribozyme cleavage rates and the actuator screen and wrote the article; and C.D.S. designed the research and wrote the article. Funding: National Institutes of Health [R01GM086663]; National Science Foundation [CBET-0917638, CCF-0943269]; Defense Advanced Research Projects Agency [HR0011-11-2-0002]; National Sciences and Engineering Research Council of Canada (fellowship to A.B.K.); Alfred P. Sloan Foundation (fellowship to C.D.S.). Funding for open access charge: Defense Advanced Research Projects Agency.

Attached Files

Published - Nucl._Acids_Res.-2012-Liang-e154.pdf

Supplemental Material - nar-01088-met-g-2012-File009.pdf


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