Welcome to the new version of CaltechAUTHORS. Login is currently restricted to library staff. If you notice any issues, please email coda@library.caltech.edu
Published January 2013 | Supplemental Material + Published
Journal Article Open

A versatile cis-blocking and trans-activation strategy for ribozyme characterization


Synthetic RNA control devices that use ribozymes as gene-regulatory components have been applied to controlling cellular behaviors in response to environmental signals. Quantitative measurement of the in vitro cleavage rate constants associated with ribozyme-based devices is essential for advancing the molecular design and optimization of this class of gene-regulatory devices. One of the key challenges encountered in ribozyme characterization is the efficient generation of full-length RNA from in vitro transcription reactions, where conditions generally lead to significant ribozyme cleavage. Current methods for generating full-length ribozyme-encoding RNA rely on a trans-blocking strategy, which requires a laborious gel separation and extraction step. Here, we develop a simple two-step gel-free process including cis-blocking and trans-activation steps to support scalable generation of functional full-length ribozyme-encoding RNA. We demonstrate our strategy on various types of natural ribozymes and synthetic ribozyme devices, and the cleavage rate constants obtained for the RNA generated from our strategy are comparable with those generated through traditional methods. We further develop a rapid, label-free ribozyme cleavage assay based on surface plasmon resonance, which allows continuous, real-time monitoring of ribozyme cleavage. The surface plasmon resonance-based characterization assay will complement the versatile cis-blocking and trans-activation strategy to broadly advance our ability to characterize and engineer ribozyme-based devices.

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 License (http://creativecommons.org/licenses/by-nc/3.0/), which permits non-commercial reuse, distribution, and reproduction in any medium, provided the original work is properly cited. Received June 9, 2012; Revised September 6, 2012; Accepted October 5, 2012. First published online: November 15, 2012. The authors thank J. Schuman of GE and M. Eckhart of the Stanford PAN facility. A.B.K. and J.C.L. designed research, performed research and wrote the article; C.D.S. designed research and wrote the article. The National Institutes of Health [R01GM086663]; the National Science Foundation [CBET-0917638, CCF-0943269]; the Defense Advanced Research Projects Agency [HR0011-11-2-0002]; the National Sciences and Engineering Research Council of Canada (fellowship to A.B.K.). Funding for open access charge: the Defense Advanced Research Projects Agency. Conflict of interest statement. The authors declare competing financial interests in the form of a pending patent application.

Attached Files

Published - Nucl._Acids_Res.-2013-Kennedy-e41.pdf

Supplemental Material - nar-01466-met-g-2012-File008.pdf


Files (7.5 MB)
Name Size Download all
5.0 MB Preview Download
2.5 MB Preview Download

Additional details

August 19, 2023
October 23, 2023