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Published September 4, 2007 | Published + Supplemental Material
Journal Article Open

A modular and extensible RNA-based gene-regulatory platform for engineering cellular function

Abstract

Engineered biological systems hold promise in addressing pressing human needs in chemical processing, energy production, materials construction, and maintenance and enhancement of human health and the environment. However, significant advancements in our ability to engineer biological systems have been limited by the foundational tools available for reporting on, responding to, and controlling intracellular components in living systems. Portable and scalable platforms are needed for the reliable construction of such communication and control systems across diverse organisms. We report an extensible RNA-based framework for engineering ligand-controlled gene-regulatory systems, called ribozyme switches, that exhibits tunable regulation, design modularity, and target specificity. These switch platforms contain a sensor domain, comprised of an aptamer sequence, and an actuator domain, comprised of a hammerhead ribozyme sequence. We examined two modes of standardized information transmission between these domains and demonstrate a mechanism that allows for the reliable and modular assembly of functioning synthetic RNA switches and regulation of ribozyme activity in response to various effectors. In addition to demonstrating examples of small molecule-responsive, in vivo functional, allosteric hammerhead ribozymes, this work describes a general approach for the construction of portable and scalable gene-regulatory systems. We demonstrate the versatility of the platform in implementing application-specific control systems for small molecule-mediated regulation of cell growth and noninvasive in vivo sensing of metabolite production.

Additional Information

© 2007 by the National Academy of Sciences. Edited by Arthur D. Riggs, Beckman Research Institute, City of Hope, Duarte, CA, and approved July 12, 2007 (received for review May 1, 2007). Published online on August 20, 2007, 10.1073/pnas.0703961104 We thank K. Hawkins for assistance with controls and HPLC experiments and data analysis; A. Babiskin (California Institute of Technology) for pRzS and assistance with quantitative RT-PCR assays; K. Dusinberre, J. Michener, and J. Liang for assistance with controls; E. Kelsic for assistance with image presentation; and Y. Chen and K. Hoff for critical reading of the manuscript. This work was supported by the Arnold and Mabel Beckman Foundation, the National Institutes of Health, and the Center for Biological Circuit Design at the California Institute of Technology (fellowship to M.N.W.). Author contributions: M.N.W. and C.D.S. designed research; M.N.W. performed research; M.N.W. and C.D.S. analyzed data; and M.N.W. and C.D.S. wrote the paper. Conflict of interest statement: The authors declare competing financial interests in the form of a pending patent application whose value may be affected by the publication of this manuscript. This article is a PNAS Direct Submission. This article contains supporting information online at www.pnas.org/cgi/content/full/0703961104/DC1. A glossary of terms is available in supporting information (SI) Text.

Attached Files

Published - WINpnas07.pdf

Supplemental Material - WINpnas07suppfig10.pdf

Supplemental Material - WINpnas07suppfig11.pdf

Supplemental Material - WINpnas07suppfig12.pdf

Supplemental Material - WINpnas07suppfig13.pdf

Supplemental Material - WINpnas07suppfig14.pdf

Supplemental Material - WINpnas07suppfig15.pdf

Supplemental Material - WINpnas07suppfig16.pdf

Supplemental Material - WINpnas07suppfig7.pdf

Supplemental Material - WINpnas07suppfig8.pdf

Supplemental Material - WINpnas07suppfig9.pdf

Supplemental Material - WINpnas07supptables1-2.pdf

Supplemental Material - WINpnas07supptext.pdf

Supplemental Material - WINpnas07table3.pdf

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Additional details

Created:
August 22, 2023
Modified:
October 16, 2023