Rationally designed families of orthogonal RNA regulators of translation

Vivek K. Mutalik; Lei Qi; Joao C. Guimaraes; Julius B. Lucks; Adam P. Arkin

doi:10.1038/nchembio.919

Rationally designed families of orthogonal RNA regulators of translation

Vivek K. Mutalik, Lei Qi, Joao C. Guimaraes, Julius B. Lucks, Adam P. Arkin^*

^*Corresponding author for this work

Chemical and Biological Engineering

Research output: Contribution to journal › Article › peer-review

147 Scopus citations

Abstract

Our ability to routinely engineer genetic networks for applications is limited by the scarcity of highly specific and non-cross-reacting (orthogonal) gene regulators with predictable behavior. Though antisense RNAs are attractive contenders for this purpose, quantitative understanding of their specificity and sequence-function relationship sufficient for their design has been limited. Here, we use rationally designed variants of the RNA-IN-RNA-OUT antisense RNA-mediated translation system from the insertion sequence IS10 to quantify >500 RNA-RNA interactions in Escherichia coli and integrate the data set with sequence-activity modeling to identify the thermodynamic stability of the duplex and the seed region as the key determinants of specificity. Applying this model, we predict the performance of an additional ∼2,600 antisense-regulator pairs, forecast the possibility of large families of orthogonal mutants, and forward engineer and experimentally validate two RNA pairs orthogonal to an existing group of five from the training data set. We discuss the potential use of these regulators in next-generation synthetic biology applications.

Original language	English (US)
Pages (from-to)	447-454
Number of pages	8
Journal	Nature Chemical Biology
Volume	8
Issue number	5
DOIs	https://doi.org/10.1038/nchembio.919
State	Published - May 2012

Funding

The authors would like to thank G. Cambray, E. Gogol, C. Liu and J. Skerker for comments on the manuscript. V.K.M. was supported by British Petroleum under contract number LB08004883 at the Joint BioEnergy Institute. A.P.A. and L.Q. acknowledge support from the Synthetic Biology Engineering Research Center under US National Science Foundation grant number 04570/0540879. J.C.G. acknowledges financial support by the Portuguese Fundação para a Ciência e a Tecnologia (SFRH/BD/47819/2008). J.B.L. acknowledges the financial support of the Miller Institute for Basic Scientific Research. This work conducted by the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the US Department of Energy under Contract No. DEAC0205CH11231.

ASJC Scopus subject areas

Molecular Biology
Cell Biology

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10.1038/nchembio.919

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title = "Rationally designed families of orthogonal RNA regulators of translation",

abstract = "Our ability to routinely engineer genetic networks for applications is limited by the scarcity of highly specific and non-cross-reacting (orthogonal) gene regulators with predictable behavior. Though antisense RNAs are attractive contenders for this purpose, quantitative understanding of their specificity and sequence-function relationship sufficient for their design has been limited. Here, we use rationally designed variants of the RNA-IN-RNA-OUT antisense RNA-mediated translation system from the insertion sequence IS10 to quantify >500 RNA-RNA interactions in Escherichia coli and integrate the data set with sequence-activity modeling to identify the thermodynamic stability of the duplex and the seed region as the key determinants of specificity. Applying this model, we predict the performance of an additional ∼2,600 antisense-regulator pairs, forecast the possibility of large families of orthogonal mutants, and forward engineer and experimentally validate two RNA pairs orthogonal to an existing group of five from the training data set. We discuss the potential use of these regulators in next-generation synthetic biology applications.",

author = "Mutalik, {Vivek K.} and Lei Qi and Guimaraes, {Joao C.} and Lucks, {Julius B.} and Arkin, {Adam P.}",

note = "Funding Information: The authors would like to thank G. Cambray, E. Gogol, C. Liu and J. Skerker for comments on the manuscript. V.K.M. was supported by British Petroleum under contract number LB08004883 at the Joint BioEnergy Institute. A.P.A. and L.Q. acknowledge support from the Synthetic Biology Engineering Research Center under US National Science Foundation grant number 04570/0540879. J.C.G. acknowledges financial support by the Portuguese Funda{\c c}{\~a}o para a Ci{\^e}ncia e a Tecnologia (SFRH/BD/47819/2008). J.B.L. acknowledges the financial support of the Miller Institute for Basic Scientific Research. This work conducted by the Joint BioEnergy Institute was supported by the Office of Science, Office of Biological and Environmental Research, of the US Department of Energy under Contract No. DEAC0205CH11231.",

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Rationally designed families of orthogonal RNA regulators of translation

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