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Design of a Toolbox of RNA Thermometers

Sen, Shaunak and Apurva, Divyansh and Satija, Rohit and Siegal, Dan and Murray, Richard M. (2017) Design of a Toolbox of RNA Thermometers. ACS Synthetic Biology, 6 (8). pp. 1461-1470. ISSN 2161-5063.

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[img] PDF ((A) RNA thermometer sequences, (B) NUPACK analysis of +C sequence, (C) activity of RNA thermometer library in cell spots, (D) correlation of sensitivities, (E) detailed analysis of a few individual thermometers, (F) quantitation of activity in cell...) - Supplemental Material
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Biomolecular temperature sensors can be used for efficient control of large-volume bioreactors, for spatiotemporal imaging and control of gene expression, and to engineer robustness to temperature in biomolecular circuit design. While RNA-based sensors, called ‘thermometers’, have been investigated in both natural and synthetic contexts, an important challenge is to design diverse responses to temperature, differing in sensitivities and thresholds. We address this issue by constructing a library of RNA thermometers, based on thermodynamic computations, and experimentally measuring their activities in cell-free biomolecular ‘breadboards’. Using free energies of the minimum free energy structures as well as melt profile computations, we estimated thata diverse set of temperature responses were possible. We experimentally found a wide range of responses to temperature in the temperature range 29 ◦C–37 ◦C, with fold-changes varying over 3-fold around the starting thermometer. The sensitivities of these responses ranged over 10-fold around the starting thermometer. We correlated these measurements with computational expectations, finding that while there was no strong correlation for the individual thermometers, overall trends of diversity, fold-changes, and sensitivities were similar. These results present a toolbox of RNA-based circuit elements with diverse temperature responses.

Item Type:Article
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URLURL TypeDescription Information Paper
Sen, Shaunak0000-0002-1412-8633
Siegal, Dan0000-0002-6821-2769
Murray, Richard M.0000-0002-5785-7481
Additional Information:© 2017 American Chemical Society. Received: October 17, 2016; Published: April 24, 2017. We thank Prof. Dr. Ralph Bock and Dr. Juliane Neupert from Max-Planck-Institut für Molekulare Pflanzenphysiologie, Potsdam, Germany, for their kind gift of plasmids used in this study (pBSU2 and pBSU9). We thank Harry Choi, Clare Hayes, Anu Thubagere, and Jongmin Kim for being gracious with their time and guidance. We are grateful to the referees for their valuable comments. This work was supported in part by the Defense Advanced Research Projects Agency (DARPA/MTO) Living Foundries program, contract number HR0011-12-C-0065 and the National Science Foundation award number 1317694. Additionally, S. S. acknowledges support through FRTA, IIT Delhi and IRD, IIT Delhi. The authors declare no competing financial interest.
Funding AgencyGrant Number
Defense Advanced Research Projects Agency (DARPA)HR0011-12-C-0065
Indian Institute of Technology DelhiUNSPECIFIED
Subject Keywords:RNA thermometers, synthetic biology, cell-free breadboard, computational design, free energies, melt profiles, temperature sensor
Issue or Number:8
Record Number:CaltechAUTHORS:20160516-100511848
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Official Citation:Design of a Toolbox of RNA Thermometers Shaunak Sen, Divyansh Apurva, Rohit Satija, Dan Siegal, and Richard M. Murray ACS Synthetic Biology 2017 6 (8), 1461-1470 DOI: 10.1021/acssynbio.6b00301
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:67121
Deposited By: Ruth Sustaita
Deposited On:16 May 2016 18:45
Last Modified:03 Oct 2019 10:03

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