article · 04/04/2018
Quantitative Characterization of Translational Riboregulators Using an in Vitro Transcription-Translation System
Résumé
Riboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo. However, riboregulators are generally designed in silico and tested in vivo, which provides little quantitative information about their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX-TL) system provides valuable information about the performances of in silico designed riboregulators. We first propose a simple model that provides a quantitative definition of the dynamic range of a riboregulator. We further characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off, in agreement with our model, and that relative dynamic ranges of riboregulators determined in vitro are in agreement with published in vivo data. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX-TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico.
Citer cet article
Senoussi, A., Lee Tin Wah, J., Shimizu, Y., Robert, J., Jaramillo, A., Findeiss, S., M. Axmann, I., & Estevez-Torres, A. (2018). Quantitative Characterization of Translational Riboregulators Using an in Vitro Transcription-Translation System. ACS Synth. Biol.. https://doi.org/10.1021/acssynbio.7b00387@article{Senoussi2018_270,
author = {Senoussi, Anis and Lee Tin Wah, Jonathan and Shimizu, Yoshihiro and Robert, Jérôme and Jaramillo, Alfonso and Findeiss, Sven and M. Axmann, Ilka and Estevez-Torres, André},
year = {2018},
month = {4},
title = {Quantitative Characterization of Translational Riboregulators Using an in Vitro Transcription-Translation System},
journal = {ACS Synth. Biol.},
abstract = {Riboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo. However, riboregulators are generally designed in silico and tested in vivo, which provides little quantitative information about their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX-TL) system provides valuable information about the performances of in silico designed riboregulators. We first propose a simple model that provides a quantitative definition of the dynamic range of a riboregulator. We further characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off, in agreement with our model, and that relative dynamic ranges of riboregulators determined in vitro are in agreement with published in vivo data. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX-TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico.},
doi = {10.1021/acssynbio.7b00387},
}TY - JOUR
AU - Senoussi, Anis
AU - Lee Tin Wah, Jonathan
AU - Shimizu, Yoshihiro
AU - Robert, Jérôme
AU - Jaramillo, Alfonso
AU - Findeiss, Sven
AU - M. Axmann, Ilka
AU - Estevez-Torres, André
PY - 2018
DA - 2018/04/04
TI - Quantitative Characterization of Translational Riboregulators Using an in Vitro Transcription-Translation System
JO - ACS Synth. Biol.
AB - Riboregulators are short RNA sequences that, upon binding to a ligand, change their secondary structure and influence the expression rate of a downstream gene. They constitute an attractive alternative to transcription factors for building synthetic gene regulatory networks because they can be engineered de novo. However, riboregulators are generally designed in silico and tested in vivo, which provides little quantitative information about their performances, thus hindering the improvement of design algorithms. Here we show that a cell-free transcription-translation (TX-TL) system provides valuable information about the performances of in silico designed riboregulators. We first propose a simple model that provides a quantitative definition of the dynamic range of a riboregulator. We further characterize two types of translational riboregulators composed of a cis-repressed (cr) and a trans-activating (ta) strand. At the DNA level we demonstrate that high concentrations of taDNA poisoned the activator until total shut off, in agreement with our model, and that relative dynamic ranges of riboregulators determined in vitro are in agreement with published in vivo data. At the RNA level, we show that this approach provides a fast and simple way to measure dissociation constants of functional riboregulators, in contrast to standard mobility-shift assays. Our method opens the route for using cell-free TX-TL systems for the quantitative characterization of functional riboregulators in order to improve their design in silico.
DO - 10.1021/acssynbio.7b00387
ER -