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Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits

RNA-based regulators are promising tools for building synthetic biological systems that provide a powerful platform for achieving a complex regulation of transcription and translation. Recently, de novo-designed synthetic RNA regulators, such as the small transcriptional activating RNA (STAR), toeho...

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Autores principales: Hong, Seongho, Jeong, Dohyun, Ryan, Jordan, Foo, Mathias, Tang, Xun, Kim, Jongmin
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8391864/
https://www.ncbi.nlm.nih.gov/pubmed/34439849
http://dx.doi.org/10.3390/biom11081182
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author Hong, Seongho
Jeong, Dohyun
Ryan, Jordan
Foo, Mathias
Tang, Xun
Kim, Jongmin
author_facet Hong, Seongho
Jeong, Dohyun
Ryan, Jordan
Foo, Mathias
Tang, Xun
Kim, Jongmin
author_sort Hong, Seongho
collection PubMed
description RNA-based regulators are promising tools for building synthetic biological systems that provide a powerful platform for achieving a complex regulation of transcription and translation. Recently, de novo-designed synthetic RNA regulators, such as the small transcriptional activating RNA (STAR), toehold switch (THS), and three-way junction (3WJ) repressor, have been utilized to construct RNA-based synthetic gene circuits in living cells. In this work, we utilized these regulators to construct type 1 incoherent feed-forward loop (IFFL) circuits in vivo and explored their dynamic behaviors. A combination of a STAR and 3WJ repressor was used to construct an RNA-only IFFL circuit. However, due to the fast kinetics of RNA–RNA interactions, there was no significant timescale difference between the direct activation and the indirect inhibition, that no pulse was observed in the experiments. These findings were confirmed with mechanistic modeling and simulation results for a wider range of conditions. To increase delay in the inhibition pathway, we introduced a protein synthesis process to the circuit and designed an RNA–protein hybrid IFFL circuit using THS and TetR protein. Simulation results indicated that pulse generation could be achieved with this RNA–protein hybrid model, and this was further verified with experimental realization in E. coli. Our findings demonstrate that while RNA-based regulators excel in speed as compared to protein-based regulators, the fast reaction kinetics of RNA-based regulators could also undermine the functionality of a circuit (e.g., lack of significant timescale difference). The agreement between experiments and simulations suggests that the mechanistic modeling can help debug issues and validate the hypothesis in designing a new circuit. Moreover, the applicability of the kinetic parameters extracted from the RNA-only circuit to the RNA–protein hybrid circuit also indicates the modularity of RNA-based regulators when used in a different context. We anticipate the findings of this work to guide the future design of gene circuits that rely heavily on the dynamics of RNA-based regulators, in terms of both modeling and experimental realization.
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spelling pubmed-83918642021-08-28 Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits Hong, Seongho Jeong, Dohyun Ryan, Jordan Foo, Mathias Tang, Xun Kim, Jongmin Biomolecules Article RNA-based regulators are promising tools for building synthetic biological systems that provide a powerful platform for achieving a complex regulation of transcription and translation. Recently, de novo-designed synthetic RNA regulators, such as the small transcriptional activating RNA (STAR), toehold switch (THS), and three-way junction (3WJ) repressor, have been utilized to construct RNA-based synthetic gene circuits in living cells. In this work, we utilized these regulators to construct type 1 incoherent feed-forward loop (IFFL) circuits in vivo and explored their dynamic behaviors. A combination of a STAR and 3WJ repressor was used to construct an RNA-only IFFL circuit. However, due to the fast kinetics of RNA–RNA interactions, there was no significant timescale difference between the direct activation and the indirect inhibition, that no pulse was observed in the experiments. These findings were confirmed with mechanistic modeling and simulation results for a wider range of conditions. To increase delay in the inhibition pathway, we introduced a protein synthesis process to the circuit and designed an RNA–protein hybrid IFFL circuit using THS and TetR protein. Simulation results indicated that pulse generation could be achieved with this RNA–protein hybrid model, and this was further verified with experimental realization in E. coli. Our findings demonstrate that while RNA-based regulators excel in speed as compared to protein-based regulators, the fast reaction kinetics of RNA-based regulators could also undermine the functionality of a circuit (e.g., lack of significant timescale difference). The agreement between experiments and simulations suggests that the mechanistic modeling can help debug issues and validate the hypothesis in designing a new circuit. Moreover, the applicability of the kinetic parameters extracted from the RNA-only circuit to the RNA–protein hybrid circuit also indicates the modularity of RNA-based regulators when used in a different context. We anticipate the findings of this work to guide the future design of gene circuits that rely heavily on the dynamics of RNA-based regulators, in terms of both modeling and experimental realization. MDPI 2021-08-10 /pmc/articles/PMC8391864/ /pubmed/34439849 http://dx.doi.org/10.3390/biom11081182 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Hong, Seongho
Jeong, Dohyun
Ryan, Jordan
Foo, Mathias
Tang, Xun
Kim, Jongmin
Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits
title Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits
title_full Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits
title_fullStr Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits
title_full_unstemmed Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits
title_short Design and Evaluation of Synthetic RNA-Based Incoherent Feed-Forward Loop Circuits
title_sort design and evaluation of synthetic rna-based incoherent feed-forward loop circuits
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8391864/
https://www.ncbi.nlm.nih.gov/pubmed/34439849
http://dx.doi.org/10.3390/biom11081182
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