Hong, Seongho, Jeong, Dohyun, Ryan, Jordan, Foo, Mathias, Tang, Xun and Kim, Jongmin (2021) Design and evaluation of synthetic RNA-based incoherent feed-forward loop circuits. Biomolecules, 11 (8). 1182. doi:10.3390/biom11081182

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Abstract

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.

Item Type:	Journal Article
Subjects:	Q Science > QH Natural history Q Science > QP Physiology T Technology > TK Electrical engineering. Electronics Nuclear engineering
Divisions:	Faculty of Science > Engineering
Library of Congress Subject Headings (LCSH):	RNA , Messenger RNA , Genetic regulation , Gene expression, Synthetic biology, Pulse generators , Gene regulatory networks
Journal or Publication Title:	Biomolecules
Publisher:	MDPI
ISSN:	2218-273X
Official Date:	10 August 2021
Dates:	Date Event 10 August 2021 Published 6 August 2021 Accepted
Volume:	11
Number:	8
Article Number:	1182
DOI:	10.3390/biom11081182
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access
RIOXX Funder/Project Grant:	Project/Grant ID RIOXX Funder Name Funder ID NRF-2019R1A2C1086830 National Research Foundation of Korea http://dx.doi.org/10.13039/501100003725 HI19C0634 Korea Health Industry Development Institute http://dx.doi.org/10.13039/501100003710 MC/PC/18073 Medical Research Council http://dx.doi.org/10.13039/501100000265 UNSPECIFIED Louisiana State University http://dx.doi.org/10.13039/100008294

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