Evaluating DFHBI-responsive RNA light-up aptamers as fluorescent reporters for gene expression
Author(s)
Climent-Catala, Alicia
Casas-Rodrigo, Ivan
Iyer, Suhasini
Ledesma-Amaro, Rodrigo
Ouldridge, Thomas E
Type
Journal Article
Abstract
Protein-based fluorescent reporters have been widely used to characterize and localize biological processes in living cells. However, these reporters may have certain drawbacks for some applications, such as transcription-based studies or biological interactions with fast dynamics. In this context, RNA nanotechnology has emerged as a promising alternative, suggesting the use of functional RNA molecules as transcriptional fluorescent reporters. RNA-based aptamers can bind to nonfluorescent small molecules to activate their fluorescence. However, their performance as reporters of gene expression in living cells has not been fully characterized, unlike protein-based reporters. Here, we investigate the performance of three RNA light-up aptamers─F30-2xdBroccoli, tRNA-Spinach, and Tornado Broccoli─as fluorescent reporters for gene expression in Escherichia coli and compare them to a protein reporter. We examine the activation range and effect on the cell growth of RNA light-up aptamers in time-course experiments and demonstrate that these aptamers are suitable transcriptional reporters over time. Using flow cytometry, we compare the variability at the single-cell level caused by the RNA fluorescent reporters and protein-based reporters. We found that the expression of RNA light-up aptamers produced higher variability in a population than that of their protein counterpart. Finally, we compare the dynamical behavior of these RNA light-up aptamers and protein-based reporters. We observed that RNA light-up aptamers might offer faster dynamics compared to a fluorescent protein in E. coli. The implementation of these transcriptional reporters may facilitate transcription-based studies, gain further insights into transcriptional processes, and expand the implementation of RNA-based circuits in bacterial cells.
Date Issued
2023-12-15
Date Acceptance
2023-11-06
Citation
ACS Synthetic Biology, 2023, 12 (12), pp.3754-3765
ISSN
2161-5063
Publisher
American Chemical Society
Start Page
3754
End Page
3765
Journal / Book Title
ACS Synthetic Biology
Volume
12
Issue
12
Copyright Statement
© 2023 The Authors. Published by American Chemical Society. This publication is licensed under
CC-BY 4.0.
CC-BY 4.0.
License URL
Identifier
https://www.ncbi.nlm.nih.gov/pubmed/37991880
Subjects
dynamics
E. coli
gene expression
reporters
RNA light-up aptamers
synthetic biology
Publication Status
Published
Coverage Spatial
United States
Date Publish Online
2023-11-22