Evaluating DFHBI-Responsive RNA Light-Up Aptamers as Fluorescent Reporters for Gene Expression.

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 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 . 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.