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 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.

Synthetic biology and bioelectrochemical tools for electrogenetic system engineering.

Synthetic biology research and its industrial applications rely on deterministic spatiotemporal control of gene expression. Recently, electrochemical control of gene expression has been demonstrated in electrogenetic systems (redox-responsive promoters used alongside redox inducers and electrodes), allowing for the direct integration of electronics with biological processes. However, the use of electrogenetic systems is limited by poor activity, tunability, and standardization. In this work, we developed a strong, unidirectional, redox-responsive promoter before deriving a mutant promoter library with a spectrum of strengths. We constructed genetic circuits with these parts and demonstrated their activation by multiple classes of redox molecules. Last, we demonstrated electrochemical activation of gene expression under aerobic conditions using a novel, modular bioelectrochemical device. These genetic and electrochemical tools facilitate the design and improve the performance of electrogenetic systems. Furthermore, the genetic design strategies used can be applied to other redox-responsive promoters to further expand the available tools for electrogenetics.

Building an RNA-Based Toggle Switch Using Inhibitory RNA Aptamers.

Synthetic RNA systems offer unique advantages such as faster response, increased specificity, and programmability compared to conventional protein-based networks. Here, we demonstrate an in vitro RNA-based toggle switch using RNA aptamers capable of inhibiting the transcriptional activity of T7 or SP6 RNA polymerases. The activities of both polymerases are monitored simultaneously by using Broccoli and malachite green light-up aptamer systems. In our toggle switch, a T7 promoter drives the expression of SP6 inhibitory aptamers, and an SP6 promoter expresses T7 inhibitory aptamers. We show that the two distinct states originating from the mutual inhibition of aptamers can be toggled by adding DNA sequences to sequester the RNA inhibitory aptamers. Finally, we assessed our RNA-based toggle switch in degrading conditions by introducing controlled degradation of RNAs using a mix of RNases. Our results demonstrate that the RNA-based toggle switch could be used as a control element for nucleic acid networks in synthetic biology applications.