Riboswitch-inspired toehold riboregulators for gene regulation in Escherichia coli

Abstract

Regulatory RNA molecules have been widely investigated as components for synthetic gene circuits, complementing the use of protein-based transcription factors. Among the potential advantages of RNA-based gene regulators are their comparatively simple design, sequence-programmability, orthogonality, and their relatively low metabolic burden. In this work, we developed a set of riboswitch-inspired riboregulators in Escherichia coli that combine the concept of toehold-mediated strand displacement (TMSD) with the switching principles of naturally occurring transcriptional and translational riboswitches. Specifically, for translational activation and repression, we sequestered anti-anti-RBS or anti-RBS sequences, respectively, inside the loop of a stable hairpin domain, which is equipped with a single-stranded toehold region at its 5' end and is followed by regulated sequences on its 3' side. A trigger RNA binding to the toehold region can invade the hairpin, inducing a structural rearrangement that results in translational activation or deactivation. We also demonstrate that TMSD can be applied in the context of transcriptional regulation by switching RNA secondary structure involved in Rho-dependent termination. Our designs expand the repertoire of available synthetic riboregulators by a set of RNA switches with no sequence limitation, which should prove useful for the development of robust genetic sensors and circuits.

Figure 1.

Design and characterization of riboswitch-inspired toehold-riboregulator controlling translational activation. (A) Scheme of a toehold riboregulator that activates translation initiation in response to trigger RNA input. In the absence of a trigger RNA (grey), the toehold hairpin (TH) confines an anti-anti-RBS sequence (purple) within its loop region. The RBS (blue) is sequestered within an anti-RBS hairpin (ARH) by an anti-RBS sequence (yellow), which prevents binding of the ribosome. Trigger RNA can initiate a TMSD process at the toehold (light blue), which releases the anti-anti-RBS sequence. The RBS sequestration hairpin is unfolded by the released anti-anti-RBS sequence and forms an anti-anti-RBS hairpin (AARH), which in turn exposes the RBS to the ribosome and allows translation of the mCherry readout (red) to proceed. (B) Predicted secondary structure and total free energy of each anti-RBS hairpin - the RBS sequence is highlighted in green. (C) Relative fluorescence intensities from in vivo measurements in the ON and OFF state for each anti-RBS hairpin, respectively. (D) Relative fluorescence intensities obtained in cell-free experiments with riboregulators in the ON and OFF state, respectively. For both the relative fluorescence/OD and fluorescence intensity data, Welch's t-tests were performed for each construct; *P < 0.05, indicating conditions where the fluorescence/OD and fluorescence intensity for the trigger RNA + condition is statistically significantly different from that of the trigger RNA-condition. Error bars represent the standard deviation (s.d.) for biologically independent samples.

Figure 2.

Design and characterization of a riboswitch-inspired toehold-riboregulator controlling translational repression. (A) In the absence of a trigger RNA (grey), the toehold hairpin (TH) constrains an anti-RBS sequence (yellow) within its loop region, the RBS is freely accessible and translational initiation is enabled. In the presence of trigger, toehold (light blue)-mediated invasion of the hairpin stem releases the anti-RBS, which leads to formation of anti-RBS hairpin (ARH) and sequestration of the RBS and thus translational repression. (B) Predicted secondary structure and total free energy of the anti-RBS hairpin. The RBS sequence is highlighted in green. (C) Relative fluorescence intensities in the ON and OFF state of the translational toehold repressor measured in vivo. (D) in vitro relative fluorescence intensities of the translational toehold repressor in its two states measured in a cell-free expression system. For both relative fluorescence/OD and fluorescence intensity data, Welch's t-tests were performed on each construct; *P < 0.05 and **P < 0.01 indicate conditions where the fluorescence/OD and fluorescence intensity for the Trigger RNA + condition is statistically significantly different from that of the trigger RNA- condition. Error bars in c, d represent the s.d. from at least three biologically independent samples.

Figure 3.

Design and characterization of riboswitch-inspired toehold riboregulator mechanisms controlling transcriptional activation via an intrinsic terminator. (A) Anti-t22 stem 1 design: In the absence of a trigger RNA (grey), a transcriptional anti-terminator (yellow) is constrained within the loop of the toehold hairpin (TH), and thus transcription is terminated by the following intrinsic t22 terminator (orange). In the presence of trigger RNA, trigger binding and TMSD opens the TH, and the released anti-terminator sequence sequesters the terminator in an alternative structure (anti-terminator hairpin, ATH), which allows downstream gene transcription to proceed. (B) Predicted secondary structure and total free energy of the anti-t22 stem, a subsequence of the t22 terminator is highlighted in orange. (C) Relative mCherry fluorescence intensities measured with the transcriptional activator in the OFF and ON state, respectively. (D) Normalized abundance of mRNA transcripts characterized by qPCR in the transcriptional OFF and ON state of the anti-t22 stem_1 activator. (E) Anti-t22 stem 2 design: In this design TMSD induced refolding of the RNA structure leads to a sequestration of the critical guanine nucleotide (indicated in blue) within the anti-anti-t22 terminator hairpin (ATH) stem, which allows downstream gene transcription to proceed. (F) Predicted secondary structure and total free energy of anti-t22 stem_2, with the sequestered t22 terminator subsequence highlighted in orange. (G) Relative mCherry fluorescence intensities and (H) abundance of mRNA transcripts measured by qPCR for the two states of the transcriptional activator. For both fluorescence/OD values and qPCR quantification, Welch's t-tests were performed on each construct; *P < 0.05 and **P < 0.01, indicate that the trigger RNA + condition is statistically significantly different from that of the trigger RNA- case.

Figure 4.

Design and characterization of transcriptional toehold riboregulators based on Rho-dependent termination. (A) Principle of a transcriptional activator: In the absence of trigger RNA (grey), the toehold hairpin (TH) confines anti-rut sequence (olive) within its loop, while the rut site (brown) is exposed to Rho factor, which terminates transcription. Upon invasion of the toehold hairpin (TH) by trigger RNA, the anti-rut sequence is released and anti- transcriptional elongation is switched ON. The design of the corresponding plasmids and gene circuits are also shown. (B) Predicted secondary structure and free energies of several variants of the anti-rut stem, where the rut sequence is highlighted in brown. (C) Relative fluorescence intensities of the transcriptional activators measured in vivo in the OFF and ON state. (D) Corresponding normalized abundance of mRNA transcripts measured by qPCR. (E) Scheme of transcriptional repression by a toehold riboregulator, which is based on the tna operon. In the absence of trigger RNA (grey), ribosomes can bind to translate the tnaC peptide, followed by stalling at the rut site (brown). This prevents Rho from binding and thus allows transcription to proceed. In the presence of trigger, translation of tnaC is disabled and Rho factor can bind to the exposed rut site and thus terminate transcription. (F) Predicted secondary structure and free energy of the anti-RBS hairpin. The RBS sequence is highlighted in green. (G) Relative fluorescence intensities and (H) mRNA abundance in the ON and OFF state of the transcriptional repressor and in the presence of 5mM tryptophan. Based on Welch's t-tests, *P < 0.05 and **P < 0.01, indicate conditions where the fluorescence/OD and qPCR quantification for the trigger RNA + condition is statistically significantly different from that of the trigger RNA- condition.

Figure 5.

A NOR logic gate based on riboswitch-inspired transcriptional and translational toehold switches. (A) The switch is composed of two modules which each control translation from an RBS. The function of the first module is to switch translation of the tnaC peptide, which results in blockage of the rut site by the ribosome (cf. Figure 4). This in turn prevents termination by Rho factor and thus allows transcription to proceed. The second module is a translational toehold repressor for the GFP reporter gene (cf. Figure 2). Binding of the two trigger RNAs lead to transcriptional termination and translational repression, respectively. (B) GFP fluorescence output of the NOR gate for different input combinations, measured by flow cytometry in the presence of 5 mM tryptophan (required for the tna operon). Given are the mean values from three biologically independent samples, error bars represent their standard deviation. (C) GFP fluorescence histograms for the NOR gate toehold riboregulator in the absence and presence of inputs obtained from a single flow cytometry run.