Engineering naturally occurring trans-acting non-coding RNAs to sense molecular signals

Abstract

Non-coding RNAs (ncRNAs) are versatile regulators in cellular networks. While most trans-acting ncRNAs possess well-defined mechanisms that can regulate transcription or translation, they generally lack the ability to directly sense cellular signals. In this work, we describe a set of design principles for fusing ncRNAs to RNA aptamers to engineer allosteric RNA fusion molecules that modulate the activity of ncRNAs in a ligand-inducible way in Escherichia coli. We apply these principles to ncRNA regulators that can regulate translation (IS10 ncRNA) and transcription (pT181 ncRNA), and demonstrate that our design strategy exhibits high modularity between the aptamer ligand-sensing motif and the ncRNA target-recognition motif, which allows us to reconfigure these two motifs to engineer orthogonally acting fusion molecules that respond to different ligands and regulate different targets in the same cell. Finally, we show that the same ncRNA fused with different sensing domains results in a sensory-level NOR gate that integrates multiple input signals to perform genetic logic. These ligand-sensing ncRNA regulators provide useful tools to modulate the activity of structurally related families of ncRNAs, and building upon the growing body of RNA synthetic biology, our ability to design aptamer-ncRNA fusion molecules offers new ways to engineer ligand-sensing regulatory circuits.

Figure 1.

The proposed riboswitch-like design for the aptamer–ncRNA fusion. (A) A riboswitch usually consists of an aptamer (black color) coupled to a structured expression platform (red color). The structural interaction between the aptamer and the expression platform could inactivate (left) or activate (right) the expression platform depending on the presence of ligand. (B) We propose to fuse the aptamer and the ncRNA (blue color) in a similar architecture. The designed structural interaction between the aptamer and the ncRNA mutation regions (black color) would inactivate ncRNA without the ligand (left). Ligand binding could eliminate such structural interactions and activate the ncRNA (right).

Figure 2.

Designed theophylline aptamer–IS10 ncRNA fusions. (A) The theophylline aptamer sequences (blue) were fused to the 5′ end of IS10 ncRNA (purple), and screening of seven designed pseudoknot mutants (Supplementary Figure S2) resulted in one functional fusion, theo–P–IS10 (P for pseudoknot). The proposed mechanism for allosteric switching of the fusion molecule is shown in the box. (B) Fluorescence assay of theo–P–IS10 using flow cytometry. The induction curves were plotted from the average values of three biological replicates at each theophylline concentration. The inset shows the cytometry histograms of three ligand concentrations (red—0.01 µM, green—100 µM, blue—2 mM), with the two black vertical lines showing the mean values of the positive and negative controls. The repression percentage between 2 mM and 0.01 µM theophylline is 83.0%, compared to 91.0% between the positive and negative controls. (C) SHAPE data of theo–P–IS10. The difference in nucleotide reactivity with and without the ligand is overlaid on a hypothesized secondary structure model of theo–P–IS10 based on Refs (21) and (24). Colors represent the changes of SHAPE reactivity upon addition of the ligand, with red colors showing positive changes (more flexible) and blues colors showing negative changes (more stable). The blue box is the known ligand-binding pocket. The red boxes are designed pseudoknot interactions. Original SHAPE data is available in Supplementary Figure S3A.

Figure 3.

Alternative designs of theophylline-sensing pT181 ncRNA fusions. (A) Screening of eight designed pseudoknot mutants (Supplementary Figure S5) results in theo–P–pT181, which displays strong theophylline-inducible repressive effects on its target. (B) An alternative design of theophylline-sensing pT181 ncRNA fusions based on the strand-exchange strategy by mutating the lower bottom stem region of the ncRNAs. The sequences of the best mutant screened from 15 mutants (Supplementary Figure S6) is shown as theo–SE–pT181 (SE for strand exchange). In vivo fluorescence data from flow cytometry is shown on the bottom. The repression percentage between 3 mM and 0.01 µM theophylline is 83.4%, compared to 86.6% between positive and negative controls. The insets show the cytometry histograms corresponding to three ligand concentrations in both (A) and (B).

Figure 4.

Orthogonal theophylline-sensing pT181 fusions based on the strand-exchange design. (A) The region that determines the target-specificity of pT181 ncRNA is in the cyan box (4), and allosteric region is in the black box. The sequences of theophylline-sensing WT pT181 and MT pT181 fusions are shown. (B) Experimental setup to test theophylline-induced target specificity of theo–SE–pT181WT and theo–SE–pT181MT. All four possible combinations between aptamer–ncRNA fusions and reporters were assayed with and without theophylline. (C) Fluorescence assay data with red bars showing (−) theophylline and blue bars showing (+) 2.5 mM theophylline.

Figure 5.

Designed MS2 coat protein-sensing pT181 ncRNA fusions. (A) Sequences of the MS2–SE–pT181 aptamer–ncRNA fusion screened from five designed mutants (Supplementary Figure S10). (B) Fluorescence assay of MS2–SE–pT181 ncRNA fusions with intracellular MS2 coat protein induced by IPTG. The repression percentage between 500 µM and 1 nM IPTG is 87.5% compared to 89.1% between positive and negative controls. The inset shows the cytometry histogram of three IPTG concentrations. (C) A NOR logic engineered from theo–SE–pT181WT and MS2–SE–pT181WT aptamer–ncRNA fusions. The two aptamer–ncRNA fusions control the same target and integrate ligand signals in a way that presence of any ligand represses the target gene expression. A theoretical NOR truth table is shown in the plot.

Figure 6.

Modularity of aptamer–ncRNAs at the molecular and network levels. (A) Combining orthogonal ncRNAs with different aptamers could provide a toolbox of orthogonally acting aptamer–ncRNA fusions that sense multiple ligands and control their cognate targets with high specificity. (B) The scalable global regulation allows regulation of multiple gene targets in the same cell by responding to a global ligand modulator. (C) The expandable signal integration allows regulation of a single gene target by integrating multiple signal inputs in a logic way. (D) The post-transcriptional disruption of ncRNA networks allows us to debug and fine-tune the performance of individual regulators in complicated networks such as transcriptional cascades.