Toehold switches: de-novo-designed regulators of gene expression

Abstract

Efforts to construct synthetic networks in living cells have been hindered by the limited number of regulatory components that provide wide dynamic range and low crosstalk. Here, we report a class of de-novo-designed prokaryotic riboregulators called toehold switches that activate gene expression in response to cognate RNAs with arbitrary sequences. Toehold switches provide a high level of orthogonality and can be forward engineered to provide average dynamic range above 400. We show that switches can be integrated into the genome to regulate endogenous genes and use them as sensors that respond to endogenous RNAs. We exploit the orthogonality of toehold switches to regulate 12 genes independently and to construct a genetic circuit that evaluates 4-input AND logic. Toehold switches, with their wide dynamic range, orthogonality, and programmability, represent a versatile and powerful platform for regulation of translation, offering diverse applications in molecular biology, synthetic biology, and biotechnology.

Figure 1. Toehold switch design and in vivo characterization

(A,B) Design schematics of conventional riboregulators (A) and toehold switches (B). Variable sequences are shown in gray, while conserved or constrained sequences are represented by different colors. (A) Conventional riboregulator systems repress translation by base pairing directly to the RBS region. RNA-RNA interactions are initiated via a loop-linear or loop-loop interaction at the YUNR-loop in an RNA hairpin. (B) Toehold switches repress translation through base pairs programmed before and after the start codon (AUG), leaving the RBS and start codon regions completely unpaired. RNA-RNA interactions are initiated via linear-linear interaction domains called toeholds. The toehold domain a binds to a complementary a* domain on the trigger RNA. Domains a and b are 12- and 18-nts, respectively. (C) Flow cytometry GFP fluorescence histograms for toehold switch number 2 compared to E. coli autofluorescence and a positive control. Autofluorescence level measured from induced cells not bearing a GFP-expressing plasmid. (D) GFP mode fluorescence levels measured for switches in their ON and OFF states in comparison to positive control constructs and autofluorescence. (E) ON/OFF GFP fluorescence levels obtained three hours after induction for 168 first-generation toehold switches. Inset: ON/OFF GFP fluorescence measured for toehold switches of varying performance levels at different time points following induction. See also Figure S1, Table S1.

Figure 2. Assessment of toehold switch orthogonality

(A) GFP fluorescence from colonies of E. coli expressing 676 pairwise combinations of switch and trigger RNAs. GFP-expressing colonies are visible as green points along the diagonal in cells containing cognate switch and trigger strands. Off-diagonal, non-cognate components have low fluorescence. (B) Crosstalk measured by flow cytometry for all trigger-switch combinations. Crosstalk was determined by taking GFP output measured for a given trigger-switch combination and dividing it by the GFP output measured for the switch with its cognate trigger. (C) Comparison of overall library dynamic range and orthogonal library size for the toehold switches and previous riboegulators. The overall library dynamic range corresponds to the minimum ON/OFF ratio to expect in a network employing this library of switches. See also Table S2.

Figure 3. Forward engineering and thermodynamic analysis of toehold switches

(A) Schematic of the design modifications made for the forward-engineered switches compared to the firstgeneration switches. (B) ON/OFF GFP fluorescence ratios obtained for the set of 13 forwardengineered toehold switches after three hours of induction. Dashed black line marks the mean ON/OFF fluorescence measured for the 168 first-generation toehold switches. Inset: Timecourse measurements for forward-engineered switches number 6 and number 9. (C) Percentage of first-generation and forward-engineered library components that had ON/OFF ratios that exceeded the value defined on the x-axis. (D) Correlation between ;G_RBS-linker and ON/OFF ratio measured for the 68 first-generation toehold switches that had an A-U base pair at the top of the hairpin stem. Inset: Schematic showing the RNA sequence range in the trigger-switch complex used to define ;G_RBS-linker. (E) Strong correlation (R² = 0.79) between ;G_RBS-linker and ON/OFF ratio measured for the complete set of forward-engineered switches. See also Figure S2, Table S3.

Figure 4. Toehold switch activated by mRNA and endogenous small RNA triggers

(A) Design schematic and putative activation pathway of the toehold switch mRNA sensors. (B) Mode GFP and mCherry fluorescence obtained from flow cytometry of three mCherry sensors in their repressed and activated states, as well as positive and negative controls. (C) ON/OFF GFP fluorescence ratios for a series of toehold switches activated by the mCherry mRNA, and cat and aadA mRNAs, which confer chloramphenicol and spectinomycin resistance, respectively. (D) Endogenous and synthetic gene networks used for sensing the ryhB sRNA. (E) Transfer function for the ryhB sensor (purple curve) as a function of ryhB inducer concentration. Output of a constitutive GFP expression cassette is shown for comparison (green curve). See also Figure S3, Table S4.

Figure 5. Synthetic regulation of endogenous genes

(A) Integration of switch modules into the genome upstream of the targeted gene (gene B) at sites H1 and H2 using λ Red recombination. Switch-edited gene B is translationally repressed, but can be activated via the cognate trigger RNA. (B) Images of uidA::Switch A and uidA::Switch B spread onto X-Gluc plates with different trigger RNAs. uidA expression like the wild-type (top left) is only observed with cognate trigger RNAs as seen by blue/green color change. (C) Images of lacZ::Switch C with different combinations of IPTG and aTc chemical inducers. lacZ::Switch C only activates with aTc-induced expression of trigger C in conditions where lacZ transcription is induced by IPTG. Wild-type lacZ (top left) is activated whenever IPTG is present. (D) Motility assays for cheY::Switch D on soft agar plates. cheY::Switch D is only able to move away from the point of inoculation at the plate center when trigger D is induced with IPTG. See also Figure S4, Table S5.

Figure 6. Simultaneous regulation of gene expression by twelve toehold switches

(A) Schematics of plasmids and ~3.4-kb polycistronic mRNAs used for multiplexing studies. Each reporter has its own switch RNA that can be independently activated by its cognate trigger RNA. (B) Percentage of cells expressing each of the four reporters for a set of 24 different trigger RNA combinations. Gray and colored circles are used to identify the particular trigger RNA being expressed and the corresponding switch RNA. See also Figure S5, Table S6.

Figure 7. A layered 4-input AND circuit

(A) Design schematic for the 4-input AND circuit consisting of three 2-input AND gates formed by three toehold switches, two orthogonal transcription factors (ECF41_491 and ECF42_4454), and a GFP reporter. (B) Complete 16- element truth table for the 4-input AND system. GFP expression from the sole logical TRUE output case with all input RNAs are expressed (far right) is significantly higher than the logical FALSE output cases where one or more input RNAs is absent. See also Figure S6, Table S7.