Short Activators and Repressors of RNA Toehold Switches

RNA toehold switches are a widely used class of molecule to detect specific RNA “trigger” sequences, but their design, intended function, and characterization to date leave it unclear whether they can function properly with triggers shorter than 36 nucleotides. Here, we explore the feasibility of using standard toehold switches with 23-nucleotide truncated triggers. We assess the crosstalk of different triggers with significant homology and identify a highly sensitive trigger region where just one mutation from the consensus trigger sequence can reduce switch activation by 98.6%. However, we also find that triggers with as many as seven mutations outside of this region can still lead to 5-fold induction of the switch. We also present a new approach using 18- to 22-nucleotide triggers as translational repressors for toehold switches and assess the off-target regulation for this strategy as well. The development and characterization of these strategies could help enable applications like microRNA sensors, where well-characterized crosstalk between sensors and detection of short target sequences are critical.


Supplemental Methods
Preparation of cell-free lysate Cellular lysate for all experiments was prepared as described by Sun et al. 1 with a few protocol modifications. Briefly, BL21 Star (DE3) lacZ cells were grown in 2xYTP medium at 37 C and 220 rpm to an optical density (OD) between 1.5-2.0, corresponding to the mid-exponential growth phase. 0.4 mM IPTG was added when the OD reached 0.4 to induce expression of T7 RNA polymerase, creating a T7 RNAP-enriched lysate. Cells were centrifuged at 2700 × g and washed via resuspension with S30A buffer (50 mM tris, 14 mM magnesium glutamate, 60 mM potassium glutamate, 2 mM dithiothreitol, and pH-corrected to 7.7 with acetic acid). These centrifugation and wash steps were repeated twice for a total of three S30A washes. After the final centrifugation, the wet cell mass was determined, and cells were resuspended in 1 mL of S30A buffer per 1 g of wet cell mass. The cellular resuspension was divided into 1 mL aliquots. Cells were lysed using a Q125 sonicator (Qsonica) at a frequency of 20 kHz and 50% of amplitude. Cells were sonicated on ice with cycles of 10 sec on and 10 sec off, delivering approximately 300-350 J, at which point the cells appeared visibly lysed. An additional 4 mM dithiothreitol was added to each tube, and the sonicated mixture was then centrifuged at 12,000 × g and 4 °C for 10 min. After centrifugation, the supernatant was removed, divided into 1 mL aliquots, and incubated at 37 °C and 220 rpm for 80 min. After this runoff reaction, the cellular lysate was centrifuged at 12,000 × g and 4 °C for 10 min. The supernatant was removed and loaded into a 10 kDa molecular weight cutoff dialysis cassette (Thermo Fisher). Lysate was dialyzed in 1 L of S30B buffer (14 mM magnesium glutamate, 60 mM potassium glutamate, 1 mM dithiothreitol, and pH-corrected to 8.2 with tris) at 4 °C for 3 h. Dialyzed lysate was removed and centrifuged at 12,000 × g and 4 °C for 10 min. The supernatant was removed, aliquoted, and stored at −80 °C for future use.

Figure S1
Schematic of trigger-mediated toehold switch activation mechanism of (A) the Series A design 2 and (B) Series B design 3 . Differences of note include the overall length of the RNA trigger, the inclusion of a 5' stability hairpin on the Series A trigger, and the length of the toehold-switch hairpin regions.

Figure S2
Truncated output triggers designed by the Series B toehold switch NUPACK code demonstrate the potential for toehold switch-mediated detection of miRNA-length triggers expressed from plasmids. Schematics above graphs indicate how the full trigger sequences were divided into shorter A and B trigger fragments for (A) SwitchA and (B) SwitchC. (-) indicates the switch-only condition in the absence of any trigger plasmid. Each reaction contains 2.5 nM of the toehold switch plasmid and 5 nM of the trigger plasmid (pTrigger). Conditions with two trigger plasmids have 5 nM of each plasmid. Error bars represent the standard deviation of technical triplicates (white circles).

Figure S3
The position-specific impacts of trigger substitutions across all positions on activation of SwitchA and SwitchB are qualitatively consistent. Mutations in the stem-binding region have a greater impact on switch activation than mutations in the trigger-binding region, expressed as the percent of activation by the consensus truncated trigger. X axis shading indicates positions within either the stem-binding region (red) or toehold-binding region (blue). Error bars represent the standard deviation of technical triplicates.

Figure S4
The impact of TriggerB wobble substitutions on SwitchB activation compared to non-wobble substitutions was less consistent than for TriggerA and SwitchA. Positions 3, 8, and 11 are all within the stem-binding region. Error bars represent the standard deviation of technical triplicates (white circles).

Figure S5
The impacts of (A) TriggerB insertions on SwitchB activation were less clear than for TriggerA, though the impacts of (B) TriggerB deletions were consistent with observations for TriggerA. X axis shading indicates positions within either the stem-binding region (red) or toehold-binding region (blue). Error bars represent the standard deviation of technical triplicates (white circles).

Figure S6
Different substitution types in TriggerB can lead to different levels of activation. Plotted are the GFP values used to calculate the percent differences shown in Figure 2 for (A) A, (B) C, (C) G, and (D) U mutations. X axis shading indicates positions within either the stem-binding region (red) or toehold-binding region (blue). Error bars represent the standard deviation of technical triplicates (white circles).

Figure S7
Different substitution types in TriggerA often lead to different levels of activation. Plotted are the percent differences in GFP expressed from SwitchA when using each possible non-wobble substitution at a given position in TriggerA compared to an arbitrarily selected baseline nonwobble substitution (y-axis) at the same position. For (A) A, (B) C, and (C) G mutations, positions in the stem region almost always have different activation for different mutations, while the toehold regions are more likely to exhibit little difference in activation between mutations. X axis shading indicates positions within either the stem-binding region (red) or toehold-binding region (blue). Error bars represent the standard deviation of the percent difference in activation between any pair of mutations at the same position.

Figure S8
Different substitution types in TriggerA often lead to different levels of activation. Plotted are the GFP values used to calculate the percent differences shown in Figure S7 for (A) A, (B) C, and (C) G mutations. X axis shading indicates positions within either the stem-binding region (red) or toehold-binding region (blue). Error bars represent the standard deviation of technical triplicates (white circles).

Figure S9
Combining multiple mutations in the same trigger can lead to unpredictable impacts on switch activation. TriggerA sequence is shown on the left, with the hyphen indicating the divide between the stem and toehold regions, and the red nucleotides representing mutations in each trigger variant. Green nucleotides represent consensus trigger nucleotides that are not mutated in each 5-mutation variant but are mutated in the 6-mutation variant. Error bars represent the standard deviation of technical triplicates (white circles).

Figure S10
The presence of multiple trigger mutations in (A) SwitchB and (B) SwitchD can lead to nonadditive effects and statistically significant activation even for high numbers of mutations in the toehold-binding region. Error bars represent the standard deviation of technical triplicates (white circles). Asterisks indicate a significant difference between a sample and the no trigger conditions as determined by the results of a two-tailed t-test (****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05).