Part:BBa_K5106016

TMSD threshold system Inhibitor under T7 promoter 

This part encodes for an RNA strand that is part of the TMSD threshold system (TTS) designed by miRADAR (WageningenUR 2024). TTS is a modular threshold system designed to provide a clear, binary output signal in response to subtle changes in input RNA concentration. It relies on Toehold-Mediated Strand Displacement (TMSD), where RNA strands A, B, and C interact to create a tuneable threshold. Strand A serves as the input, binding to strand B via a toehold and displacing strand C once the concentration of A exceeds the threshold of free strand B. Displaced strand C then triggers strand D, which contains an inducible spinach aptamer that refolds into a fluorescent form, generating a measurable output. This system ensures precise detection by only producing a signal when RNA levels surpass a set threshold, making it ideal for cell free diagnostic applications.

This part specifically (BBa_5106012 under a T7 promoter), referred to as Strand B or the inhibitor is central to the TMSD reaction, acting as the scaffold that interacts with both strands A and C. In the initial state, strand B is annealed to strand C, and present in an unbound form, creating the threshold. Strand B has a free region known as the toehold, which is available for partial binding by strand A. This toehold region is critical because it enables strand A to initiate the displacement reaction by providing an initial binding site. The free strand B's role is to sequester all strand A below the level of the threshold. Once A exceeds this tuneable threshold concentration, strand B facilitates the competitive interaction between strand A and strand C, mediating the switch from an "OFF" to "ON" state in response to changes in strand A concentration, by releasing the output strand C.

Usage and Biology

When developing sensors for miRNA, for example for diagnostics, it is important to keep in mind that a certain miRNA is always present in e.g. blood, but can be dysregulated with certain disease. Even though these dysregulations are quite subtle, these can be used to diagnose patients. It is, however, really important to be able to differentiate between healthy levels of miRNA and disease levels. That is why miRADAR (WageningenUR 2024) developed a toehold mediated strand displacement (TMSD) threshold system (TTS). With this system you can get a binary (being ON or OFF) output signal, based on a chosen threshold concentration of a specific miRNA.

Overview TMSD threshold system mechanism

In TMSD there are at least three strands of RNA present: A, B and C (Figure 1). In the initial state, strand B and C are annealed, however strand A has a higher affinity for B. Because there is a free region of RNA on strand B, strand A can partially bind. This is known as the toehold, and is essential to stabilise the kinetic bottleneck in the reaction. When the toehold is at least six bases long, the forward reaction kinetics in the exchange reaction improve, with equilibrium constant K increasing up to the power six.¹ Once partially bound, strand A can compete with strand C, ending in strand C being displaced from strand B. To create a TMSD threshold you need three RNA strands where all binding affinities are controlled so that the binding energy of RNA pairs is ranked: AB>BC>CD, with D being an external output receptor for C. This is because A must anneal stronger to B, otherwise a very large ratio of A:C is required to displace C.

Figure 1: Schematic overview of toehold mediated strand displacement (TMSD) with labels A, B, C.

This alone does not create a threshold, for this we need a surplus of strand B. By creating a pool of free strand B, low levels of strand A will bind to these, not releasing any of strand C. By controlling the concentration of free B, a threshold can be created. In the situation where there is less A present than B, indicated as the OFF state (Figure 2a), A binds to B, but due to the excess of B, no C is released. When A is present in higher concentrations (Figure 2b), the unbound B is sequestered and C is displaced from B. This means output signal C is only released when A is present in concentrations above the set threshold.

Figure 2: Overview threshold function by TMSD between A, B and C, with a surplus of B. a: strand A is present in lower concentrations than strand B, all C remains bound to B, meaning no output signal C is released; B: A is present in excess of strand B. This causes all B to be sequestered and C to be released, which is the output signal.

The slope of this threshold is dependent on the ratio between B and C. If the ratio of B to C is 1:1 when any A is present, C will be released, causing a linear relation between A and C (Figure 3a). However, when the ratio of B is increased, a larger fraction of A is required to bind the free B than to release the C (Figure 3b). The smaller the fraction of A that releases C becomes, the more the output signal starts to resemble a full binary signal. Computer modelling has shown that a B:C ratio larger than ~3 can create a near binary output response (Figure 3c).

Figure 3: Computer model of effect of B:C ratio on steepness of output (C) compared to input (A). Simulation with C set to 0.5 in all situations, and concentration of B: 0.1, 0.5, 2.5 for each graph a,b and c respectively. When the concentration of A increases the output C will increase, the slope is determined by the ratio of B:C. Above a ratio ~3 this becomes a near perfect transition.

This system allows for an easy and modular system for thresholding in cell-free systems. The RNA sequences can be easily designed based on a desired input and/or output sequence, and the threshold can be altered by simply changing the concentration RNA strand B. The output signal C can modified for any downstream detector (D), which can then detect the output signal of the threshold system C. This downstream detector D can be a toehold or an inducible fluorescent aptamer, thereby giving a visible signal in a Cell-free test.

Experimental Design and Results

For establishing the threshold of miRNAs in our test, we used a Toehold-Mediated Strand Displacement (TMSD) threshold system, further referred to as TTS. The TTS system, consisting of the four different parts, acts as one part collection, which consists of BBa_K5106015, BBa_K5106016, BBa_K5106017, and BBa_K5106018. After we designed and confirmed the sequences of the TTS in NUPACK, we converted them into DNA and combined them with a T7 promotor. This allowed us to order the sequences as DNA oligos and transcribe them using in vitro transcription (IVT) back into RNA. The T7 promotor allows RNA polymerase to bind and amplify the RNA sequence at 37°C. This was done in a thermocycler to ensure optimal temperature and minimal evaporation. RNA from IVT was directly loaded in a 96-black clear bottom plate for fluorescence detection. No purification was performed as this caused irredeemable misfolding of the fluorescent aptamer. Spinach-DFHBI-1t fluorescence was measured at 470/505 nm at 25°C, 30 minutes after adding the RNA fragments to allow the mixture to reach equilibrium.

Alternatively, RNA was transcribed in a plate reader at 37°C to directly measure the fluorescence and thereby the concentration of RNA in the system. DNA with IVT buffer and enzymes was directly loaded in a 96-well black clear bottom plate for fluorescence detection. To obtain a fluorescent signal, C and D had to be co-transcribed to result in the correct folding of the fluorescent aptamer. Spinach-DFHBI-1t fluorescence was measured at 470/505 nm at 37°C in 30-minute intervals, to reduce photobleaching.

Figure 4: Fluorescent signals of spinach-DFHBI-1T after IVT. IVT was performed in a plate reader at 470/505 nm fluorescence measurement over 7 and a half hours with 30-minute intervals. All measured samples contained 1x T7 buffer with associated IVT reagents as specified in protocols and 10μM DFHBI-1T. Measurements shown of a negative control (pink), IVT reaction TTS part C (Yellow), IVT reaction of TTS part D (Green), in vitro co-transcription reaction of TTS part C and D (Blue), spinach positive control (Purple).

We observed that fluorescence was produced in the presence of TTS part D alone (Figure 4), this background signal was to be expected. Since it was less than 20% of the signal of CD, this was deemed acceptable. Notably more fluorescence was observed when part C and D were co-transcribed in the same system, indicating that co-transcription is necessary to ensure correct folding. When fragment C is produced separately, no fluorescent output signal was observed as expected. This was also the case for the negative control. The positive control sample consisting of the spinach-2 fluorescent aptamer also showed no fluorescence. This was hypothesised to be caused by misfolding of the RNA during IVT. This was shown by heat denaturation of the spinach-2 aptamer, after which the RNA was able to refold, and fluorescence was restored (not shown).

Figure 5: Introduction of part A increases fluorescence. Fluorescence measurement at 470/505 nm of CD in the presence of 8 μL IVT product A (green), compared to only CD (pink) and a blank measurement (blue).

After the first experiment in which we showed that TTS part C and D can produce fluorescence, RNA strand A was added to the reaction mix. This was to produce a threshold, opposite of that originally designed, where the subsequent addition of B would inactivate the fluorescence. Since A should only have affinity for strand B, no change was expected. However, an almost 3-fold higher fluorescent signal was observed after the addition of A (Figure 5). Since A itself cannot bind and stabilise DFHBI-1T, it was speculated that it might have affinity for D.

Figure 6: NUPACK analysis indicates AD has affinity when concentration D exceeds C.

This was confirmed by a NUPACK analysis (Figure 6), which indicated that A could bind with D. This was missed since in previous analyses C and D were expected, and therefore modelled, to be at equal concentration. If the concentration of D is greater than that of C, A can bind to D. Since NUPACK showed these criteria are essential for this reaction to occur, we must assume this is what caused the increase in fluorescence due to the addition of A (Figure 6). This is in line with protocols about IVT, which states that longer RNA sequences transcribe more efficiently than short RNA sequences. Therefore we hypothesise that; 1: Due to the longer DNA sequence, fragment D has been transcribed more.; 2: Due to the antisense compatibility A can bind to D (when C is present in smaller concentrations than D), and induce fluorescence.

Figure 7: Effect of B on CD (+A) fluorescence. Fluorescence measurement at 470/505 nm of the TTS system of TTS system with fluorescence of the same amount of co-transcription product of part C and D, with 8 μL A and varying amounts of part B added: blank (purple), control (CD without A or B) (Yellow), CD + A + 4 μL B (Pink), 8 μL (Blue), 20 μL (green).

To test the full functionality of the TTS system, we investigated how well part B could inhibit the fluorescent signal caused by part C,D and A. This is a simplified (and reverse) version of the TTS system, as it was unsure whether C could again bind to D and produce fluorescence after release by B. This system was predicted give a similar threshold reaction, however the threshold would be based on the concentration A, and the signal would be inhibited once the concentration of B exceeded that of A.

Since the binding affinity between part B and C (and especially A and B) is higher than between part C and D, addition of part B should lead to a decrease in the observed fluorescence. The fluorescence of CD was measured at 470/505 nm with varying concentrations strand B (Figure 11). It was observed that fluorescence indeed went down over time with the addition of part B. In addition, fluorescence went down to a plateau that was also determined by the concentration of inhibitor B, whereby adding more B resulted in lower fluorescence. This indicated that part B can indeed inhibit fluorescence. part C as expected, and can thus be used for creating the TTS system together with part C and D.

However, since the fluorescence of all wells dropped immediately without any threshold function being observed, therefore we hypothesise that C/A has not saturated all of D, therefore when any B is present, CD/AD pair is split up and fluorescence decreases.

Altogether, we showed that fluorescence caused by the secondary structure formed by part C and D together works. In addition, inhibition of part B is also successful. The next step would be proving that the inhibition of B, only occurs once the threshold of A is exceeded. Secondly it must be shown that A can release part B again as required for the complete TTS system.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

References

1. Zhang, David Yu, and Erik Winfree. ‘Control of DNA Strand Displacement Kinetics Using Toehold Exchange’. Journal of the American Chemical Society 131, no. 47 (2 December 2009): 17303–14. https://doi.org/10.1021/ja906987s.
2. Confavreux, Christian, Sandra Vukusic, Thibault Moreau, and Patrice Adeleine. ‘Relapses and Progression of Disability in Multiple Sclerosis’. New England Journal of Medicine 343, no. 20 (16 November 2000): 1430–38. https://doi.org/10.1056/NEJM200011163432001.
3. Wang, Tianhe, and Friedrich C. Simmel. ‘Switchable Fluorescent Light‐Up Aptamers Based on Riboswitch Architectures’. Angewandte Chemie International Edition 62, no. 41 (9 October 2023): e202302858. https://doi.org/10.1002/anie.202302858.