In order to achieve our goal, we integrated and designed a series of RNA logic gates. Then, we improved them and finally made them work in practical applications, such as detecting viruses.

Figure 1. Project profile.

1. The ON RNA Switch—Toehold

1.1 Single Hairpin Structure Toehold Switch

Toehold switch, a single hairpin structure, is composed of cis-acting element RNA hairpin and trans-acting factor trigger RNA (Figure 2). The switch RNA hairpin contains the start codon (AUG) and ribosome binding site (RBS), forming a bulge and a loop of the hairpin, respectively. The binding of a trigger RNA to the toehold sequence allows for a branch migration process, exposing AUG and RBS for translation initiation. ^[1]

Figure 2. The toehold switch.

1.2 Inhibitory Hairpin Structure Toehold

To increase the dynamic range of toehold switch, the inhibitory hairpin is introduced before a single hairpin structure toehold, called the double hairpin structure toehold (Figure 3). The inhibitory hairpin can reduce leakage and increase the ON/OFF ratio. When the spacing a is sufficiently long, trigger B has a high chance of base-pairing with the primary switch even in the absence of trigger A (as shown in the upper route in Figure 3), making the circuit behave like a one-input switch with a secondary tuning knob for the other input. When the unpaired region, a, is short (* is long), as shown in the second situation in Figure 3, trigger B would need trigger A-hairpin binding to expose the unpaired, full-length toehold sequence of the primary hairpin, making the circuit behave like a two-input AND gate.^[2]

Figure 3. Inhibitory hairpin structure toehold.

2. The OFF RNA Switch—3WJ Repressor

Three-way junction(3WJ) repressors switch RNA employs an unstable hairpin secondary structure that contains an RBS in the loop region and a start codon in the stem region. Despite its high secondary structure, this unstable hairpin was previously demonstrated to be translationally active in toehold switch mRNA sensors (Figure 4). However, When the trigger RNA is expressed, the trigger will bind to the switch RNA. The resulting trigger–switch complex has a stable 3WJ structure that effectively sequesters the RBS and the start codon within the loop and stem of the switch RNA, respectively, and strongly represses translation. ^[3]

Figure 4. 3WJ switch

3. Logic Gates

Based on the above RNA switch elements, we constructed different logic gates with trigger RNA as the input signal, protein as the output signal, and switch RNA as the intermediate processing element.

We use E. coli BL21 Star (DE3) (low RNase E activity) as the biological chassis. The reporter circuit with switch RNA and gfp gene is inserted into the pACYC184 plasmid and triggers are inserted into the pUC19 plasmid to conduct experimental verification of the logic gates (Figure 5).

Figure5. The plasmids that were used for the construction of the logic gates.

Here shows the different types of RNA logic gates. In the truth table, 'A' and 'B' are the triggers and the value '1' means the presence of input, '0' means no input. For output, the expression level of GFP represents two states: 1 (high expression) and 0 (low expression).

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In Alexander A. Green, Jongmin Kim, et al., 2017^[5], they used toehold to construct RNA-only nanodevices to evaluate complex logic in living cells. They demonstrated that their devices can realize AND, OR and NIMPLY logic gate. In Jongmin Kim, Yu Zhou, et al., 2019^[3]. They constructed 3WJ repressors and integrated them into biological circuits that can execute universal NOT, NAND and NOR logic in E.coli.

So far, the construction of RNA-only IMPLY, XOR and XNOR logic gate have not been realized. Here, we designed IMPLY and XOR gate to fill this gap. The following are details about these design logic gates.

IMPLY

We combined the 3WJ switch and toehold switch to realize the IMPLY Boolean calculation (Figure 6). When no trigger is expressed, this logic gate behaves just like a 3WJ switch. When trigger A is expressed, the trigger will bind to the switch RNA. The binding of trigger RNA to the toehold sequence allows for a branch migration process, exposing AUG and RBS for translation initiation ^[1]. When trigger B is expressed, the trigger will bind to 3WJ switch RNA. The resulting trigger–switch complex has a stable 3WJ structure that effectively sequesters the RBS and the start codon within the loop and stem of the switch RNA, respectively, and strongly represses translation. When both rigger A and B are expressed, the binding of trigger RNA to the toehold sequence allows the ribosome to bind to the former RBS and break open the 3WJ stable hairpin.

Figure6. IMPLY gate

XOR

The XOR was inspired by the NIMPLY gate, consisting of a toehold switch and two triggers. The trigger’s core sequence is the same, and at the triggers’ both ends, there are the nucleotide-binding domains. When input one of these triggers, the switch can turn ON. Moreover, when input these two triggers simultaneously, they can pair together and form a ring in the middle. As a result, the switch will still be in the OFF state.

Figure 7. XOR gate

4 Improvements

4.1 Stability

When it comes to RNA, we all know that the stability of RNA is a key factor affecting the mechanism of its interaction. Therefore, we hope to optimize the ON/OFF ratio of the toehold switch by improving the stability of Trigger RNA. After interviewing teachers and consulting many pieces of literature, we learned that it can be achieved by adding an RNA stability hairpin at the 5 ' end ^[4], because that can block the degradation mediated by RNase E.

4.2 Versatility

We found a certain difference in the threshold value of different logic gates, which is not conducive to combining different types of logic gates. To improve the generality of these logic gates, it is necessary to find a universal threshold. Therefore, we have developed a SOFTWARE that can calculate the common threshold, which can give the normalized parameters of fluorescence intensity and the calculated threshold, so that the designer can easily find the common threshold of different logic gates and better combine different types of logic gates.

4.3 Composability

Incorporation of ribo-computing devices into sophisticated layered circuits will require systems that can provide RNAs as output species. Thus, we will use σ factors to achieve this function. The σ factors have several highly orthogonal pairs, so that this functionality can be implemented.

4.4 Multiple-input

On the basis of single-input logic gates and two-input logic gates, we also designed the three-input logic gates (Figure 8) and four-input logic gates (Figure 9). These enrich the types of our logic gates.

Figure 8. Three-inputs logic gates

Figure 9. Four-inputs logic gates

4.5 Swapping Gates

When we interviewed the professors, they provided us a new and interesting idea about swapping gates. We thought about that and here shows our two way to achieve that.

In the first one, we used a trigger to control the transformation between two logic gates. For example, we combined the inhibitory hairpin constructed switch with the 3WJ repressor, which achieved the transformation between IMPLY and NOT. If input the trigger A, it is an IMPLY gate. If not, it is a NOT gate(figure 8).

Figure 10. IMPLY and NOT swapping gate

In the second one, we used different triggers to control the transformation. For example, we combined the single hairpin constructed switch and the inhibitory hairpin constructed switch to achieve the transformation between AND gate and OR gate. When input the trigger B, it is OR gate. When trigger A is absent, it behaves like an AND gate(Figure 9).

Figure 11. AND and OR swapping gate

Using these methods, we also achieved the transformation between NAND and IMPLY, IMPLY and AND, IMPLY and OR.

5 Application

As a basic logic gate element, our project can be applied to many aspects. Here just show two applications.

5.1 Arithmetic Units

We construct the half-adder and the subtractor with the AND, XOR and NIMPLY gates. The half-adder is capable of carrying out simple binary mathematical operations necessary for digital computing. The subtractor can be done by adjusting the concentration of trigger RNAs. In the future, they can be used in the more complicated calculations.

5.1.1 Half-adder

We used the AND gate and XOR gate to make up the half-adder. A half adder is a type of adder, an electronic circuit that performs the addition of numbers. The half adder is able to add two single binary digits and provide the output plus a carrying value. It has two inputs, called A and B, and two outputs S (sum) and C (carry).

When there is only one input, the reporter of AND gate is not expressed, and the reporter of XOR gate is expressed. When there are two inputs, the reporter of AND gate is expressed, and the reporter of XOR gate is not expressed.

Figure 12. Half-adder circuit diagram

5.1.2 Subtractor

We used the NIMPLY gate to make up the subtractor. The subtractor can be done by adjusting the concentration of trigger RNAs.

We use inducers to turn on trigger expression, use inducer concentration to represent the input trigger concentration, and use fluorescent protein expression levels to react and output results. When two triggers are expressed at the same time, trigger S1 and trigger S2 will combine to form an RNA double strand that cannot be turned on by RNA Switch. Only when trigger S2 is expressed, the downstream gene expression of RNA Switch will be turned on. This subtracter can simulate S2-S1=Ps, and Ps is the fluorescence intensity of the reporter gene.

Figure 13. Abstractions of subtractor

5.2 Virus Detection

Our logic gates also can be used to detect various viruses. The use of logic gates can reduce false negative and false positive of virus tests and also makes it possible to detect multiple viruses simultaneously.

5.2.1 Reduce False Negative

A false negative in a test can often cause people to ignore the danger. For viruses that often produce new mutated individuals, testing for a single fragment is likely to result in omissions. Using OR gate can effectively solve this problem, we can select a specific segment from the original virus and the novel virus for testing, as long as any segment exists, the reporter gene can be expressed.

Figure 14. Demonstration of OR gate

5.2.2 Reduce False Positive

False positives are another major challenge in virus testing. Many homologous viruses have a large number of highly similar or even identical conserved fragments. We found that the use of the AND gate containing an inhibiting hairpin increased the specificity of the detection. We designed the binding domain to inhibit the hairpin based on the same sequence of the homologous virus, and designed the second hairpin based on the specific fragment of the target virus we wanted to detect.

Figure 15. Demonstration of AND gate

5.2.3 Multi-virus Detection

In complex environments, there is often more than one virus. For example, Noroviruses (NoV), Hepatitis A (HAV), Coronavirus, and other pathogenic viruses often exist in the hospital environment. This can easily lead to alimentary infection, respiratory infections, operation slices infection, and other hospital infections.

The programmability of RNA-based regulatory elements is based on the sequence complementary pairing, which gives the biosensor good specificity. By designing particular regulatory elements, we could detect multiple viruses simultaneously in an orthogonal way. Furthermore, as mentioned above, by combining different types of logic gates, we can improve the biosensor performance by avoiding false-negative and false-positive reports. According to the expression of different logic-gate reporters, we can determine the presence of various viruses.

Reference

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[2] Kim, S.-J., Leong, M., Amrofell, M. B., Lee, Y. J., & Moon, T. S. (2019). Modulating responses of toehold switches by an inhibitory hairpin. ACS Synthetic Biology. doi:10.1021/acssynbio.8b00488

[3] Kim, J., Zhou, Y., Carlson, P. D., Teichmann, M., Chaudhary, S., Simmel, F. C., … Green, A. A. (2019). De novo-designed translation-repressing riboregulators for multi-input cellular logic. Nature Chemical Biology. doi:10.1038/s41589-019-0388-1

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[7] Moon, T. S., Lou, C., Tamsir, A., Stanton, B. C., & Voigt, C. A. (2012). Genetic programs constructed from layered logic gates in single cells. Nature, 491(7423), 249–253. doi:10.1038/nature11516

[8] Jongmin Kim, Elisa Franco,RNA nanotechnology in synthetic biology,Current Opinion in Biotechnology,Volume 63,2020,Pages 135-141,ISSN 0958-1669, https://doi.org/10.1016/j.copbio.2019.12.016.

[9] Song, T., Garg, S., Mokhtar, R., Bui, H., & Reif, J. (2016). Analog Computation by DNA Strand Displacement Circuits. ACS Synthetic Biology, 5(8), 898–912. doi:10.1021/acssynbio.6b00144

[10] Reif, J. H. (2009) Encyclopedia of Complexity and Systems Science, pp 5466– 5482, Springer.

Team:OUC-China/Design