RIBOTOX

Silver HP

Design

MicroRNA Biomarkers

MicroRNAs (MiRNAs) are small single-stranded non-coding RNAs found in eukaryotes and some viruses that function in post-transcriptional gene regulation^[1]. MicroRNAs circulating in human serum have been shown to be upregulated in certain conditions, such as Preeclampsia, allowing them to be used as biomarkers in disease detection^[2].

After searching through many studies for miRNAs that are upregulated in the serum of Patients with Preeclampsia, we settled on miR-210-3p and miR-517-5p to detect. These are upregulated at an average of roughly 4-fold in patients with the condition, and the studies investigating each of their expression levels have confirmed their potential to be used as biomarkers to test for Preeclampsia ^[3] ^[4] .

Toehold Switches

When few microRNAs are present, the ribosome binding site of an mRNA coding for luciferase is folded up, so the ribosome cannot unzip the long stem and move down the strand to the start codon to initiate translation.

However, when a microRNA binds to the trigger site, the switch unfolds, decreasing the length of the stem, and exposing the start codon, so translation of leuciferase - a reporter protein - can begin.

We designed composite parts containing toehold switches which are activated in the presence of miR-210-3p and miR-517-5p. The switches are downstream of a T7 promoter and downstream of the reporter protein firefly luciferase, the expression of which can be quantifiably measured using a luminometer when its substrate, D-luciferin, is added.

Our design evolution consisted of three generations of toehold switches to detect our two target miRNAs.

Our first generation of switches (gen1) were designed to be highly specific, by incorporating an anti-miRNA, and detecting the complex of anti-miRNA and the miRNA trigger.

However, after a consultation with Dr Alex Green (see Integrated Human Practices page), we were advised to pursue a more efficient and simpler ‘gen2’ switch, which detected just the miRNA triggers. Our gen3 switch uses AND-gate logic and can detect both miRNAs simultaneously by creating an anti-miRNA which binds together the two triggers, and that complex causes the switch to unfold.

Gen2 Toehold Switches

Design Methodolgy

We used the software package NUPACK to help design our gen2 switches in-silico. NUPACK is able to predict the interactions of the secondary structure of one or more nucleic acid strands. And so, we used the programme to model and perfect our switches.

At first, we designed our switches base by base, checking the minimum free energy (MFE) structure, using NUPACK, to ensure that our switch had a strong hairpin structure in the ‘off’ state and was properly unfolded in its ‘on’ state.

We were successful in designing multiple switches that had MFE structures fulfilling this criteria, but multiple bases had low probabilities of being in their respective positions.

Therefore, we used the NUPACK API to generate a Python program to test a randomly generated list of one-hundred-thousand linker regions (which did not contain any stop or start codons) and simulated a thousand probabilistic secondary structure samples for each, showing which switch had the highest probability of unfolding in the presence of our miRNA triggers, but not in their absence.

The Design

Toehold switches contain multiple parts, namely: the trigger binding site, the ribosome binding site (RBS), the seven amino acid long linker sequence and the protein coding sequence.

The trigger binding site on each toehold switch is the reverse complement to the trigger RNA strand. As the miRNAs are both 22 nucleotides long, the trigger site is also 22 nucleotides long. The trigger binding site makes up the lower stem in the ‘off’ state as well as part of the toehold base. We were advised to ensure the trigger site was >10 nucleotides long by Dr Wooli (see Integrated Human Practices page).

The RBS (AGAGGAGA) was taken from Dr Alex Green and is a constant region throughout all our parts. Our linker sequence varies between switches. The linker sequence provides structure to the switch in both ‘on’ and ‘off’ states. We had to ensure that the linker sequence did not bind with any other parts of the toehold switch. The software that we created helped ensure that we selected the most appropriate linker sequence to fulfil our criteria.

The start codon (AUG) is left unpaired in both ‘on’ and ‘off’ state. In order to achieve that, a wobble pair is placed on the opposite side of the stem. This increases the minimum free energy structure of the OFF state to ensure the MFE structure of the ON state is lower, such that the structure will change shape in the presence of the trigger miRNA.

There was a fine balance between the length of the base and the length of the stem: The longer the stem of the toehold switch in the ‘off’ state, the more energy is required to overcome the intermolecular forces and so there is lower leakage. However, the longer the base of the switch, the easier it is for an RNA strand to bind and so there is greater expression of the protein.

Gen3 Toehold Switch

Helical representation of AND-gate in ‘ON’ state after dual miRNA and anti-miRNA complex has bound to the trigger binding site of the toehold switch

Our third generation of toehold switch uses AND-gate logic to detect the presence of our two target miRNAs in one switch.

This switch employs an anti-miRNA with three regions; two hybridization domains that are complementary to each of our trigger RNAs and the anti-miRNA’s own trigger site. This 29 nucleotide long strand has a 13 nucleotide long binding site for the miRNA 210-3p and an 11 nucleotide long binding site for miRNA 517-5p. This complex of the 3 RNA strands is necessary to bind to the toehold switch’s trigger binding site. We had to ensure that each of these trigger binding sites was >10 nucleotides on the advice of Dr Bae Wooli.

For the AND gate, we were unable to use the NUPACK API as the minimum free energy structure would show each strand binding, independent of the position of the other strands. Therefore, we designed the anti-miRNA and the toehold switch base by base. To ensure that the switch would unfold, we had to ensure that the difference in MFE structure between the ‘off’ and ‘on’ state was such that unfolding was energetically favourable when the anti-miRNA-miRNA complex had formed. In order to help us achieve this energetically favourable state we replaced an adequate amount of C-G bonds with U-G bonds to raise the free energy of the MFE structure of the OFF state.

This table shows the homologous RNA compared to their percentage activation of the toehold switch. This switch comes up with 98% specificity and therefore we can be confident in how little leakage we expect due to homologous RNAs in a given blood sample.

The second way which we can increase our specificity is by amplifying the RNA concentrations. (Information about testing switches in Silico can be found on Model page )

Isothermal Amplification

As seen in our Measurement page , we found it would be difficult to discriminate between leaky expression of our toehold switches and <2.25M concentrations of miRNAs, and there is no data giving concentrations of our trigger miRNAs online.

Therefore, to ensure miRNA concentration is high enough for them to be detected by the toehold switches, we decided to find a way to amplify our microRNAs. Not only is PCR difficult to perform on microRNAs due to their short length (~22 nucleotides), we wanted to find a solution that would allow amplification to take place isothermally, in a single tube, increasing the accessibility of our tests, especially in countries with less access to expensive equipment such as thermocyclers.

With further research and contacting, we came across Dr O’Sullivan, a researcher in isothermal amplification strategies. Dr O’Sullivan introduced us to Recombinase Polymerase Amplification (RPA), a single tube, isothermal alternative to PCR, which can amplify dsDNA strands5. So, before RPA could work, we had to find a way to reverse transcribe miRNA into DNA. And for this we decided to use miRPA.

As seen in the diagram above, two DNA probes, one with 5’ phosphorylation, bind to the miRNA, and are ligated together by DNA ligase. Then, primers are added, with DNA polymerase, and complementary strands to the ligated probes are synthesised. Then, RPA can take place: primers, associated with recombinase protein so they can dislodge the strands, replicating them in a similar method to PCR, but as no heat cycles are required to break up the strands, the process can take place isothermally.

In order for the miRNA to be detected, we decided to use ‘asymmetric RPA’: an excess of forward primers are added (usually 5x the amount), so an excess of the strand that was originally miRNA form, so there is now ssDNA with the miRNA code in DNA. This can be detected by our toehold switches.

In order to design probes for miRPA, we designed another python script using Nupack’s design functions in its API to find probes which would bind to the miRNAs, but have overhangs which didn’t bind within themselves, to ensure primers could easily anneal to them. We then tested, using Nupack, the specificity of these probes and found that they had a less than 1% likelihood of binding to the closest human miRNA homologs in the correct shape. This demonstrated the increase in specificity that using miRPA could add to our test.

Refrences

David P. Bartel (2018). Metazoan MicroRNAs [Online]. Available at: https://doi.org/10.1016%2Fj.cell.2018.03.006 [Accessed 19 October 2021]
Condrat, C.E. et al., 2020. miRNAs as Biomarkers in Disease: Latest Findings Regarding Their Role in Diagnosis and Prognosis. Cells, 9(2), p.276. Available at: http://dx.doi.org/10.3390/cells9020276 [Accessed 19 October 2021].
Reza M. A. N., Kolsoum S. et al., 2019. Quantification of circulating miR-517c-3p and miR-210-3p levels in preeclampsia. Pregnancy Hypertension, Volume 16, 2019, Pages 75-78, ISSN 2210-7789. Available at: https://doi.org/10.1016/j.preghy.2019.03.004 [Accessed 19 October 2021]
Condrat, C.E. et al., 2020. miRNAs as Biomarkers in Disease: Latest Findings Regarding Their Role in Diagnosis and Prognosis. Cells, 9(2), p.276. Available at: http://dx.doi.org/10.3390/cells9020276 . [Accessed 19 October 2021]

Team:City of London UK/Design