iGEM 2024 — Team wageningenur

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{% block title %}Notebook{% endblock %}
{% block lead %}Describe how and why you chose your iGEM project.{% endblock %}
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# Notebook

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* [May](#may)
* [June](#june)
* [July](#july)
* [August](#august)
* [September](#september)

# May

**Week 1-2** - 10th of May to 31st of
May

These weeks started with defining and dividing the subprojects. After
joining the safety tours on the labs of all chair groups, we were ready
to do the first lab experiments. In addition to a transformation of the
iGEM Ptet promoter, we tried to add an extra part to make Ptet\*. In
these weeks, the constructs of the Gate RNA plasmids and trigger RNA
plasmids were designed *in silico*. We also made competent cells
for the paper-based constructs.

Besides the experiments in the lab, we also started with our dry-lab
projects. When access to the virtual machine was obtained, we started
with analysing and testing SwitchMi Designer and started experimenting
with the NUPACK software. We also researched possible optimisation
algorithms and feed forward loops, after which we made an object
function. For our circuit boolean model, we created and simulated AND
circuits and created a test to check the production of GFP using this
boolean model.

# June

**Week 3** - 3rd of June to 9th of
June

In this week, we cloned the LacZ construct under the T7 and p70a
promoters and verified its production. We started with designing the
primers to make the TMSD parts and designing the first DNAzymes. A
literature study on RNA sponges was started as well. The AND and OR gate
plasmids were made with overlap extension PCR and golden gate cloning.
This method was also used to make our first toehold switch (CLSB-UK),
and the same was tried for the trigger plasmids for the toehold gates.
In addition, we made heat shock competent "Marionette" *E.coli*
cells, which later were used for *in vivo* experiments. This week
we were also ready to sequence the modified Ptet promoter. When we saw
there was a missing part, we designed primers to fix it.

We also continued with building our models. We continued working on
the feed forward loops and made the first version of our network
building algorithm.

**Week 4** - 10th of June to 16th of
June

In this week, we mainly focused on tutorials. As the input RNA and
AND gate inserts were incorrect, we also did some troubleshooting and
repeating of these experiments. The overlap extension PCR was repeated
for the AND gate, after which we also designed new primers. We performed
a golden gate cloning experiment of the first toehold and finished the
plasmid construct for it.

By now, we understood SwitchMi Designer and started working on
improving the software tool. We added some code to obtain a figure of
the secondary structure of the toehold switches.

**Week 5** - 17th of June to 23rd of
June

After designing them, we were ready to construct the DNAzyme
components and the TMSD parts. For the input plasmids of the toehold
gates a golden gate, transformation, and a colony PCR was preformed.
However, in the sequencing results, we saw that the promoters were
incorrect. A PCR was preformed on the AND and OR gate plasmids, after
which they were cloned. After the literature study, we also started with
the design of the sponges strategy with BioBricks. The marionette strain
was also transformed with our first toehold switch plasmid. By adding
the missing tet, the trigger plasmid was also established for the first
toehold switch.

For the *in silico* toehold design, we continued working on
the code to make the figures with the secondary structure. We also used
synthetic data on our first feed forward models, which could form the
basis for experimental design later on. By using adjacency matrices, we
build a new version of the network building algorithm.

**Week 6** - 24th of June to 30th of
June

This week, we performed our first test of the DNAzymes. We also
finished the production of the TMSD parts. For our amplification method,
we started in the lab testing NASBA with QUBIT. Qubit was bound to the
primers which caused the signal to be too high. Therefore, we decided to
test NASBA in steps. Even though, we obtained the correct AND gate, we
needed to do some troubleshooting with the OR gate sequence. We also
redesigned the sponge strategy and ordered ologo’s for these
experiments. After repeatedly transforming the marionette strain, we
still found our plates to be overgrown. When researching, we found a
problem in the resistance of the strain. Since a different resistance
gene was used on the trigger plasmids, we now first transformed this
into marionette.

After HP talked to some stakeholders, we decided to focus on
diagnosing relapsing-remitting multiple sclerosis. Therefore, we could
start search for data on this disease. We also started on modelling the
TMSD to predict the optimal parameters to be used in the lab. By
expanding the network building algorithm, we created an algorithm that
can generate all possible combinations of logic gate circuits.

# July

**Week 7** - 1st of July to 7th of
July

As the DNAzyme did not work, we continued troubleshooting them. We
had to design and order new primers for LacZ. As NASBA needed to be
tested in steps, we also designed new constructs for this experiment.
The contructs of TMSD were made, so the transcription of them was tested
*in vitro*. This production of the AND, and OR gates, including
both the LIRA and toehold based gates, continues with PCR and cloning,
and the produciton of the corresponding trigger plasmids needed some
more troublehooting and designing. The sponges were made with golden
gate cloning, after which a transformation and colony PCR were
preformed. As the marionette strain was found to have a resistance
problem, we recloned the toehold switch construct to a kanamycin
Resistance plasmid. The marionette strain with trigger was made
competent with an electroshock and the new toehold switch plasmid was
added.

The data found for relapsing remitting MS was researched and
processed according to the paper it was from. We continued with the
model for the TMSD. After checking the conditions, we also started with
making an optimisation algorithm for the circuit design.

**Week 8** - 8th of July to 14th of
July

This week, we were mainly busy with troubleshooting the performed
experiments. We continued our experiments with testing and
troubleshooting the DNAzymes. This is also the case for the sponge
constructs. The construction of the paper-based constructs were also
wrong so we continued with troubleshooting these for the next four
weeks. The aptamer for the TMSD was optimised and the final NASBA
constructs were ordered. The production of the different toehold logic
gates also needed troubleshooting and redesign for most constructs.

In our dry lab, we researched the possibility to add a model
predicting the ON/OFF ratios of the toehold switches, continued with the
TMSD model and generated the first version of the optimisation algorithm
for the circuit design.

**Week 9** - 15th of July to 21st of
July

For the DNAzymes we tested different buffers and designed
alternatives DNAzyme options. After successful cloning of the miRNA, we
still had to redesign the golden gate cloning for the sponges. We were
also still in the process of troubleshooting the paper-based constructs.
This week, we optimised the IVT for the TMSD. For the first step of
NASBA, we made a positive control with PCR, and tried reverse
transcription, which gave some unclear results indicating some problems.
In the toehold AND gates new RBS sequences were designed to replace the
old once, and PCR and cloning was performed for this. For our first
toehold switch, we prepared buffers and M9 medium and our first plate
reader experiment was performed. However, no signal was seen.

This week, we continued with the miRNA expression data and filtered
it according to the paper the data is from. The TMSD model was also not
finished, so we continued with that. A second version of the
optimisation algorithm was made by using pyPESTO and latin hypercube
sampling.

**Week 10** - 22nd of July to 28th of
July

For the RNA sponges, we performed a golden gate method with 4
templates instead of 9. We were still troubleshooting the paper-based
constructs. Because we wanted to use a different aptamer, we also
redesign the other TMSD parts. The reverse transcription and RTPCR of
NASBA were repeated, but the results were still unclear. In addition,
IVT was tested, but did not yet work

For the TMSD model, we found the optimal parameters to be used in the
lab. We also optimised the model for a more complex system. As the
optimisation algorithm was finished, we could find the optimal circuit
design.

**Week 11** - 29th of July to 4th of
August

In this week, we repeated the golden gate cloning for the sponges but
now with different ratios of the components. We were also still in the
process of troubleshooting the paper-based constructs. For the TMSD
reaction the *in vitro* transcription was tested and for NASBA
the step of reverse transcription was tested with an *in vitro*
transcription reaction. However, for the later the reaction did not
perform properly. To produce a postive control for the toehold tests, a
PCR was performed on only and RBS with GFP. By this time we were also
able to do some tests in a PURE system and do some test with the kit
provided by Promega.

# August

**Week 12** - 5th of August to 11th
of August

The miRNA that will bind to the sponges were synthesised. For the
sponges, we tried to find the best template this week. We continued with
troubleshooting the DNAzymes. For the PURExpress system we experimented
with lower reaction volumes. As the promega kit was unable to give us a
proper output, we cloned a chromoprotein as an alternative construct and
tested different variables for Promega. To troubleshoot NASBA we used
different primers. However, the reverse transcription only worked when
we used a lower concentration of only the reverse primer. We also
amplified the LIRA AND gate after which it was cloned. A plate reader
experiment was also done for the toehold switch with different promoters
(p105 and p100) after a positive control was constructed.

In the dry-lab, we continued working on our models. We also performed
a random forest model to obtain the most important miRNA in MS.

**Week 13** - 12th of August to 18th
of August

As the golden gate reactions for the sponges did not work, we
redesigned the experiment to make a one pot reaction. After some time of
troubleshooting the DNAzymes, we found that an old bottle of
H2O2 was the problem. The NASBA step of reverse
transcription was repeated for an MS related miRNA. After the promoters,
Psyn and Ptet, arrived we were able to perform a golden gate and finish
the input plasmid and the gates. We repeated the golden gate to change
the RBS and we added a hairpin to the AND gates. With a plate reader
experiment, we checked the leakiness of the correct gates. Using a PCR,
we remove extra bases from the trigger. We also fixed to Ptet promoter
on the positive control plasmid of the toehold switch. The
transformation for the production of the chromoprotein for Promega
testing was not yet successful.

To find miRNA to distinguish between MS and healthy control, we used
another classification technique (histgram gradient based
classification). However, this was not used further. The code was also
improved to enforce a better miRNA annealing to the predicted toehold
switches. We also build a model that was able to predict the TMSD
reaction in the lab. We compared the model output with the data obtained
in the lab. The boolean network for the prediction of the AND gate was
also converted to a continuous model.

**Week 14** - 19th of August to 25th
of August

This week the first golden gate one pot reaction was performed on the
sponges with a two step protocol. As the DNazymes of the paper don’t
work due to hybridisation of H3 and H4, new DNAzymes were designed such
that it anneals to the linear domain of the toehold switch. For the
amplification of the MS miRNA, an *in vitro* transcription on the
RT PCR product was performed. A colony PCR shows all AND gate and input
plasmids are correct. By amplifying the plasmid backbone with GFP, we
reduced the amount of constructs in the golden gate reaction. This was
done to troubleshoot the wrong gates. We also build a plasmid for a
trigger RNA and toehold switch plasmid for a second toehold. Primers to
make toehold switches targeting an MS miRNA and to add a hairpin to the
trigger plasmid were also designed. We repeated production of
chromoprotein for *in vitro* testing, but again did not work, so
we designed new priemrs for gibsom assembly for this

This week, we designed toehold switches targeting MS miRNAs *in
silico*. We continued with the analysis of the data obtained in the
lab with the model. The model predicting logic gates switches was also
made and simulated.

**Week 15** - 26th of August to 1st
of September

We unfortunately decided to stop working on the sponges, due one of
the team members getting ill. In this week, we finished the production
of all different RBS an hairpin sequences for the AND gates. We also
troubleshooted the modular input RNA and reconstructed the toehold
gates. With overlap extension PCR we obtained a plasmid for the toehold
switches detecting an MS-miRNA. Since new PURE kits arrived, we could
perform a pretreatment for the paper test, including cutting out some
discs, and drying these. We also tested the first paper discs using the
positive control plasmids. During this week, we also performed overlap
extension PCR to construct all 3 toehold switches targeting the MS
miRNA, and cloned the chromoprotein.

We found an MS-specific miRNA combination with our models and
continued working on the TMSD models optimising the parameters. We also
fitted the data obtained in the lab of the logic gate circuits to the
model predictions.

# September

**Week 16** - 2nd of September to 8th
of September

The construction of RNA switches, trigger and DNAzymes parts were
finalised. An *in vitro* transcription was performed for TMSD and
the RNA folding was optimised. A RT transcription was performed on the
MS miRNA of which an PCR was performed. In addition, the final IVT test
for NASBA was performed, and larger reaction volumes were tested for
this. For the toehold AND gates, double transformations of both trigger
and circuit plasmids of marionette strain were performed, of which the
colonies were used for plate reader experiments.In addition. Overlap
extension PCR was performed to obtain the MS trigger plasmid after which
an PCR was performed. To make the toehold switch plasmid for the *in
vitro* experiments, a golden gate was used. The final toehold
plasmids, including one AND gate, were also tested *in vitro*
(PURExpress) for a qualitative test.

To take other disease into account in our test, we searched for miRNA
expression data for mimic diseases. We also performed a parameter space
exploration for TMSD.

**Week 17** - 9th of September to
15th of September

In one of the last lab weeks, we tested the RNA DNAzymes in tubes and
tested out first toehold switch on paper. For the TMSD reaction, we
tested the optimisation of the folding, the CD activation and the B
inhibition. We also tested the limit of the detection of our MS miRNA
for RT. We also tried to replace our inducible promoter for the trigger
plasmids with an constitutive promoter, as a control for the failing
*in vivo* experiments. For the AND gate, we changed the copy
number of the plasmid and the degradation tag after GFP. Plate reader
experiments were performed on the gates. The trigger RNA plasmid for the
LIRA gates were cloned. We also finished the trigger constructs and
transformed everything in the marionette strain with multiple methods
for the MS toehold. As there were no colonies visible, we decided to
stop the *in vivo* experiments.

**Week 18** - 16th of September to
22nd of September

In our final lab week, we did the final tests with the DNAzyme. We
also tried to quantify the toehold output and performed an accuracy
essay for the toehold switch. In addition, the majority of our team was
working on the wiki from this point onward.

The data obtained in the lab on the AND gate was fitted to the data
obtained from our toehold circuit model.

**Week 19** - 23rd of September to
29th of September

While most wiki pages were finalised in this week, we still found
some time to use our circuit model to gain more insights into the
leakiness of the toehold switches and circuits.

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# Engineering

**The main goal of our iGEM project was to design an accurate
and accessible diagnostic test to simplify the diagnosis of multiple
sclerosis (MS). We divided our main goal into sub-parts to work
efficiently. Our team investigated microRNA (miRNA) amplification,
designed an RNA threshold system, and designed and tested *in
vitro* miRNA-targeting toehold switches. Throughout our project, we
used the Design-Build-Test-Learn (DBTL)-cycle to effectively work
towards our goals. The two DBTL-cycles that we would like to highlight
are the cycle of making accurate miRNA-targeting toehold switches and
the cycle of developing an RNA threshold through toehold-mediated strand
displacement.**

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miRNA detection

Threshold detection

* [miRNA-targeting toehold
  switches](#mirna-targeting-toehold-switches)
  + [Introduction](#introduction)
  + [Round 1](#round-1)
  + [Round 2](#round-2)
  + [Round 3](#round-3)

# miRNA-targeting toehold switches

![image](https://static.igem.wiki/teams/5106/engineering/dbtlcycle.webp)

We performed three iterations of the engineering cycle,
focusing on the computational design of miRNA-targeting toehold switches
and their performance *in vitro*. During the first and second
cycle, we further improved the software programme SwitchMi Designer,
built by [the 2021 Uparis\_BME team](https://2021.igem.org/Team%3AUParis_BME/Drylab), to enable the miRNA and toehold
switch to fully hybridise. During the third cycle, we measured the
performance of the designed toehold switches over time and fitted the
results against our model to gain insight on leakiness of the toehold
switch output.

## Introduction

miRADAR focused on miRNAs as potential biomarkers to diagnose MS
patients. In our [test design](https://2024.igem.wiki/wageningenur/design#detection-of-target-mirnas), we
used toehold switches to detect RNA levels that are associated with MS.
For a proof-of-concept of our test, we computationally designed
miRNA-targeting toehold switches and tested the predicted designs in the
lab. We specifically focused on detecting hsa-miR-484 since this miRNA
is shown to be significantly upregulated in patients with
relapsing-remitting MS (RRMS).[1](#ref-regev_identification_2018)
The SwitchMi Designer software tool, built by the [Uparis\_BME team of
2021](https://2021.igem.org/Team%3AUParis_BME/Drylab), generates toehold switch sequences targeting miRNAs. We
predicted three toehold switch sequences for hsa-miR-484 using this tool
and verified these in NUPACK, a web-based software which predicts how
two sequences hybridise based on base-paring and minimum free energy
(MFE) calculations.[2](#ref-fornace_nupack_2022)  NUPACK generates a figure which
shows the predicted secondary structure of the hybridised miRNA and
toehold switch. We found that not all predicted toehold switch sequences
fully anneal to the miRNA (Figure [1](#fig:toeholdSwitchMi)). A
fully annealed miRNA results in a larger difference in MFE between the
native secondary structures of the toehold switch and miRNA, and the
hybridised structure. A large MFE difference makes hybridisation
energetically favourable, which is expected to improve the performance
of the toehold switch *in vitro*. Therefore, we aimed to first
improve the SwitchMi Designer software tool to ensure complete
hybridisation of the miRNA and the toehold switch, before testing the
designs *in vitro*.

![](https://static.igem.wiki/teams/5106/engineering/toeholdswitchmi.webp)

NUPACK hybridisation analysis for hsa-miR-484 and a toehold
switch predicted by SwitchMi Designer. hsa-miR-484 (shaded) partially
anneals to the toehold switch sequence. The colour of the nucleotides
indicates the probability of the nucleotide’s position in the complex
from low (blue) to high (red) probability.

## Round 1

**Design**
The SwitchMi Designer tool allows the user to set restrictions to the
length of the miRNA sequence that anneals to the paired and unpaired
regions of the toehold switch (Figure [2](#fig:toeholdStructure)).
However, we found that defining these parameters leads to predicted
toehold switch sequences that do not always fully anneal to the miRNA.
Therefore, we decided to replace these parameters with a fixed
requirement in the code of the software that takes into account the
total length of the miRNA to anneal to the toehold switch.

![](https://static.igem.wiki/teams/5106/engineering/toeholdstructure.webp)

Schematic representation of a miRNA (green) and toehold
switch (light and dark purple), showing the top- and bottom stem, the
bulge, the loop, and the linker. Our adjusted software scans the miRNA
sequence to find the stem base (orange box), after which the miRNA
sequence is split downstream of the stem base (dotted line). The
downstream miRNA part anneals to the unpaired region of the toehold
switch and the upstream miRNA part anneals to the paired region of the
toehold switch.

**Build**
To design the toehold switch, the SwitchMi Designer tool scans the miRNA
sequence to find two A or T nucleotides and one G or C nucleotide
together, to form the base of the bottom stem (stem base) (Figure [2](#fig:toeholdStructure)). We kept this first step.
Then, we adjusted the software to split the miRNA sequence downstream of
the stem base into two parts (Figure [2](#fig:toeholdStructure)). The 3’ end of the miRNA sequence anneals to the unpaired
region of the toehold switch while the 5’ end of the miRNA sequence
anneals to the paired bottom stem of the toehold switch, thereby opening
up the bottom stem (Figure [2](#fig:toeholdStructure)).
We coded the software to then implement the reverse complement sequence
of both parts of the miRNA in the
toehold switch design.

**Test**

![](https://static.igem.wiki/teams/5106/engineering/barsnupacknobind-1.webp)

NUPACK hybridisation analysis of the trigger miRNA
hsa-miR-484 (Trigger) and three predicted toehold switch sequences
(Toehold 1-3), provided by the improved software. The concentration
(\muM) of each (hybridised) component
or complex present in the system is shown. A higher concentration of a
component or complex indicates a more favourable state.

**Learn**
We found that a very small amount of the miRNAs annealed to the
predicted toehold switches. Complete hybridisation would lead to a lower
MFE compared to the native secondary structures of the toehold switches
and miRNA, and thus a favourable hybridisation reaction. Therefore, it
was assumed that complete hybridisation was not possible with these
toehold switches. Based on our results, we revisited the software. Round
one revealed that the software joined the two reverse complement
sequences of the miRNA parts in an inside-out manner, which led to an
incorrect miRNA-recognition sequence in the toehold switch. We focused
on resolving this error to work towards software that predicts toehold
switch sequences that fully anneal to the miRNA.

## Round 2

**Design**
The software should design toehold switches for the trigger miRNA
sequence by correctly implementing the reverse complementary miRNA
sequence in the predicted toehold switch sequence. We aimed to implement
this requirement while maintaining the [basic
requirements](https://2024.igem.wiki/wageningenur/toehold-model#software-tools-for-the-design-of-mirna-detecting-toehold-switches) of the software.

**Build**
We adjusted the model to first generate the reverse complementary
sequence of the whole miRNA sequence. This sequence was then used as
input for finding the stem base and implementing the sequence in the
paired and unpaired region of the predicted toehold switch sequence.

**Test**
After adapting the software, we again predicted three toehold switch
sequences for hsa-miR-484 using the software tool. NUPACK analysis
showed that all predicted sequences fully annealed to the miRNA sequence
(Figure [4](#fig:ToeholdsNupackFullBind)). In addition, all
miRNA annealed to a toehold switch during hybridisation analysis in
NUPACK (Figure [5](#fig:BarsNupackBind)). This indicated a clear
improvement compared to the toehold switches in round 1, which did not
have a favourable hybridisation reaction, leading to free trigger in
solution (Figure [3](#fig:BarsNupackNoBind)).

![](https://static.igem.wiki/teams/5106/engineering/toeholdsnupackfullbind-1.webp)

NUPACK hybridisation analysis for hsa-miR-484 and three
toehold switches (a,b, and c) predicted by our improved software tool.
hsa-miR-484 (shaded) fully anneals to the toehold switch sequences. The
colour of the nucleotides indicates the probability of the nucleotide’s
position in the complex from low (blue) to high (red).

![](https://static.igem.wiki/teams/5106/engineering/barsnupackbind-1.webp)

NUPACK hybridisation analysis of the trigger miRNA
hsa-miR-484 (Trigger) with three predicted toehold switch sequences
(Toehold 1-3), provided by the improved software. The concentration (nM)
of each complex present in the system is shown.

**Learn**
Round two revealed that our software can successfully predict toehold
switch sequences that fully anneal to the miRNA hsa-miR-484 and that the
hybridisation reaction is energetically favourable. Based on these
findings we decided to test our toehold switches *in vitro*, to
confirm their ability to be accurately activated by annealing to the
trigger miRNA.

## Round 3

**Design**
We tested two of our three predicted toehold switches in the lab, which
we refer to as toehold switch A (THA) [(BBa\_K5106001)](https://parts.igem.org/Part%3ABBa_K5106001) and
toehold switch B (THB) [(BBa\_K5106002)](https://parts.igem.org/Part%3ABBa_K5106002). Our
third *in silico* tested toehold switch had a less desired native
secondary structure. Here, we aimed to design an accurate toehold switch
with a fast visible output signal, and a high signal-to-noise ratio. The
toehold switches were tested *in vitro*. This required a T7
promoter ([BBa\_K1614000](https://parts.igem.org/Part%3ABBa_K1614000)) upstream of the toehold switch sequence, a reporter gene
downstream of the toehold switch, and a downstream T7 terminator([BBa\_K5106019](https://parts.igem.org/Part%3ABBa_K5106019)). *LacZ*([BBa\_K1444017](https://parts.igem.org/Part%3ABBa_K1444017))
was chosen as reporter gene since it provides a clear visible output
signal which promotes the [accessibility](https://2024.igem.wiki/wageningenur/description#accessible) of
the test.

**Build**
 For each toehold switch we built a [plasmid](https://2024.igem.wiki/wageningenur/experiments#cell-free-in-vitro-test)
containing the composite parts [(BBa\_K5106004)](https://parts.igem.org/Part%3ABBa_K5106004) and
[(BBa\_K5106005)](https://parts.igem.org/Part%3ABBa_K5106005)
for THA and THB respectively, under control of the T7 promoter and T7
terminator. We used [the PURE cell-free system](https://2024.igem.wiki/wageningenur/experiments#cell-free-in-vitro-test) to test the toehold
switches *in vitro.* A negative control which did not contain the
trigger miRNA was added to investigate the possible leakiness of the
system.

**Test**

![](https://static.igem.wiki/teams/5106/engineering/graph-th-ab-570-2.svg)

Performance over time of our designed toehold switches THA
and THB *in vitro*. Absorbance at 570 nm over time (min) for
cell-free reactions with THA and THB constructs, with (+) and without
(-) miRNA trigger added. \*\*: *p* < 0.01.

**Learn**
Round three revealed that THA and THB can be activated by the trigger miRNA to produce \beta-galactosidase, although
THB showed more effective activation. THB was however
leaky since the toehold switch without trigger miRNA also showed an
increase in absorbance. This means that translation of *LacZ* is possible
without miRNA trigger present. We wrote a [model](https://2024.igem.wiki/wageningenur/model#data-fitting) to quantify
the leakiness of THB and fit the model to the results obtained for THB,
with and without trigger miRNA. We found that the 35.8 % of the signal
output of THB is caused by leakiness. To further improve our toehold
switches, we aim to minimise the leakiness in the future. This could be
done by optimising the toehold switch structure to get a more
energetically favourable native secondary structure of the toehold
switch. The possibility of the toehold switch linearising by chance,
enabling translation, could then be minimised. This may be achieved by
elongating the stem of the toehold switch for instance.

References

(1)

Regev, K.; Healy, B. C.; Paul, A.; Diaz-Cruz,
C.; Mazzola, M. A.; Raheja, R.; Glanz, B. I.; Kivisäkk, P.; Chitnis, T.;
Jagodic, M.; Piehl, F.; Olsson, T.; Khademi, M.; Hauser, S.; Oksenberg,
J.; Khoury, S. J.; Weiner, H. L.; Gandhi, R. Identification of
MS-Specific Serum miRNAs in an
International Multicenter Study. *Neurology Neuroimmunology &
Neuroinflammation* **2018**, *5* (5), e491. <https://doi.org/10.1212/NXI.0000000000000491>.

(2)

Fornace, M. E.; Huang, J.; Newman, C. T.;
Porubsky, N. J.; Pierce, M. B.; Pierce, N. A. NUPACK:
Analysis and Design of Nucleic
Acid Structures, Devices, and
Systems, 2022. <https://doi.org/10.26434/chemrxiv-2022-xv98l>.

[Proof of concept](proof-of-concept)

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# Experiments

# Amplification and threshold detection of miRNAs

We discuss why amplification is necessary and what the steps/
experiments involved to amplify the specific miRNA sequences are. We also
discuss the importance of the threshold system and how we built it.

[Click to read more...](#amplification)

# Detection of target miRNAs

We discuss our strategy to detect miRNAs from the threshold system
using toehold switches and the AND gate. We also explain the
construction strategies used for making our detection system
modular.

[Click to read more...](#toehold-switches-mirna-detection-system)

# Paper test platform and output signal

We discuss details of experiments carried out to test our detection
system *in vitro* on a paper-based platform.

[Click to read more...](#cell-free-in-vitro-test)

{% endblock %}
{% block page\_content %}

* [**Amplification
  and threshold detection**](#amplification-and-threshold-detection-system)
  + [**Amplification**](#amplification)
  + [**Threshold detection
    system**](#threshold-detection-system)
* [**Toehold
  switches: miRNA Detection System**](#toehold-switches-mirna-detection-system)
  + [**The
    Backbone**](#the-backbone)
  + [**The
    toehold**](#the-toehold)
  + [The AND
    gates](#the-and-gates)
  + [Flow of
    experiments](#flow-of-experiments)
* [Cell free *in-vitro* test](#cell-free-in-vitro-test)
  + [Paper-based testing](#paper-based-testing)
  + [*In vitro*
    tests](#in-vitro-tests)
  + [DNAzymes](#dnazymes)

In patients with autoimmune diseases, the levels of specific
microRNAs(miRNAs) in the blood are different. The concentrations of
these miRNAs can increase or decrease during the onset of the disease.
These changes in concentration can be used to diagnose patients having
an autoimmune disease.[1](#ref-junker_emerging_2011)
We planned to develop a minimally invasive, cell-free paper-based
diagnostic tool that can detect these disease-specific miRNAs. For this,
we divided our project into three sub-categories as mentioned in the [description](https://2024.igem.wiki/wageningenur/description#toehold-switches).
The categories are (i) specific amplification and threshold detection of
miRNAs, (ii) a modular tool to detect miRNAs(our detection system), and
(iii) the design of the test platform and signal output. Below you can
find the experimental strategy that we used in each of the three
sections to make our diagnostic tool turn from an idea to a reality.

# **Amplification and threshold detection system**

## **Amplification**

We understood from our Human practices that similar miRNAs can change
concentrations in multiple diseases. That’s why, we first used a [machine
learning algorithm](https://2024.igem.wiki/wageningenur/dry-lab-overview#finding-the-combination-of-mirnas-specific-for-rrms) to identify the miRNA combination that can
distinguish relapsing-remitting MS patients from healthy people.

The concentrations of miRNAs in the blood range from fM to pM.[2](#ref-gines_emerging_2020)
To enable our toehold switches to detect the selected miRNAs, we aim to
incorporate a sequence-specific amplification step. We decided to use
Nucleic Acid Sequence-Based Amplification [(NASBA)](https://2024.igem.wiki/wageningenur/design#amplification-and-threshold-selection-of-mirnas) as our
amplification technique. NASBA produces a large amount of RNA, which
consists of the selected miRNA sequence, flanked by universal sequences
on both sides. This product is obtained through a cycle of 2 reactions:
reverse transcription and *in vitro* transcription. We wanted to
prove that the two NASBA reactions work to amplify our miRNAs.

The [reverse
transcription](https://2024.igem.wiki/wageningenur/protocols#In-vivo)  was performed first. During reverse transcription, the
Reverse Transcriptase (RT) enzyme uses an RNA template to produce a
complementary DNA (cDNA) strand. Subsequently, the RNase H domain of the
RT degrades the miRNA in the RNA/DNA duplex (Figure [1a](#fig:RT1)). We designed
a reverse stem-loop primer based on Mader et al., 2012, that has a
six-nucleotide overlap with our miRNA ‘has-miR-484’; which is found to
be upregulated during relapsing-remitting MS.[3](#ref-mader_universal_2012)
The annealing region of this primer serves as the starting point for the
synthesis of the cDNA strand by RT. To confirm that the reverse
transcription was successful, we performed a [PCR](https://2024.igem.wiki/wageningenur/protocols)  with the
reverse transcription product as a template. A PCR product can be
obtained only if cDNA was produced during the reverse transcription
reaction (Figure [1b](#fig:RT1)). In addition, we performed an assay to
determine the limit of detection (LOD) for reverse transcription. Since
this is the first essential step in NASBA, it is valuable to find the
lowest miRNA concentration that allows the reaction to work. For this
assay, we performed reverse transcription in triplets with miRNA
concentrations ranging from 70 nM to 7 fM, while the stem-loop primer
concentration was held constant. The reaction products were used as a
template for a PCR. We loaded the samples on the gel for analysis of the
limit of detection for reverse transcription.

![](https://static.igem.wiki/teams/5106/experiments/rt1-1.webp)

Schematic of reverse transcription experiments. a;
After, the stem-loop primer anneals to the selected miRNA, the reverse
transcriptase synthesises a cDNA strand, starting from the annealing
point of the stem-loop primer (light blue) to the miRNA (pink). The
RNase H domain degrades the miRNA in the DNA/RNA duplex. b; PCR is
performed with the reverse transcription product as a template. The
forward primer contains a T7 promoter sequence for in vitro
transcription (dark green).

Secondly, we tested the *in vitro* transcription. During
*in vitro*  transcription, a T7 RNA polymerase transcribes a DNA
template, starting from the T7 promoter sequence. In the one-pot NASBA
reaction, this promoter sequence is incorporated in front of the miRNA
sequence by the reverse transcriptase enzyme when adding a primer with
the T7 promoter to the mixture (Figure [2](#fig:NASBA)). Since we focused on the sub-reactions,
we prepared the DNA template separately. We performed the PCR using the
cDNA strand as a template, with a forward primer including the T7
promoter sequence, and a reverse primer complementary to the cDNA strand
(Figure [1b](#fig:RT1)). During this PCR, the T7 promoter
sequence was added to the cDNA strand upstream of the miRNA sequence.
Then, we performed the *in vitro* transcription by adding the
obtained DNA template to the *in vitro* transcription reaction
mixture with the T7 RNA polymerase. After overnight incubation, we
carried out [denaturing gel
electrophores](https://2024.igem.wiki/wageningenur/protocols) and concentration measurements to confirm if the
*in vitro* transcription was successful. Finally, we studied the
effect of [DNase
I treatment](https://2024.igem.wiki/wageningenur/protocols) and [RNA
purification](https://2024.igem.wiki/wageningenur/protocols) on RNA stability and yield, by loading a part of the
product on gel after each step. These downstream processes may be
necessary for the use of the amplified miRNA sequence in our test.

![](https://static.igem.wiki/teams/5106/experiments/rt2-2.webp)

NASBA method: Reverse transcription followed by in vitro
transcription

## **Threshold detection system**

Once the specific sequence is amplified, the concentration of miRNA
is enough to be detected by the detection system. However, the miRNAs
are always present in the blood, and the only difference between MS
patients and healthy individuals is the change in concentration (Figure
[3a](#fig:TMSD1)). To detect upregulated levels of
miRNAs during a disease, we must detect them above a certain threshold.
Toehold switches, our detection systems, resulting in an output signal
that scales linearly with the input signal (Figure [3b](#fig:TMSD1)). Thus, we
need a tuneable system that gives an output only above a certain
threshold concentration, before we can use it in our detection
circuit.

![](https://static.igem.wiki/teams/5106/experiments/tmsd1-1.webp)

a: Schematic overview of RNA dysregulation, and the
effect of a threshold on the output of the system.; b: Comparison
between a linear response from a toehold switch, and what an ideal
binary signal would look like.

One method to construct such a tuneable threshold is by utilizing the
properties of Toehold Mediated Strand Displacement [(TMSD)](https://2024.igem.wiki/wageningenur/design#amplification-and-threshold-selection-of-mirnas). TMSD is
based on the concept that two annealed RNAs can be replaced by another
molecule that has a higher affinity. For TMSD, three RNA molecules are
needed: the molecule of interest A (which is amplified in the previous
step), and two partially complementary strands B(antisense strand) and
C(Input for detection system). Initially, B and C are bound. Besides
this, there is an excess of B present equivalent to the threshold limit.
Once the amplified product A is added to the reaction, it starts binding
to free B. As there is no displacement of C, there is no input for the
detection system, hence no output. Once all the free B’s are bound by A,
i.e. concentration of A is above the threshold, A starts displacing C
because A has a stronger affinity to B. Displaced C will trigger the
detection system and output will be observed (Figure [4](#fig:TMSD2)). In this way, a threshold can be added,
based on the amount of B and C RNA present, that results in a sharp
transition.

![](https://static.igem.wiki/teams/5106/experiments/tmsd2-2.webp)

Schematic overview of toehold mediated strand
displacement with labels A, B, C.

In the chosen system, the output C was visualised by using an
inducible spinach aptamer (D) produced by Wang et al. (2023).[4](#ref-wang_switchable_2023)
Spinach is an RNA structure that can stabilise the side group of a
fluorophore molecule (DFHBI), which allows it to fluoresce. The
inducible spinach structure (Figure [5](#fig:TMSD3)) contains a
misfolded spinach aptamer, that upon binding a trigger (in this case C),
can refold into a fluorescing structure that can be quantified in a
fluorescence microplate reader. By measuring the fluorescence in a plate
reader, the output of C can be detected. Since the structures of D and C
are set due to the refolding requirements of D, A and B had to be
adapted to comply with these requirements.

![](https://static.igem.wiki/teams/5106/experiments/triggerable-spinach-sequence-1.webp)

Inducible spinach sequence from Wang et al. 2023.[4](#ref-wang_switchable_2023)
Part of the spinach aptamer sequence shown in green is trapped in a
hairpin in the normal state. When a trigger RNA (C) is present, the
structure (D) refolds and the spinach region can refold into a
fluorescent form.

[NUPACK](https://www.nupack.org/) was used to model affinities of desired pairs and prevent
undesired binding between unrelated RNA sequences. Based on a template
form of D (an inducible spinach aptamer in this context) and the
associated trigger C from a paper by Wang et al. (2023),[4](#ref-wang_switchable_2023)
the template parts A and B were designed. Based on sequence C, an
antisense structure B was designed with a greater affinity to BC than CD
and can generate a sharp threshold with binding affinities that follow
AB>BC>CD. C was elongated to improve the hybridisation between AB
and consequent TMSD. Strand B was designed complementary to C, with an
additional toehold and elongated region to hybridise with A (Figure [6](#fig:TMSD4)).

![](https://static.igem.wiki/teams/5106/experiments/tmsd4-3.webp)

NUPACK thermodynamic equilibrium complex prediction of
RNA structures at 25C. a: This equilibration indicates the ON state. In
this equilibration the initial concentration of A is 2 \muM, B 1 \muM, and C and D are 0,5 \muM. This graph shows that A binds to all B,
releasing C to bind with D, as can be seen in green. b: This represents
the OFF state. The initial concentration of A is 1 \muM, B is 2 \muM, and C and D are again at 0,5 \muM. Since A is not present in excess, C
remains bound to B, and cannot bind to D. D remains unbound to C as can
be seen in grey, and therefore does not give any signal (in this example
fluorescence).

After we designed and confirmed the sequences 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 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.

# **Toehold switches: miRNA Detection System**

The detection of miRNA is done by toehold switches and logic gates. In our
final test, the output from the threshold system (C) will act as the
trigger for the detection system.

We aimed to build and test these toehold switches *in vitro*.
We did this by cloning the toeholds and gates into a plasmid and testing
expression *in vitro*. Firstly, we designed the plasmid
containing toeholds and gates *in silico*. We wanted to make a single
standardized modular plasmid. Doing so would help replace the toehold
switches with other toehold switches detecting different miRNAs specific
for other diseases. We made some cloning strategies to make this
possible. The plasmid was divided into two parts:
1. The backbone with a
reporter gene (*lacZ*)
2. The detection system (toeholds and AND gate)
(Figure [7](#fig:T1))

![](https://static.igem.wiki/teams/5106/experiments/t1.webp)

Modular plasmid construction design for the detection
system. The backbone will consist of and be amplified with T7 promoter
and LacZ at 5’ and 3’ ends respectively. So, the 4nt overhang sequences
for 5’ and 3’ ends at the end of T7 and the AND gate were selected as
modular single-stranded DNA overhangs.

We considered the backbone with *lacZ* gene as the constant
part and the toehold switches as a variable part which can be replaced
with another fragment if required. To make the plasmid modular, the
single-stranded DNA overhangs (Sticky ends) of the backbone should
remain constant. *lacZ* is a part of the plasmid backbone and is
placed immediately next to the toehold switches. So, the first four
nucleotides of *lacZ* were decided as the modular single-stranded
DNA overhangs on the 3’ end of the AND gate. The 5’ end of the AND gate
consists of the T7 promoter. As the T7 promoter is required for *in
vitro*  testing, it will be a fixed part of the backbone (Figure [8](#fig:Insilico)). So, the 4nt
overhang sequences for 5’ and 3’ ends at the end of T7 and the AND gate
were selected as modular single-stranded DNA overhangs (Marked blue T7
and red for *lacZ*  in (Figure [7](#fig:T1))).

![](https://static.igem.wiki/teams/5106/experiments/t2.webp)

Map of the constructed AND gate plasmid. The plasmid
contains the AND gate in between a T7 promoter and terminator, followed
by a lacZ reporter gene. It also contains a kanamycin resistance gene as
a selection marker.

## **The Backbone**

Initially, we did not have the backbone with the required constructs
like T7 promoter, T7 terminator, and *lacZ* gene. So, we
amplified them separately and later added them to the backbone along
with the toehold switches. As the T7 promoter was small, we amplified it
along with the backbone. The T7 terminator and the *lacZ* gene
were amplified separately. We did the amplification using PCR. All
primers were flanked by BsaI restriction sites so that the amplified
fragments could directly be used during the Golden Gate reaction.

## **The toehold**

We constructed the toeholds using overlap extension PCR (OE-PCR) to
test them *in vitro*. The whole construct was produced by
designing four primers, with partial overlap. To perform the OE-PCR,
these four primers are used at the same time using a [Q5 PCR program](https://2024.igem.wiki/wageningenur/protocols), with a Tm of 65°C, followed by DNA extraction of the product.
The same PCR program is then used again on the product, together with
the first and last primer, to amplify the whole construct. At the same
time, the backbone is amplified. Both the construct and the backbone are
flanked by BsaI sites. The region where the enzyme cuts overlaps with
the backbone. Thus, the construct could directly be used in a golden
gate reaction with a linearized SEVA backbone (Figure [9](#fig:T3)).

![](https://static.igem.wiki/teams/5106/experiments/646e486e-6e39-42b5-9f63-a3d6de69ffb5.webp)

Example of toehold construct and overlap extension PCR
primers.

We designed the toeholds specific to hsa-miR-484. This miRNA was
shown to be upregulated in the blood of multiple sclerosis patients.
Three toeholds were designed to detect this miRNA namely Toehold A,
Toehold B, and Toehold C. Toehold A and B were designed using our
[toehold designer](https://2024.igem.wiki/wageningenur/toehold-model), and Toehold C was designed using only NUPACK. In the
lab, we used a similar method of construction for all the toeholds as
mentioned above.

## The AND gates

During the onset of a disease, several disease-specific miRNAs vary
in their concentration. To increase the accuracy of our test, detecting
multiple disease-specific miRNAs is advantageous. Therefore, we want to
use toehold switches performing logic gate functions such as an AND
gate. These AND gates can detect multiple miRNAs and express output only
in the presence of all the miRNAs. For our test, we constructed a
two-input AND gate that detects two different trigger RNAs.

A two-input AND gate is a single toehold switch that gets activated by
two trigger RNAs. The input for the respective toehold switch is a
combination of two input RNA molecules that are partially complimentary
to each other. These partially complementary inputs hybridize to form an
input trigger RNA that can anneal to the trigger binding region of the
toehold switch. Once the trigger RNA is bound to the trigger binding
region, the loop in the toehold switch opens up and the RBS is available
for the ribosome to bind, allowing gene expression. The absence of any
one trigger RNA will not lead to the unfolding of the loop structure,
thus causing no gene translation (Figure [10](#fig:AND)).

![](https://static.igem.wiki/teams/5106/experiments/andgatemechanism.webp)

Working of AND gate from Green et al 2017.[5](#ref-green_complex_2017) Input A1 and A2 partially hybridize with each other forming a single trigger RNA. This trigger RNA binds to the trigger binding region of the toehold and opens up the loop in the toehold switch exposing the RBS. This allows gene expression.

Instead of directly ordering the AND gate, we constructed it using Q5
high-fidelity PCR. The AND gate was divided into two parts. Both the
parts were amplified with Q5 PCR separately. Primers were constructed in
such a way that the 3’ end of the first part and the 5’ end of the
second part have the same single-stranded overhangs. Thus, after the
BsaI cut during a Golden gate reaction, these single-stranded DNA
overhangs can anneal making one complete AND gate (Figure [11](#fig:T4)).

![](https://static.igem.wiki/teams/5106/experiments/t4.webp)

Construction of AND gate. AND gate was divided into two
parts. Each part was amplified separately. We constructed the primers
were constructed in a way that complementary overhangs would be created
after digestion by BsaI enzymes (marked as red). This would allow both
parts to anneal to each other.

## Flow of experiments

Two parts of the AND gate and the backbone along with the reporter
gene were amplified using Q5 high-fidelity PCR. DNA was extracted using
Gel electrophoresis and purified using Zymoclean™ Gel DNA Recovery Kit.
All amplified fragments were ligated using the Golden Gate method. We
directly ordered trigger RNAs for testing the toeholds and AND gate
*in vitro*  (Figure [12](#fig:kette)).

![](https://static.igem.wiki/teams/5106/experiments/plasmid-kette.webp)

Construction of AND gate. AND gate is divided into two parts with overhangs complementary to each other. The ends of both parts have complimentary overhangs with the backbone.

# Cell free *in-vitro* test

Our diagnostic test relies on cell-free transcription and
translation. To test the functionality of our detection system, we used
the commercial PURExpress *In Vitro* Protein Synthesis Kit [(NEB
E6800)](https://www.neb.com/en/products/e6800-purexpress-invitro-protein-synthesis-kit) to perform *in vitro* experiments. The kit contains
two solutions and control DHFR Plasmid. This is a reconstituted form of
gene expression that works by isolating the necessary components, such
as ribosomes and amino acids, purifying them, and assembling them into a
reaction mixture.[6](#ref-cui_cell-free_nodate)
It consists of two solutions, which can be supplemented with other
reagents like substrate or RNase inhibitor. PURExpress is based on the
T7 RNA Polymerase, which has a high specificity for the T7 bacteriophage
promoter. To test this system, a plasmid was assembled containing the T7
promoter, the *lacZ* gene and the T7 terminator. *lacZ*
was selected as the output gene (Figure [13](#fig:Map)). This gene encodes the enzyme \beta-galactosidase, which catalyses the
hydrolysis of the yellow Chlorophenol Red-b-D-Galactopyranoside (CPRG)
into Chlorophenol red. If the gene is expressed, the colour changes from
yellow to purple, which can be measured at 570 nm.

![](https://static.igem.wiki/teams/5106/experiments/map-of-the-constructed-positive-1.webp)

Map of the constructed positive control plasmid. The
plasmid contains a T7 promoter and terminator, as well as an ampicillin
resistance gene as a selection marker

The plasmid encoding \beta-galactosidase was assembled using HiFi
Assembly (NEB, E2621), with the DHFR control plasmid from the PURExpress
kit as the backbone. We designed primers to create homology arms for
both the backbone and the full LacZ gene. The plasmid was assembled and
transformed into E. coli DH10\beta and
miniprepped.

The PURExpress reactions were carried out according to the
manufacturer’s instructions, but we scaled down the volume from 25 \muL to 2 \muL. By working in smaller volumes, fewer
materials are required per test. This means that the production is more
cost-efficient. To prevent the breakdown of RNA, an RNase inhibitor was
supplemented (Promega, 2515). Additionally, the substrate CPRG was added
to the reaction mixture. The reaction was tested at the recommended 37°C
and at room temperature. This same method was used to test toehold
switches and logic gates.

## Paper-based testing

Our test platform is paper-based. A paper-based method has several
advantages, such as low production costs, easy storage, and stability of
biochemical components.[7](#ref-pardee_paper-based_2014) Therefore, we wanted to
integrate cell-free expression with a paper matrix. This method was
described previously by Pardee et al. (2014), which we also used for our
test.[8](#ref-park_detection_2021)
First, the cellulose filter paper is treated with 5% BSA, to prevent any
unwanted side reactions due to pH-dependent charges. After drying, the
filter paper is cut into 2 mm discs using a biopsy punch, and these
disks are placed in a 384-well plate. Next, cell-free reactions are
assembled by combining the two PURExpress solutions, containing the
cell-free reaction components, with the substrate, the DNA template, and
the RNase inhibitor. These reactions are applied to the discs. Then, the
plate is dipped in liquid nitrogen to flash-freeze the discs, after
which it is freeze-dried overnight. After 24h, the discs can be
rehydrated with nuclease-free water with or without trigger miRNA. The
plate is then incubated at 37°C for two hours. Colourimetric output can
be seen by the eye; a negative result will remain yellow, and a positive
result will be purple (Figure [14](#fig:Workflow)).

![](https://static.igem.wiki/teams/5106/experiments/workflow-of-miradar-test-platform.webp)

Workflow of the preparation of the miRADAR test
platform. First, the necessary transcription and translation components
(ATP, ribosomes, nucleotides, amino acids and T7 RNA polymerase are
shown) are combined with the logic circuit and applied to a paper disc,
supplemented with the substrate CPRG and RNase inhibitor. The disc is
then freeze-dried for storage and distribution. To reactivate the test,
the discs are rehydrated with amplified miRNA after which the reaction
proceeds.

By assembling the tests in 384-well plates, they can easily be stored
and distributed at room temperature. Furthermore, no biotic components
are present, contributing to the biosafety of the product.

## *In vitro* tests

To evaluate the performance of the MS-specific toehold switches and
the AND gate, we performed *in vitro*  tests over time. As
explained previously, the toehold switches and the AND gate were put
under the control of the T7 promoter and terminator. Synthetic input RNA
was added to imitate the final testing environment. The lacZ gene (Figure [15](#fig:CF1)) was
used as the output protein for the toehold switch and the AND gate
(Figure [11](#fig:T4))(Figure [15](#fig:CF1)).

![](https://static.igem.wiki/teams/5106/experiments/cf1.webp)

Map of the constructed toehold switch plasmid. The
plasmid contains the toehold switch in between a T7 promoter and
terminator, followed by a lacZ reporter gene. It also contains a
kanamycin resistance gene as a selection marker.

Cell-free reactions were set up in a 5 \muL volume. The toehold switch and AND gate
plasmids were supplied at a final concentration of   30 ng. For all
samples, 1 \muL of trigger RNA was
added at 5 \muM. For the AND gate, 1
\muL was added to both triggers. The
reactions were assembled in PCR tubes and incubated at 37°C. At eight
time points, a 0.5 \muL sample was
taken from the reactions and diluted 151x in a 96-well plate. The
absorbance was measured at 570 nm with a plate reader.

## DNAzymes

As an alternative to the conventional enzymatic reporter signal, we
investigated the use of DNAzymes, short ssDNA oligonucleotides that form
catalytically active structures. Especially the G-quadruplex (G4) /
hemin DNAzyme has been gaining increasing recognition in
biosensing/nucleic acid sensing platforms. This quadruplex contains four
stretches of three guanines, a G4 sequence, and forms a tertiary
structure which is able to interact with hemin as a cofactor (Figure [16](#fig:D1)). It acts as a horseradish
peroxidase-mimic, catalysing H2O2-mediated oxidations of various
substrates. For colourimetric measurements, 2,2-azino-bis
(3-ethylbenzothiazoline-6-sulfonic acid) can be used to turn the
colourless ABTS^2- to the
green-coloured ABTS^- radical.

![](https://static.igem.wiki/teams/5106/experiments/d1.webp)

Illustration of G-quadruplex. Blue circles indicate the
stretches of guanines, that form a tertiary structure that allows the
quadruplex to interact with the cofactor hemin.

A common approach for designing such a DNAzyme-biosensor is by
splitting the G4 sequence, usually in a 1:3 fashion, and extending these
with probe sequences that hybridise with their target adjacently, which
thereupon reconstitutes the DNAzyme (Figure [17](#fig:D2)).

![](https://static.igem.wiki/teams/5106/experiments/d2.webp)

Mechanism of DNAzymes. Trigger (mi)RNA opens up the
stem-loop of the toehold switch, allowing the G4 probes to hybridise
with the unfolded RNA structure.

For the detection of the activated toehold switch, we designed our
probes by splitting the EAD2 sequence [(BBa-K3343000)](https://parts.igem.org/Part%3ABBa_K3343000)  and
attaching 15 – 20 nucleotides complementary to the sequences that are
normally sequestered in the hairpin / stem-loop, namely the RBS and
start codon (Figure [18](#fig:D3)).

With this approach, we could theoretically at least leave out the
protein translation aspects of the cell-free test which is the most
component-heavy and energy-dependent part of a PURE/cell-free
transcription and translation (TXTL) system. The toehold switch protocol
is to be followed suit, but without the translation components of the
cell-free test. The *in vitro* transcription (IVT) can be
conducted with the switch DNA template, trigger RNA, and probes
present.

![](https://static.igem.wiki/teams/5106/experiments/d3.webp)

NUPACK model results showing correct hybridisation of
nucleic acid strands at equal concentrations at standard conditions. The
toehold switch is shown in orange; Trigger miRNA (hsa-miR-484) is shown
in blue; G4 probes are shown in green. Note: this only accounts for RNA:
RNA hybrids, not RNA: DNA.

References

(1)

Junker, A.; Hohlfeld, R.; Meinl, E. The
Emerging Role of microRNAs in Multiple
Sclerosis. *Nat Rev Neurol* **2011**, *7*
(1), 56–59. <https://doi.org/10.1038/nrneurol.2010.179>.

(2)

Gines, G.; Menezes, R.; Xiao, W.; Rondelez, Y.;
Taly, V. Emerging Isothermal Amplification Technologies for microRNA Biosensing: Applications to Liquid
Biopsies. *Molecular Aspects of Medicine* **2020**,
*72*, 100832. <https://doi.org/10.1016/j.mam.2019.11.002>.

(3)

Mader, A.; Riehle, U.; Brandstetter, T.;
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[Wet lab results](wet-lab-results)

[Engineering](engineering)

{% endblock %}