iGEM 2022 — Team thessaloniki

#### iGEM Thessaloniki 2022

###### 20 Dec.

![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-note-21.jpeg)

Our first meeting as
 a new team & first
brainstorming session

###### Weeks: 1-11

Brainstorming Sessions
 First meetings with professors
Guidance from former igemmers

![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-note-13.jpg)

###### Week: 12

First meeting w. PI

![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-note-19.png)

###### Weeks: 13-14

Meeting with the Rector of A.U.Th.
 Our first workshop
Meetings with professors

![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-note-18.jpg)
![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-note-17.jpg)

###### Weeks: 15-16

Meetings, meetings,
 meetings,
 more iggemers

![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-note-3.png)

###### Weeks: 17-20

First co-PI meeting,
 Lab training, meetings

![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessalonik22-labtraining.jpg)
![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-note-16.jpg)

###### Week: 21

Lab training, More meetings

![]( https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-note-7.jpg)

###### Weeks: 22-24

First meeting with mentor

First conferences & events, and Tv interviews

![]( https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-mentor.png)
![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-note-12.jpeg)

###### Weeks: 25-27

First meeting with iGEM Crete

###### Weeks: 28-30

First meeting with iGEM Toulouse

Greek Meet-up:

![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-note-2.png)
![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-aegean.png)

###### Weeks: 31-34

Interlab Experiments

Troubleshooting &
 meetings with professionals

![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-note-4.jpg)

###### Weeks: 35-38

First Theriac experiments

Troubleshooting meetings
 with professors

![](https://static.igem.wiki/teams/4141/wiki/wiki/notebook/thessaloniki22-note-6.png)

###### Weeks: 39-42

Theriac Experiments

Researcher's Night & Astroparty

![]( https://static.igem.wiki/teams/4141/wiki/wiki/education-communication/thessalonik22-researcher-snight3.jpg)
![]( https://static.igem.wiki/teams/4141/wiki/wiki/education-communication/astroparty-psari-06536.jpg)

###### Weeks: 43-45

Wiki Freeze

#### Jamboree

{% extends "layout2.html" %}
{% block title %}Engineering Success{% endblock %}
{% block page\_content %}

* [Introduction](#intro)
* [Engineering Cycle 1](#cycle1)
  + [Design](#design)
  + [Build](#build)
  + [Test](#test)
  + [Learn](#learn)
* [Engineering Cycle 2](#cycle2)
  + [Design](#design2)
  + [Build](#build2)
  + [Test](#test2)
  + [Learn](#learn2)

Engineering Success

## Introduction

Engineering success is a vital part of our project this year. It ensembles the basis of the design of our hairpins and the procedures we used to test them.

Our engineering cycle started with a simple idea:

* Design: of the Y-shaped hairpins - the molecules responsible for the detection of two cancer biomarkers (two microRNAs)
* Build: the Y-shaped hairpins using our algorithm
* Test: the Y-shaped hairpins using microRNAs and monitoring their response and stability
* Learn: analyze the results and improve our Y-shaped hairpins

In our Y hairpins, a bulge of 4 unpaired base pairs was designed in order to create the desired secondary structure with the necessary stability. We ordered our sequences and conducted native gel electrophoresis to check our design in the lab. The results showed that the Y-shaped hairpins with the bulge of 4 bases had low stability, opening in the presence of just one microRNA. The high leakage significantly reduces the specificity of Theriac which would cause severe side effects if administrated in patients. Thus, we redesigned the Y hairpin with fewer unpaired bases (3 bases bulge) and the results were much better.

![](https://static.igem.wiki/teams/4141/wiki/wiki/engineering-success/engineering-cycle.png)
Figure 1. The engineering cycles on the Y structure hairpins

## Engineering Cycle 1

## Design

Theriac consists of two types of hairpins. The Y-hairpin is designed to detect 2 microRNAs and release an initiator, a single-strand DNA that activates Theriac’s next set of hairpins (HCR-hairpins). Only when both of the miRNAs are detected, the reaction is triggered (Figure 2).

![](https://static.igem.wiki/teams/4141/wiki/wiki/engineering-success/thessaloniki22-ymechanism.jpg)
Figure 2. The reaction of Y-shaped hairpin with the 2 miRNAs

![](https://static.igem.wiki/teams/4141/wiki/wiki/model/design-of-the-molecules/thessaloniki22-model-design-1.png)
Figure 3. The structure of Y-hairpin

The design of the Y-hairpin is based on the literature review and the parameter of their stability. This three-way DNA junction, as it is shown in Figure 3, has on the Y1 strand of DNA a group of unpaired bases, called bulge. Bulge was added in order to increase the Y hairpin's stability in presence of metal ions. In our first attempt at Y-hairpin design, we selected the length of the bulge to be equal to 4. We did bibliographic research, which demonstrated that the secondary structure of the hairpins would be stable enough with 4 bulges. After our research, we designed 2 different Y-shaped hairpins each one with 4 bulges.

## Build

The design was made with the creation of a python code, which computes for different combinations of DNA bases (adenine, cytosine, guanine and thymine) on the bulge, the stability of the Y-hairpin structure by the determination of Gibbs energy with Nupack package. The more negative the energy Gibbs is, the more stable is the molecule. The code has as output the most stable Y hairpin, with the more negative Gibbs energy. One of the main parameters of the build step of the Y hairpin is the length of the bulge. All the design parameters can be found on our
 [**model page.**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27model%27%29%20%7D%7D) Defining the length of the bulge as 4 in the Y hairpins, using the online platform of Nupack, we get the two structures below (Figure 4 and Figure 5). These two differ on the bases that consist the bulge.

![](https://static.igem.wiki/teams/4141/wiki/wiki/engineering-success/thessalonik22-figure-4-y-hairpin-named-ya-with-bulge-of-4-bases.jpg)
Figure 4. Y-hairpin (named Ya) with bulge of 4 bases

![](https://static.igem.wiki/teams/4141/wiki/wiki/engineering-success/thessaloniki22-figure-5-y-hairpin-named-yb-with-bulge-of-4-bases.jpg)
Figure 5. Y-hairpin (named Yb) with bulge of 4 bases

## Test

The goal of our lab experiments was to test if the designed Υ-hairpins had high stability, especially while being hybridised with the complementary microRNAs. For this purpose, we used native gel electrophoresis, where we loaded the 2 different Y-hairpins with different conditions:

* Y-hairpin alone
* Y-hairpin with miR-21
* Y-hairpin with miR-10b
* Y-hairpin with both microRNAs

For the native gel electrophoresis, we used polyacrylamide gel, which is used in order to separate short nucleic acids with a range of 1-1000 base pairs

We also chose native electrophoresis, which means we didn’t use any denaturant. Denaturants added to the gel make nucleic acids single-stranded and since we aimed to observe the secondary structure of the Y-hairpins after specific reactions, we used native/non-denaturing electrophoresis. The strategy we followed was to load the wells with the 4 above-mentioned conditions for each hairpin.

## Learn

During the separation of the nucleic acids that were loaded into the gel’s wells, we observed in some cases leakage. The results of our experiments demonstrated that the Y hairpins with a bulge of 4 bases are not stable enough. When both Ya and Yb were incubated with miR-10b, the secondary structure of the Y hairpins was destroyed. More specifically, instead of two bands, three bands were noticed (cell number 3 of both gels). This means that, after connecting miR-10b to its complementary sequence, the Y-hairpin loses its secondary structure or that miR-10b might attach to the complementary sequence of miR-21 as well.

Such leakage is not an option when it comes to therapeutic molecules. Thus, we did again bibliography research to define the reasons that led to this problem. We found out that in some cases molecules with 3 bulges might be more stable.

For more details and pictures of the gels visit our [**Results page.**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

## Engineering Cycle 2

## Design

Bibliographic research indicated that we should try Y hairpins with a bulge of 3 unpaired bases. So we redesigned 2 hairpins with a different combination of 3 bulge bases. Another difference between these molecules was the length of the
toehold on the Y1 strand, where the first reactant microRNA is binding.

## Build

In this step, we did some changes to the parameters of the design code. Especially, we changed the code part for the length of the bulge and the length of the toehold was reduced by one base. For the visualization of our new Y-hairpins, we used the online platform of Nupack (Figure 6 and Figure 7).

![](https://static.igem.wiki/teams/4141/wiki/wiki/engineering-success/figure-6-y-shaped-hairpin-named-yc-with-a-bulge-of-3-bases-and-a-bigger-by-one-base-toehold.jpg)
Figure 6. Y-shaped hairpin (named Yc) with a bulge of 3 bases and a bigger, by one base, toehold.

![](https://static.igem.wiki/teams/4141/wiki/wiki/engineering-success/figure-7-y-shaped-hairpin-named-yd-with-a-bulge-of-3-bases.jpg)
Figure 7. Y-shaped hairpin (named Yd) with a bulge of 3 bases.

## Test

To test the stability and secondary structure of the redesigned Y-hairpins, we used the same lab experimental procedure, native gel electrophoresis with acrylamide gel. In this way, we could visualize the results and be able to compare the differences.

## Learn

Our results indicated that the Y-hairpins were more stable with 3 bases of bulge, after comparing all the results from our experiments. At our last native acrylamide gel electrophoresis, we also tested the Ya-hairpin again, to make the comparison of the bulges easier. For this purpose, we loaded the Y-hairpins with the following conditions:

* Y-hairpin alone
* Y-hairpin with miR-21
* Y-hairpin with miR-10b
* Y-hairpin with both microRNAs

We first added Yd, Yc and then Ya hairpin. For Yc and Yd hairpins, we noticed less leakage on the gel, which means that their secondary structures were more stable. Also, between them, the hairpin with the bigger by one base toehold indicates hybridization in a bigger percentage (Yc).

For more details and analytical results of our experiments visit our [**Results page.**](%7B%7B%20url_for%28%27pages%27%2C%20page%3D%27results%27%29%20%7D%7D)

{% endblock %}


{% extends "layout2.html" %}
{% block title %}Results{% endblock %}
{% block lead %}You can describe the results of your project and your future plans here.{% endblock %}
{% block page\_content %}

* [Y Hairpins](#yhairpin)
  + [Bulge 4](#bulge4)
  + [Bulge 3](#bulge3)
  + [Hybridization Temperature](#temperature)
  + [Selection](#selection)
* [HCR Hairpins](#hcrhairpins)
  + [Fluorescence](#fluorescence)
  + [Gel Electrophoresis](#electrophoresis)
  + [Initiator Quantification](#quantification)
  + [Transfection in cancer cells](#transfection)
* [Future vision](#future)

Results

## Y Hairpins

## Bulge 4

The first experiments of our team were carried out on the Y hairpins to evaluate their design and functionality. After the hairpins’ preparation, it was examined whether the complementary sequences for miR-21 and miR-10b are de-hybridized when miR-21 and miR-10b are present. Unfortunately, we observed leakage. When both Ya and Yb were incubated with miR-10b, the secondary structure of the Y hairpins was destroyed. More specifically, instead of two bands, three bands were noticed (cell number 3 of both gels). This means that, after connecting miR-10b to its complementary sequence, the Y-hairpin loses its secondary structure or that miR-10b might attach to the complementary sequence of miR-21 as well.

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/thessaloniki22-figure-1-ya-hairpin-testing-polyacrylamide-gel-12.jpg)
Figure 1: Ya hairpin testing (polyacrylamide gel 12%)

In Figure 1 the wells are loaded with the Ya hairpin, which has a bulge with 4 unpaired bases:

1. Ya
2. Ya + miR-21
3. Ya + miR-10b
4. Ya + mirR-21 + miR-10b

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/thessaloniki22-figure-2-yb-hairpin-testing-polyacrylamide-gel-12.jpg)
Figure 2: Yb hairpin testing (polyacrylamide gel 12%)

In Figure 2 the wells are loaded with the Yb hairpin, which also has a bulge with 4 unpaired bases:

1. Yb
2. Yb + miR-21
3. Yb + miR-10b
4. Yb + miR-21 + miR-10b

From the second well, which is loaded only with the Ya we observe the creation of 2 bands, which means that the Ya structure is not very stable, since its secondary structure is not stable enough. In the third well, there are 3 bands indicating the full opening of the Ya only in the presence of one microRNA (miR-10b). The Yb hairpin structure was opened from miR-10b and had leakage when it was mixed in the well with miR-21. Due to these experimental results, we decided to redesign our Y-shaped molecules, to minimise the leakage and to improve their stability.

## Bulge 3

After redesigning the Y hairpins, with a bulge of 3 unpaired bases, laboratory experiments were conducted to examine the outcome of the hairpin’s hybridisation reaction and their interactions with the complementary sequences for miR-21 and miR-10b.

In the following gel, we have loaded the Yc and Yd hairpins (3 bulge) to check if we would receive better results. We also loaded the Ya hairpin, to compare the native gel electrophoresis results of the hairpins with different bulges.

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/thessaloniki22-figure-3-yd-yc-and-ya-polyacrylamide-gel-electrophoresis-12.jpg)
Figure 3: Yd, Yc and Ya polyacrylamide gel electrophoresis (12%)

1. Yd
2. Yd + miR-21
3. Yd + miR-10b
4. Yd + miR-21+ miR-10b
5. Yc + miR-21
6. Yc
7. Yc + miR-10b
8. Yc + miR-21+ miR-10b
9. Ya
10. Ya + miR-21
11. Ya + miR-10b
12. Ya + miR-21 + miR-10b
13. Initiator sequence

From these hairpins, Yc seems to have the best results, with almost no leakage, because in the absence of microRNAs there is only one band. In the presence of only one microRNA, there are 2 bands, while when both miR-21 and miR-10b are present there are 3 bands.

## Hybridization Temperature

According to the literature, the optimum temperature for the hybridization of Y-hairpins is the melting temperature minus 5 or 10 degrees Celsius. To find the best possible hybridization temperature, samples of hairpins were incubated at
45°C and 50°C degrees and then separated with native gel electrophoresis to identify the best hybridization. The results show that
45°C degrees are the best temperature for hybridization, as the band appearing in the gel is more intense in the hybridized part.

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/ya-yd-temp.jpg)
Figure 4: Yd at 45°C and 50°C degrees Celcius

1. Yd
2. Yd & miR-21
3. Yd & miR-10b
4. Yd & miR-21 & miR-10b
5. Yd
6. Yd & miR-21
7. Yd & miR-10b
8. Yd & miR-21 & miR-10b

The results showed that incubation in 45°C has better results because the band is a bit more intense.

## Selection of the best Y hairpin

The next step of our research was the selection of the appropriate Y-hairpin. The hairpins were hybridized at 45°C celsius and incubated with miR-21 and miR-10b. Then a native gel electrophoresis experiment was conducted. The experiments showed that the hairpin Yc had the highest hybridization rates without showing high leakage when incubated with one of the miR-21 & miR-10b because the band was more intense.

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/yc-yd-selection.jpg)
Figure 5: Yc and Yd comparison (polyacrylamide gel 12%)

## HCR Hairpins

In the following experiments, we wanted to check the hybridization of the HCR hairpins, our second type of hairpins. Normally, the hairpins should hybridize and stick together, giving a large molecule, with a secondary structure. HCR hybridization was checked by both fluorescence measurement and native gel electrophoresis.

## Via fluorescence

To study hybridization by fluorescence, the sequences of Y and HCR hairpins were used and incubated with SYBR green I. SYBR green I has the ability to bind mainly to the double-stranded regions of the molecules, producing fluorescence. When the hybridization of the hairpins, which are mainly composed of single-stranded sites, happens, a large double-stranded molecule with small single-stranded protrusions is produced, (mainly at the sites where siRNAs are released). Therefore, in the wells where hybridization reactions are about to happen, according to our design, we expect higher fluorescence values (Figure 6).

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/figure-6-fluorescence-result-of-stat3-and-hif1a-hairpins.png)
Figure 6: Fluorescence result of STAT3 and HIF1a hairpins (excitation 490 nm, emission 520 nm). H=HCR hairpin and i=initiator (low affinity - zero binding black plate).

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/figure-7-fluorescence-results-of-stat3-and-plk1-hairpins.png)
Figure 7: Fluorescence results of STAT3 and PLK1 hairpins (excitation 490 nm, emission 520 nm). H=HCR hairpin and i=initiator (low affinity - zero binding black plate).

Comparing Figure 6 and Figure 7, we found out that the HCR hairpins with the siRNAs for STAT3 and HIF-1a (SH set) work best than the set for STAT3 and PLK1 (SP set). In the SP set, H1 and initiator have a higher signal than SH set, suggesting a better activation of the procedure. Nevertheless, in the SH set, HCR hairpins with initiator have a higher fluorescence signal when compared to the hairpin alone and the hairpins in the presence of microRNAs. This suggests that the hairpins hybridize as supposed to and the leakage in the presence of the microRNAs is small.

The measurement was performed on 2 types of plates, low and high-affinity plates. Figures 6 and 7 show the results from the low-affinity plates, while Figures 8 and 9 show the results from the high-affinity plate. The reason we did both measurements was the fact that high-binding plates might affect the secondary structure of our hairpins due to the bonds forming between plates and molecules. However, the results were similar on both plates, suggesting that the affinity of the plates doesn’t affect the reaction and the molecules can effectively interact with each other.

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/figure-8-fluorescence-results-of-stat3-and-hif-1a-hairpins.png)
Figure 8: Fluorescence results of STAT3 and HIF-1a hairpins (excitation 490 nm, emission 520 nm). H=HCR hairpin and i=initiator (high affinity - binding black plate).

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/figure-9-fluorescence-results-of-stat3-and-plk1-hairpins.png)
Figure 9: Fluorescence results of STAT3 and PLK1 hairpins (excitation 490 nm, emission 520 nm). H=HCR hairpin and i=initiator (high affinity - binding black plate).

## Via native gel electrophoresis

To confirm the fluorescence results and the hybridization of the HCR hairpins we designed and conducted a native gel electrophoresis (12% polyacrylamide gel). At the results we expected to observe a large band high on the gel and a smaller band lower, coming from the released of the siRNA molecules.

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/hcr-gel.jpg)
Figure 10: Results of hairpins gel electrophoresis (12% polyacrylamide gel)

1. SiRNA sequence for STAT3 and PLK1
2. H1+H2+i (sp set)
3. H1+H2+H3 (sp set)
4. H1+H2+H3+i (sp set)
5. H1+H2+H3+H4+i (sp set)
6. H1+H2+H3+H4 (sh set)
7. H1+H2+H3+H4+i (sh set)
8. SiRNA sequence for STAT3 and HIF-1A
9. H1+H2+i (sh set)
10. H1+H2+H3 (sh set)
11. H1+H2+H3+i (sh set)

Interestingly enough, the results were different than the expected! The electrophoresis showed that SP set is better than SH. In the wells with the SH set of hairpins, there is no band in the upper section of the gel. This suggests that the higher fluorescence observed with SYBR Green I, was due to hybrids between 2 hairpins and not the whole system. SH hairpins didn’t work as expected. On the other hand, the SP set had promising results, with both an upper, a middle, and a higher band suggesting that it works as designed.

## Initiator Quantification

To proceed with the validation of Theriac’s mechanism, we concluded that for the rest of the experiments we should use the SP set of hairpins. The first step towards recognizing the limitations of our system was to quantify the HCR hairpins. We wanted to find out the best concentration for Theriac to work. According to the literature, most researchers used a concentration of 200 nM of HCR hairpins and 50 nM of microRNAs. For this reason, we used the serial dilutions from 12.5 nM to 12800 nM concentration of HCR hairpins. Based on the results of Figure 11, we concluded that the best concentration is 100-300 nM of hairpins.

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/figure-11-quantification-of-hcr-hairpins-of-sp-set.png)
Figure 11: Quantification of HCR hairpins of SP set.

From the values presented in this diagram, we have previously removed the blind sample. Based on the saturation (before the diagram transforms into a straight line), the ideal concentrations are between 100-300 nM of the HCR hairpins.

The next important step is the quantification of the microRNAs. We chose to proceed with the concentrations of 100 nM for the HCR hairpins. According to the literature, the ideal concentration of microRNAs - initiator is 50 nM. To test the sensitivity of our hairpins, the maximum and the minimum concentration they can detect, we used serial dilutions from 6.25 nM to 6400 nM concentration of initiator.

Based on the results of Figure 12, we concluded that the maximum concentration that our hairpins can quantify is 800 nM of initiator, while the minimum is approximately 400nM. Our hairpins can be activated with lower levels of initiator, too, but they can not quantify these levels. However, although the design of our hairpins needs to be improved, this is not a problem for Theriac’c future implementation.

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/figure-12-quantification-of-micrornas-using-hcr-hairpins-of-sp-set.png)
Figure 12: Quantification of microRNAs using HCR hairpins of SP set.

From the values presented in this diagram, we have previously removed the blind sample.

## Transfection in cancer cells

Theriac consists of different hairpins with different sizes and characteristics. For the future implementation of Theriac is essential to provide some preliminary results of its transfer into cancer cells with a nanocarrier. Thus, as a follow-up to our experiments, we tried to transfer our molecular mechanism to U-87 and U-251 cell lines. The transfection was performed using the Xfect kit via lipid nanocarrier. U-87 MG is a cell line with epithelial morphology that was isolated from malignant gliomas from a male patient and formed clusters if left to grow. On the other hand, the U251 cell line is a human glioblastoma astrocytoma delivered from a malignant tumor by explant technique (figure 13 & figure 14).

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/figure-13-image-of-the-u87-cells-that-our-hairpins-were-transferred-to.jpg)
Figure 13: Image of the U87 cells that our hairpins were transferred to.

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/figure-14-image-of-the-u251-cells-that-our-hairpins-were-transferred-to.jpg)
Figure 14: Image of the U251 cells that our hairpins were transferred to.

Our H4 hairpins had a fluorochrome on their 3’ end. This fluorochrome is called  [**ATTO 633**](https://www.atto-tec.com/ATTO-633.html)  with excitation at 629 nm and emission at 657 nm, red fluorescence.

The transfection of the cells was successful, the hairpins were transferred into the cells, however, the transfection rate success was low. Images 15 and 16 from the fluorescence microscope show the nanocarriers loaded with hairpins that emit red.

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/figure-15-images-of-transfected-with-theriac-u-87-cells-red-fluorescence.jpg)
Figure 15: Images of transfected with Theriac U-87 cells (red fluorescence).

![](https://static.igem.wiki/teams/4141/wiki/wiki/results/figure-16-image-of-transfected-with-theriac-u-251-cells-red-fluorescence.jpg)
Figure 16: Image of transfected with Theriac U-251 cells (red fluorescence).

Optical and fluorescence microscopy images were taken using a Nikon DS-Fi3 microscope camera. These results indicate that the loading of our molecular mechanism in a lipid nanocarrier could be possible. Further research and laboratory tests with alterations of the parameters, should be conducted, in order to identify the best possible concentration per hairpin, in the nanocarrier.

## Future vision

### Viability assay & HCR selection

One of the primary experiments we want to do is that of cell survival. Normally, the molecular mechanism of Theriac should release siRNA sequences for STAT3 and PLK1 or HIF-1A. These proteins are overexpressed in glioblastoma and, according to the literature, inhibition of their expression results in apoptosis and cell death. In continuation of the experiments, we would like to see the results of the effect of these molecules in cancer cell lines and, based on these results, select the appropriate/best HCR set.

### Transfection of different concentrations of hairpins in cancer cells

It would also be interesting to study the concentration of hairpins that can be loaded onto a nanocarrier and transferred into cancer cell lines. We would like to find out the maximum amount that can be loaded, as well as the optimal concentration that will have the desired effects.

### Nanocarrier experiments

Except for the right concentration, the selection of the appropriate vehicle for transfection is also an important part of the equation. For our next steps, we want to test the transfection rate of a magnetic nanocarrier, coated with a PEG layer, which we hope will provide a safe transportation of the molecules and, at the same time, a signal to MRI in order to monitor the transfection.

### Other RNA hybridizations

After a literature search, we discovered that there are microRNAs molecules that are similar sequences to miR-21 & miR-10b. We would therefore like to test whether these molecules are able to activate our molecular mechanism and release the siRNA sequences. Furthermore, we would like to incubate our hairpins in tumour cell serum to see if the microRNAs they produce are capable of activating the system in vivo.

{% endblock %}