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.

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

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.

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.

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.

Figure 5: Yc and Yd comparison (polyacrylamide gel 12%)

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).

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).

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.

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).

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.

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.

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.

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).

Figure 13: Image of the U87 cells that our hairpins were transferred to.