Overview
Theriac tries to inactivate some crucial for tumor growth proteins by exploiting the overexpression of specific biomarkers in glioblastoma cells. In order to succeed, it recruits some small therapeutic molecules, siRNAs, which have the ability to block the expression of certain proteins and degrade them.
Figure 1: Pipeline of Theriac’s proof of concept.
Is the Y-shaped hairpin hybridised and activated by miR-10b and miR-21?
The first participant of our molecular mechanism is the Y hairpin. Y hairpin is designed to identify the sequences of miR-21 and miR-10b, microRNAs.
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 order to prove that Theriac works, we conducted the following experiments/procedures:
The first step towards Theriac proof of mechanism, is the detection of the biomarkers and the release of the initiator. The two biomarkers the Y-hairpin is partially complementary with are miR21 and miR10b. When these are hybridised, a single stranded molecule, designed as an initiator, is released.
We verified that the Y-hairpins we designed and the one we chose, work the way they were supposed to. The experiments involved hybridisations, incubations, and native gel electrophoresis. The samples used to load every well of the gel, had different composition, in order to study and observe all the possible stages and hybridisations that could happen in between the molecules. Our results proved that the hybridisations were successful, the microRNAs were hybridised with the Y-hairpin and the initiator was released.
Is the HCR hairpin activated by initiator?
During our experiments, we examined the activation of our HCR hairpins. HCR hairpins should only be activated after the release of the initiator. The initiator has a very similar sequence with miR-21 and miR-10b, thus we tested our hairpins in absence of the initiator and in the presence of each microRNA, too. To do so we used fluorescence measurement and native gel electrophoresis and compared the results of HCR + initiator vs HCR + miR-21 vs HCR + miR-10b. The STAT3-PLK1 set of our hairpin gave signal in the presence of the initiator, while in presence of microRNAs the leakage was small. To learn more about our experiments, visit our Results page.
Figure 2: Activation of HCR hairpins by the initiator.
Does it form a tetrahybrid that releases siRNA sequences?
The final step of our therapeutic approach was the testing of the formation of the tetrahybrid and the release of siRNAs. To do so we did native gel electrophoresis with different conditions in each well. The conditions used are:
- SiRNA sequence for STAT3 and PLK1
- H1+H2+i (sp set)
- H1+H2+H3 (sp set)
- H1+H2+H3+i (sp set)
- H1+H2+H3+H4+i (sp set)
- H1+H2+H3+H4 (sh set)
- H1+H2+H3+H4+i (sh set)
- SiRNA sequence for STAT3 and HIF-1A
- H1+H2+i (sh set)
- H1+H2+H3 (sh set)
- H1+H2+H3+i (sh set)
We noticed that the STAT3-PLK1 HCR hairpins set formed tetrahybrids because 3 bands were shown in the gel (for detailed results check our Results page ).
Figure 3: Release of siRNA molecules after the hybridization of HCR hairpins.
Can hairpins be transferred in living cells?
Getting our first positive results on the function of the molecular mechanism of Theriac, gave birth to the idea of expanding our research to in vivo experiments. The first question that has arisen is whether our hairpins can be loaded onto a lipid nanocarrier and be transferred to a living cell. Glioblastoma cancer cell lines U-87 and U-251 were selected for that purpose and a primary attempt of transfection took place.
Even thought the transfection rate was low, we can claim with relative certainty that our sequences were transferred into living cancer cells, bringing us one step closer to our goal, seeing how Theriac affects glioblastoma cells.
Figure 4: Transfection of cancer cell with HCR hairpins.
Parts
BBa_K4141000:miR-21 complementary sequence from Y-shaped hairpin
BBa_K4141001:miR-10b complementary sequence from Y-shaped hairpin
BBa_K4141002:Initiator sequence from Y-shaped hairpin
BBa_K4141003:H1 sequence from HCR (STAT3-PLK1)
BBa_K4141004: H2 sequence from HCR (STAT3-PLK1)
BBa_K4141005:H3 sequence from HCR (STAT3-PLK1)
BBa_K4141006: H4 sequence from HCR (STAT3-PLK1)
