Results | thessaloniki

Overview

Due to the multifaceted design of NeuroMuSceteer each component had to be first validated individually before we could test them all as a unit. In this page we provide a cursory view of our experimental proof of concept for the following;

T structure and H hairpins hybridization

After successfully designing and in silico testing both types of hairpins for the mechanism, it was necessary to validate their predicted function in the lab. This means assessing their hybridization of T-structures and subsequent release of the initiator, its hybridization to the HCR hairpins and the formation of the final four-part hybridisation chain reaction that releases the therapeutic molecules. After molecule behavior is verified via fluorescence and native gel electrophoresis it is important to determine the ideal quantity for their activation.

T structure and H hairpins hybridizations

Before proceeding with our experiments we wanted to check the quality of our materials. Therefore, we conducted electrophoresis to verify their size and purity (fig 1).

Figure 1:Quality testing. In this figure (L): ladder, (1): H1, (2): H2, (3): H3, (4): H4, (5): T1, (6): T2, (7): T3, (8): miR-219a-5p, (9): miR-338-3p, (10): miR-125a-3p, (11): miR-146a-5p.

1.T-structure polyacrylamide gel electrophoresis (12%)

Our first experiment was to perform a 12% polyacrylamide gel electrophoresis in which we evaluated whether our designed T structure succeeds in being de-hybridized in the presence of miR-125a-3p & miR-146a-5p in order to release the initiator. According to fig.2 it is evident that our original T structure is disordered in the presence of the two microRNAs (cells number 2 and 3). In addition, in cell number 4, which includes the T-structure and both miRNAs we can see a lower band which could be the released initiator.

Figure 2:Y hairpin testing (polyacrylamide gel 12%). In this figure wells are loaded with (1): T-structure, (2): T-structure + miR-146a-5p, (3): T-structure + miR-125a-3p, (4): T-structure + miR-125a-3p + miR-146a-5p.

2. Fluorescence experiments

We then performed fluorescence experiments from which we performed HCR Hairpin Testing and checked both the quantification of the initiator and Hairpins. According to Fig.3 and knowing from the literature that the dye we used has better binding affinity with double stranded molecules, which is confirmed by the results as both initiator and water have exceptionally low concentrations, we obtain the following data. We see that in the quantification of Hairpins we have excellent yields at 800nM concentration while satisfactory yields are also obtained at concentrations of 400nM, 200nM and 6400nM (second diagram). About the quantification of initiator, we can observe that no significant changes are presented when we change its quantity, therefore its concentration does not affect the yield (third diagram). From the HCR Hairpin Testing we see that there is a difference between the four Hairpins (4H) and the four Hairpins in the presence of initiator (4H+i). This shows us that they interact with each other and that HCR can be started.

Figure 3:Fluorescence experiments to test the HCR Hairpin formation and the effect of the concentration of the Hairpin and the initiator in the yield. In this figure H=H1, 2H = H1+H2, 3H = H1+H2+H3, 4H = H1+H2+H3+H4.