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.
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).
Figure 2. The reaction of Y-shaped hairpin with the 2 miRNAs
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. 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.
Figure 4. Y-hairpin (named Ya) with bulge of 4 bases
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.
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).
Figure 6. Y-shaped hairpin (named Yc) with a bulge of 3 bases and a bigger, by one base, toehold.
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.
