To ensure the validity of our approach, we engaged dry lab validation in all along our project steps. Initially, we settled a plan for validating the biological systems in our approach. Our plan is to visualize the protein structure, measure the affinity of sensing parts to their ligands, and simulate the human physiological environment to predict its effect on our different biological parts.Hence, we started our dry lab validation of our receptor by homology modelling to visualize our receptor parts:
After providing the 3D structure, we need to ensure these parts function, which is binding to the VEGFA extracellulary, by performing molecular docking simulation using alpha fold 3 server between VEGFR1 and VEGFA, and between the VEGFR1 and VEGFA to compare between their binding affinities.
The alignment plot indicates a positive diagonal alignment between the 3D structure and the experimental structures which reflect favorable protein structure.
The alignment plot shows positive alignment between the 3D structure’s amino acids and the experimental structures in the diagonal line indicating good protein structure.
The step preceding molecular docking is molecular dynamics in which we expose the receptor ligand complex to a microenvironment similar to that of the human body. Therefore,we have performed molecular simulation for VEGFR1-VEGFA and VEGFR2-VEGFA, and the results were as follow:
Being simulated in normal physiological conditions by amber notebook, VEGFR-1 shows an initial rise in RMSD from 2Å to 6Å which indicates changes in surrounding environment and conditions. After that, a state of fluctuation and deviation , respectively, indicating a stable protein with deviation less than 5 Å.
Being simulated in normal physiological conditions using the Amber Molecular Dynamics Package, the VEGFR-2 shows an initial rise in RMSD from 2Å to 5Å which indicates changes in surrounding environment and conditions. After that, a state of narrow fluctuation and deviation , respectively, indicating a stable protein with deviation not more than 2 Å.
Conclusively, It is not an easy decision to choose the optimum external domain for our receptor. Therefore, we went through multiple dry lab steps for validation and simulation. Despite the validity of the previous results, we have neglected the effect of the receptor’s internal domain. Thus, we still need to perform more validational steps in the presence of the internal domain.
In our design TEV protease is divided into two domains N and C in which each domain is presented in a different receptor chain. After the receptor dimerization, the TEV protease will fuse to be active and perform its function. Indeed, we performed molecular docking to test this fusion:
The alignment plot reflects that TEV protease’s 3D structure has positive alignment with the experimental structures used in AlphaFold 3. The results indicate a favourable protein structure.
We aim to make our project modular through providing different TCS mutants that could be an alternative for other applications of our platform. Firstly, we tested the TEV protease binding affinity with two available variants of the TCS: (Q.L), (QG). Then, we induced random mutations on them to be tested against the TEV protease. As a consequence, the induced mutation in TCS (Q.L) ,was (Q 6 G), recorded binding stability with TEV (ΔG) of -9.0 (kcal mol-1), while the non mutated variant TCS(Q.L) recorded (ΔG) of -9.3 (kcal mol-1). On the other site, the mutant form of TCS (Q.G), was (E 1 D), recorded (ΔG) of 10.1(kcal mol-1), while its non mutated variant TCS (Q.G) recorded (ΔG) 10.1(kcal mol-1). These minimal differences in the binding affinity could alter the receptor sensitivity based on the designer needs.
dCas9 is the main actor in our system as it will target Yes Associated protein-1 (YAP-1) to increase its expression. Yet, dCas9 is divided into two domains C and N, each one is expressed in different receptor chains. The dCas9 assembly is conditioned by its release and depends on their binding affinity.However, we put in our consideration the gRNA as an external factor that could affect their binding. Accordingly, we performed a molecular docking for the dCas9 two domains with and without the gRNA.
The alignment plot shows positive alignments between its amino acid residues and the experimental structures.This indicates a valid dCas9 domains binding. The stability of binding was calculated using the Prodigy Haddock Webserver. It gave us ΔG of -43.8 (kcal mol-1) which is considered very high binding stability.
The alignment plot in gRNA presence is near to that of dCas without gRNA which reflects that gRNA didn’t affect the domains binding state.
Our receptor is composed of two chains each one of them are composed of internal and external domains. Additionally, there are two choices to be used in the receptor’s external domain VEGFR1 and 2, and two choices to be used with them in the internal domain CdCas9 and NdCas9. The combination between the internal domain and the external domain generates 4 possibilities: VEGFR1-CdCas9, VEGFR1-NdCas9, VEGFR2-CdCas9, and VEGFR2-NdCas9. In which, all of them were tested against their target VEGFA.
In conclusion, we had a lot of variants to use in the single domain as demonstrated above. The VEGFR2-CdCas9 records the highest binding stability with their ligands. But it is not a conclusive result, because our project is composed of two chains, we need to test the two chains binding with the same ligand VEGFA.
The variance between the receptor’s chains led us to multiple combinations. Thus, the binding affinity changes according to the combined chains. Therefore, we ran a docking simulation between the two receptor chains and VEGFA in different combinations to reach our receptor’s final form.
Finally, we reached the optimum combination for our receptor. Previous results declared that the (VEGFR1-Cdcas9 – VEGFR2-Ndcas9) is the best combination as it recorded binding stability of -13.7 (kcal mol-1).
Our dCas-9 system is responsible for YAP-1 expression enhancement. According to our design, after the assembly of dCas-9 domains, the gRNA navigates them to the YAP-1 gene. We have designed 58 different gRNA using CRISPR ON online software tool. Then, we chose the lowest three gRNA off-targeting designs using CRISPR OFF online software tool and tested their stability by RNAfold online software tool. This multi-step approach led us to the best safe gRNA design with minimal off-targeting effect.
As shown above, gRNA-3 recorded (-13.90 kcal/mol) which is the most stable variant. Among all variants, despite the minimal difference between their MFE, we have chosen the most stable one to reduce the off-targeting effect of our dCas-9 system.
We amplified our gBlocks ordered from IDT by using specific forward and reverse primers that were designed using benchling and purchased from IDT. This step was performed to reach sufficient concentration before flanking our gBlocks with the desired restriction site required for the cloning step.
The initial concentration of the fragment after amplification and gel purification was measured through Nanodrop spectrophotometer which showed that the concentration was still low for the restriction enzyme-based cloning process. Therefore, we performed another cycle of PCR and gel electrophoresis.
Figure.(1) This figure illustrates 0.8 % agarose gel electrophoresis of PCR; each well contains one part in the following order, as shown in the following table
| Well ID | Part name | Length |
|---|---|---|
| P1 | GFP | 804 |
| P2 | Mcherry | 769 |
| P3 | TCS(Q.G) | 576 |
| P4 | TCS(Q.L) | 576 |
| P5 | HU.gRNA | 585 |
We obtained V1 and V2 from Addgene which was a Gift from Tudor Fulga bio labs performed in LB agar with 100 mg Ampicillin didn’t show any bacterial growth
We repeated an overnight culture of V1 and V2 and also didn’t show any detected colonies.
Therefore, we adapted a classic technique to clone V1 and V2 plasmids by adding nutrient agar free of antibiotics to the stabbed culture obtained from Addgene; this was done to give the bacteria a chance to flourish with the effect of antibiotic load.
After that the nutrient agar which was added previously was picked into LB agar containing ampicillin 100mg and incubated for overnight culture.
The next morning V1 and V2 plasmid finally showed successful colony growth in LB agar media with ampicillin.
Anyway, the colonies were picked and subcultured followed by extraction with a miniprep purification kit followed by PCR validation of the colonies and we ran gel electrophoresis of the amplified fragments beside the vector.
Gel isolation was done by gel purification Kit and their concentration was measured by Nanodrop spectrophotometer.
We grafted the previously amplified fragments by using restriction enzyme-based cloning into V1 and V2 according to our experimental design.
As we digested both pV1 and its inserts including GFP, and HU.gRNA with EcoRl (Hi-Fi) and Kasl (Thermo fisher) then we ran agarose gel electrophoresis for the digestion step products followed by their extraction and measuring their concentration.
Then the ligation was mediated by Taq DNAligase and the ligated mixture was applied to gel electrophoresis after PCR amplification to the inserted fragments to confirm the grafting of our fragment.
The final ligated form of plasmid V1 was transformed into electro-competent cells DH5 alpha and saved in glycerol stock -80C for long-term storage till the transfection phase following preparation of the second plasmid.
V2 was digested with its insert mcherry using EcoRl (Hi. Fi) followed by agarose gel electrophoresis for the digestion step products then isolated from the gel by gel purification kit then measured their concentration with a Nanodrop spectrophotometer.
Ligation of V2
Then the ligation was mediated by Taq DNA ligase and the ligated mixture was applied to gel electrophoresis after PCR amplification to the inserted fragments to confirm the grafting of our fragment.
We saved the final plasmid construct in glycerol stock -80C for long-term storage till the transfection phase following the preparation of the second plasmid.
ARMED FORCES COLLEGE OF MEDECINE
AFCM EGYPT iGEM 2024
