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dCas9(C)_NLS-Syn-VEGFR-1 (VEGF-R1, N-TEV, NLS, TCS(Q,G),HA,dCas9(C),VP64,GFP)

Part Description

In our first receptor chain, we've engineered a system that responds to tissue injury. An external domain, VEGF-R1, is attached to an internal domain composed of N terminal domain of TEV protease, a nuclear localization signal (NLS), a TEV cleavage site(TCS(Q,G)), and dCas9(C) which is linked to transcription activator VP64

Usage

this is our receptor's first chain. our receptor is activated after binding of VEGF to the external domain which is designed to attach specifically to it. after activation the two domains of TEV dimerizes forming catalytically active TEV protease which will cleave the two chains at TCS. upon cleavage of the two chains the two domains of dCas9 dimerize and is released attached to transcription activator to be guided to its direction

This figure illustrates variant of our receptor's first chain where N-TEV is to it. . .

Dry lab Characterization

we had the chance to match the external domains with different internal domain components to select single suitable receptor chain. The whole chain affinity is affected by the internal domain, thus we had to try VEGFR1Cdcas with VEGFA:

VEGFR1Cdcas-VEGFA

The interaction between the chain composed of VEGFR1 as an external domain and Cdcas9 as an internal domain with VEGFA yields ΔG of -11.2 kcal mol-1 .

Then we have made a comparison between the four receptor chain variants’ binding stability with VEGFA.

This figure shows that VEGFR2Cdcas-VEGFA complex has the highest stability among other variants and VEGFR1NdCas9-VEGFA complex has the lowest stability among other variants .

The final form of our receptor is composed of two chains, each chain is built of internal and external domains, so we validated these interactions by calculating the binding affinity between the two chains and VEGFA, which simulate the final design of our receptor.

(VEGFR1-Cdcas9 – VEGFR1-Ndcas9) with VEGFA

The figure displays the interaction between two receptor chains and VEGFA, The (VEGFR1-Cdcas9) chain appears in the red colour, and the (VEGFR1-Ndcas9) chain appears in the blue colour. The VEGFA is in green colour. The calculated binding stability (ΔG) of the combination is -12.9 kcal mol-1 .

The final form of our receptor is composed of two chains, each chain is built of internal and external domains, so we validated these interactions by calculating the binding affinity between the two chains and VEGFA, which simulate the final design of our receptor.

(VEGFR1-Cdcas9 – VEGFR2-Ndcas9) with VEGFA

The figure displays the interaction between two receptor chains and VEGFA, The (VEGFR1-Cdcas9) chain appears in the red colour, and the (VEGFR2-Ndcas9) chain appears in the blue colour. The VEGFA is in green colour. The calculated binding stability (ΔG) of the combination is -13.7 kcal mol-1 .

The receptor chains’ affinity could be affected by the internal domains interactions with the external domains, so we compared between the receptors’ variants to choose the best receptor design in our project.

The figure shows that the combination of VEGFR1-Cdcas9 and VEGFR2-Ndcas9 took the upper hand among other variants .

Characterization by Mathematical Modeling

The model provides the interaction kinetics of VEGFR1 external domain upon binding of VEGF to it , the result shows satisfactory binding affinity and stability based on parametric values from literature

Graph (1). illustrates the decreasing of VEGFR2 (Red line) upon binding of VEGF (A) to form VEGF-VEGFR2 complex (R2A) (Yellow line) which increases till its binding to VEGFR1 (R1), so (R1) also decreases after will ( Black line). To finally form a fitted ligand receptor complex (R2AR1) (Blue line) .

The continuation of the first model provides the activation kinetics of the TEV protease which occurs subsequent to the binding of VEGF to our receptor allowing the dimerization process for our receptor chains to take place. The result shows sufficient TEV protease activation based on parametric values from literature

Graph (2). Illustrates the dimerization level (Blue line) that reaches (16) upon binding of VEGF to its receptor to activate TEV protease (Red line), The activation level of TEV protease reaches (14) to release d-Cas9 system .

The continuation of the second model provides the activation kinetics of the d-Cas9 system which occurs subsequent to cleavage activity of TEV protease after its activation. The result shows increase in d-Cas9 activity which implies successful cleavage of the TEV protease for releasing the N and C terminal of the d-Cas9 system and its assembly based on parametric values from literature

Graph (3). Illustrates the released d-Cas9 system that activation reaches (220), upon activation of TEV protease .

The continuation of the third model provides the activation kinetics of VP64 transcription activator which occurs subsequent to releasing of the d-Cas9 system to initiate the transcription of the YAP-1.The result shows increase in transcription activation level of YAP-1 which implies successful VP64 activation based on parametric values from literature

Graph (4). Illustrates the relation between activation level of VP64 transcription activator (Yellow line) for increasing the transcription level of YAP-1 (Black line) .

References

White C, Rottschäfer V, Bridge LJ. Insights into the dynamics of ligand-induced dimerisation via mathematical modelling and analysis. J Theor Biol. 2022 Apr 7;538:110996. doi: 10.1016/j.jtbi.2021.110996. Epub 2022 Jan 24. PMID: 35085533.

Mac Gabhann F, Popel AS. Dimerization of VEGF receptors and implications for signal transduction: a computational study. Biophys Chem. 2007 Jul;128(2-3):125-39. doi: 10.1016/j.bpc.2007.03.010. Epub 2007 Mar 24. PMID: 17442480; PMCID: PMC2711879.

Paththamperuma C, Page RC. Fluorescence dequenching assay for the activity of TEV protease. Anal Biochem. 2022 Dec 15;659:114954. doi: 10.1016/j.ab.2022.114954. Epub 2022 Oct 18. PMID: 36265691; PMCID: PMC9662696.

Morita S, Horii T, Kimura M, Hatada I. Synergistic Upregulation of Target Genes by TET1 and VP64 in the dCas9-SunTag Platform. Int J Mol Sci. 2020 Feb 25;21(5):1574. doi: 10.3390/ijms21051574. PMID: 32106616; PMCID: PMC7084704.

Sequence and Features