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Latest revision as of 08:28, 30 September 2024

MMP9 Nanobody3- NSP3

Part Description

Our engineered switch contains a nanobody that recognizes MMP9, an enzyme elevated in injured cells. This nanobody is linked to NSP3A protein, which is attached to the cap from the other end.

Usage

Our TID device contains NSP3A protein linked to specific sensors (nanobodies) that detect MMP9, a protein elevated within cells after tissue injury. TID switches from off state to on state when MMP9 is present in the cell cytoplasm. This triggers the circularization of YAP-1 mRNA, which is essential for protein production

this figure illustrates the structure of NSP3A-MMP9 Nanobody which is attached to the cap of our switch .

Dry lab Characterization

We measured the effect of the nanobodies on the NSP3A-CAP binding stability, so we measured NSP3A-CAP before and after binding to nanobody1 and nanobody3

NSP3A-CAP

this figure illustrates The estimated binding stability (ΔG) between NSP3A and the Cap binding protein at the 5’ end of the mRNA equals -13.8 kcal mol-1 .

NSP3A-Cap-NB1

this figure illustrates The estimated binding stability (ΔG) between NSP3A and the Cap binding protein, in the presence of nanobody1 , at the 5’ end of the mRNA equals -27.8 kcal mol-1. .

NSP3A-Cap-NB3

this figure illustrates The estimated binding stability (ΔG) between NSP3A and the Cap binding protein, in the presence of nanobody 3 , at the 5’ end of the mRNA equals -34.9 kcal mol-1. .

Then we compared between the previous three states of the 5’ prime end and we conclude that Nanobodies presence stabilized the proteins at the 5’ end.

this figure shows that adding the NB1 to the NSP3A and the Cap increased their binding stability (ΔG) from -13.8 kcal mol-1 to -27.8 kcal mol-1. While adding the NB3 intensified their binding stability (ΔG) from -13.8 kcal mol-1 to -34.9 kcal mol-1 Therefore, we used the NB3 at the 5’ end to increase its stability, and NB1 at the 3’ prime end putting its high binding stability with MMP9 in our consideration .

Characterization by Mathematical Modeling

The model provides the interaction kinetics of MMP-9 to both Nanobody-3 NSP3A from the cap side and Nanobody-3 MCP from MS2 aptamer side to form a binding complex to activate our TID switch. The result shows an increase in the binding complex upon MMP-9 interaction based on parametric values from literature.

Graph(1). Illustrates the relation between decreasing free MMP-9 (Blue line) upon their binding to nanobody-3 NSP3A (orange line) and nanobody-3 MCP (Red line) at the same time, which results in forming a binding complex ( Green line) .

Experimental Characterization

It is considered that manipulating the mRNA circularization process could be a promising approach to gain better control over translational initiation. Circularization and translation of this mRNA transcript would rely on a TID that imitates the natural PABP role by binding both the aptamer-based control region and any preinitiation complex member. TID consists of fusing the MCP to different eIF4F-binding proteins (eIFBPs). by sitting HHR-binding sites between aptamer region and the poly (A) region to remove the poly (A) tail through ectopic co-expression of HHR-binding sites, Due to high background activity of natural poly A tail located downstream of the synthetic aptamer region, which may attract endogenous factors (such as native PABP) to form a closed-loop configuration and activate translation even in the absence of TIDs, so SEAP expression from poly(A)-deficient mRNA showed markedly reduced background levels and increased TID-dependency.

(a) HEK-293 cells were co-transfected with a SEAP expression vector and the production of proteins from mRNA modified with a 3' UTR carrying a specific aptamer for RNA-binding proteins and a site for pre programmed poly (A) removal via HHR relies on the existence of TIDs made up of various RBPs linked to different eIFBPs as (PABP-MCP, pSL1315; eIF4G-MCP, pSL154; MCP-eIF4E, pSL1316; MCP-NSP3A, pSL95; MCP-VPg, pLYL47). Although this approach may improve control that relies on TID, it also carries possible drawbacks as Trans-removal has high background activity in ­ -shRNA on the other hand (b) cis-removal of poly A tail showed that MCP-NSP3A has the highest SEAP so it is the most preferred and MCP-Coh2 protein incapable of initiating translation (pSL674, negative control) .

In this case shows treatment potential of TID-based protein sensors in vivo, tumor xenografts were created by stably expressing EGFP-NS3a (H1) in epithelial B16-F10 cells, exemplifying a malignant cell signature featuring the presence of a specific target protein in the cytosol. Mice received daily intratumoral injections of plasmid mixtures encoding for an MCP-LaG16/ (ANR) 8-NSP3A-based EGFP-NS3a (H1) sensor driving translation and in situ production of a pro-apoptotic Bax protein. The experiment showed no significant activation of apoptosis and rapid tumor growth was observed in mice implanted with native B16-F10 cells not expressing EGFP-NS3a (H1) and activation of apoptosis in mice treated with the genetic sensor indicating negligible background Bax expression under a potentially “normal” cell signature. Importantly, effective protein levels of Bax detected in tumors correlated with the cell lysis profile.

The gene circuit is created to trigger apoptosis specifically in cells that produce the EGFP-NS3a (H1) protein. It is made up of three parts: 1-(H1) protein which detected by a sensor 2-Bax is effector EGFP-NS3a gene which induce Apoptosis 3-Translation element which present in malignant cells and induce Apoptosis .

In the First Experiment: EGFP-NS3a (H1)-specific Activation of Apoptosis

As (b, c) Daily changes of tumor size were assessed by calculating volume. show that the tumor size increased in the control group and slightly maintained or not increased in the treated group due to apoptosis due expression of Bax protein. (d) Mice harboring subcutaneous B16-F10 EGFP-NS3a (H1)-derived tumors received local injections of pcDNA3.1 (+) (negative control, n = 5 mice per group) showed no translation of Bax protein so no apoptosis occurs and no decrease in tumor size on the other hand group injected with plasmid DNA mixture comprising pSL831 (PhCMV-mBax-(MS2-box) 24-HHR-pA), pSL776 (PhCMV-MCP-LaG16-pA) and pSL582 (PhCMV-(ANR) 8-NSP3A-pA) (treatment group, n = 5 mice per group) showed translation of Bax protein so apoptosis occurs and decrease in tumor size .

In the second experiment: No Activation of Apoptosis in EGFP-NS3a (H1)-deficient Tissues

(E, f) showed that both group Control and treatment group have the same tumor volume, which indicated that no apoptosis and due to absences of Bax protein. (g) Western Blot showed No activation of apoptosis by a PhCMV-driven EGFP-NS3a (H1) sensor in EGFP-NS3a (H1)-deficient tissues. Mice harboring subcutaneous B16-F10-derived tumors received local injections of pcDNA3.1 (+) (negative control, n = 5 mice per group) or plasmid DNA mixture comprising pSL831/pSL776/pSL582 (treatment group, n = 5 mice per group). So in the absences of translation element EGFP-NS3a (H1) protein in both groups the sensor didn’t activate so no translation of Bax which is the effector gene so no apoptosis occurs in both .

Reference

Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Fry TJ, Orentas R, Sabatino M, Shah NN, Steinberg SM. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. The Lancet. 2015 Feb 7;385(9967):517-28.

Shin YJ, Park SK, Jung YJ, Kim YN, Kim KS, Park OK, Kwon SH, Jeon SH, Trinh le A, Fraser SE, Kee Y, Hwang BJ. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins. Sci Rep. 2015 Sep 16;5:14269. doi: 10.1038/srep14269. PMID: 26373678; PMCID: PMC4571616.

Sequence and Features