Part:BBa_K5036042
YAP-MS2(x16)-HHR-poly A tail
Part Description
This composite part includes the YAP-1 coding region, followed by 16 copies of MS2 which in turn followed by the HHR enzyme, and then a poly A tail. This structure ensures that the switch is activated only in response to tissue damage, as indicated by elevated MMP9 levels, resulting in the production of YAP-1 protein.
Usage
Our switch contains YAP-1 coding region and incorporates 16 copies of MS2, enhancing mRNA stability and binding strongly to MCP, which is linked to the MMP9 Nanobody. The HHR enzyme is designed to mediate self-cleavage to remove the poly A tail, preventing unwanted circularization and the premature production of YAP-1. This ensures that YAP-1 production occurs only in response to tissue damage, as indicated by elevated MMP9 levels.
this figure illustrates the structure of our TID switch which is composed of YAP-1 coding region,16 copies of MS2, HHR and poly A tail. .
Dry Lab Characterization
Our priority is to conserve the mRNA stability in the presence of all these modifications. Therefore, we started our switch’s dry lab validations using the RNA fold online tool that predicts the RNA secondary structure and calculates its stability. Despite the importance of the poly A tail in the RNA stability, it is responsible for mRNA basal activity which could interfere with our project’s safety. Indeed, we compared our mRNA stability with and without the poly A tail
mRNA-MS2- poly A tail binding stability
Mountain Plot
Secondary Structure
this figure shows that the mRNA with the poly A tail records Minimal Free Energy (MFE) of -586.80 kcal/mol which is considered a stable structure in the presence of the poly A tail .
mRNA-MS2 binding stability
Mountain Plot
Secondary Structure
this figure shows that The mRNA MFE without poly A tail records -561.40 kcal/mol which is a minor decline than that of the Poly A tail mRNA .
mRNA-Poly A tail binding stability
Mountain Plot
Secondary Structure
this figure shows thatThe mRNA with the poly A tail MFE records -239.80 kcal/mol which is markedly lower than the recorded score in the presence of the MS2 reflecting our new hypothesis validity .
For further validation, we measured mRNA stability in the absence of both Poly A tail and the MS-2
mRNA only binding stability
Mountain Plot
Secondary Structure
this figure shows that The mRNA alone without poly A tail and MS2 MFE records -205.00 kcal/mol .
Then we compared the previous possible conditions to find the most responsible part for mRNA stability
As appears in the figure, mRNA-MS2 with and without poly A tail shows minimal change in their minimal free energy (MFE) while the mRNA without MS2 records almost the half of the MFE value of the mRNA combined with MS2. Obviously, the MS2 aptamers are responsible for around 50% of the mRNA stability. This encourages us to give away the poly A tail , using HHR to limit the poly A tail basal activity.
Characterization by Mathematical Modeling
The model provides the result of HHR action on our switch which is preventing the basal activity of our switch in absence of the MMP-9 , so once MMP-9 increases, its presence will initiate the switch circulation for translation of YAP-1. It is based on parametric values from literature. note we neglected the basal activity that may be due to cross talk between different MMPs
Graph(1). Illustrates the relation between circulation form activity of our switch (Yellow line) and their ability for YAP-1 production (Black line) upon their binding to MMP-9 .
Illustrates the absence of basal activity of the switch through the inability of the switch to circulate (Yellow line) resulting in zero production of YAP-1 (Black line) .
Experimental Characterization
Manipulating the circularization of mRNA may be a promising strategy for improving 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 consist of fusing the MCP to different eIF4F-binding proteins (eIFBPs). The relative fold changes between basal TID-independent expression and TID-mediated upregulation could be further fine-tuned by increasing the number of aptamer repeats placed downstream of the protein-coding region as shown in C
(Shao J, Li S, Qiu X, Jiang J, Zhang L, Wang P, Si Y, Wu Y, He M, Xiong Q, Zhao L, Li Y, Fan Y, Viviani M, Fu Y, Wu C, Gao T, Zhu L, Fussenegger M, Wang H, Xie M. Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells. Cell Res. 2024 Jan;34(1):31-46. doi: 10.1038/s41422-023-00896-y. Epub 2024 Jan 4. PMID: 38172533; PMCID: PMC10770082.)
(c) Correlation between aptamer region size and TID-mediated SEAP expression. HEK-293 cells were transfected with SEAP expression vectors containing different tandem repeats of the MCP-specific MS2-box aptamer ((MS2-box)8, pSL515; (MS2-box)12, pSL1284; (MS2-box)16, pSL516; (MS2-box)24, pSL468) and constitutive expression vectors for either MCP-NSP3A (ON; pSL95) or an MCP-Coh2 protein incapable of binding eIF4F (OFF; pSL674), SEAP expression in culture supernatants was scored at 48 h post transfection and showed that SEAP expression is highest in case MS2-Box copy number 24 which is *80.1 fold compared to control (MCP-Coh2) .
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.
(Shao J, Li S, Qiu X, Jiang J, Zhang L, Wang P, Si Y, Wu Y, He M, Xiong Q, Zhao L, Li Y, Fan Y, Viviani M, Fu Y, Wu C, Gao T, Zhu L, Fussenegger M, Wang H, Xie M. Engineered poly(A)-surrogates for translational regulation and therapeutic biocomputation in mammalian cells. Cell Res. 2024 Jan;34(1):31-46. doi: 10.1038/s41422-023-00896-y. Epub 2024 Jan 4. PMID: 38172533; PMCID: PMC10770082.)
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
Kawamura, Kunio & Ogawa, Mari & Konagaya, Noriko & Maruoka, Yoshimi & Lambert, Jean-François & Ter-Ovanessian, Louis & Vergne, Jacques & Herve, Guy & Maurel, Marie-Christine. (2022). A High-Pressure, High-Temperature Flow Reactor Simulating the Hadean Earth Environment, with Application to the Pressure Dependence of the Cleavage of Avocado Viroid Hammerhead Ribozyme. Life. 12. 1224. 10.3390/life12081224.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12INCOMPATIBLE WITH RFC[12]Illegal NheI site found at 1696
Illegal NotI site found at 1757 - 21INCOMPATIBLE WITH RFC[21]Illegal BglII site found at 921
Illegal BamHI site found at 1782
Illegal XhoI site found at 1770 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 335
Illegal NgoMIV site found at 530
Illegal NgoMIV site found at 1733
Illegal AgeI site found at 1460
Illegal AgeI site found at 1503
Illegal AgeI site found at 1795
Illegal AgeI site found at 2013 - 1000COMPATIBLE WITH RFC[1000]
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