Part:BBa_K5036023
MS2(x16)-HHR
Part Description
MS2 is a small viral protein which forms the outer shell of the MS2 bacteriophage. Its ability to bind to specific RNA sequences has made it a valuable tool for studying RNA biology and gene expression and it is frequently used in combination with the MS2 system to purify and analyze RNA-protein complexes. this part contain 16 repeats of MS2. while HHR is a type of self-catalytic RNA molecule that has been engineered to cleave specific RNA targets which is essential for various biological processes, such as gene regulation and viral replication.
Usage
MS2 is used to enhances stability through its interaction with MCP, which in turn binds to the MMP9 nanobody. HHR folds spontaneously and cleaves itself to remove the poly A tail, preventing the switch from unwanted circularization and thus stopping unintended translation
this figure illustrates the structure of MS2 and HHR in our switch .
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.
We measured the effect of the nanobodies on the MCP-MS2 binding stability, so we measured MCP-MS2 before and after binding to nanobody1
MCP-MS2 binding stability
Alignment Plot
3D structure of MCP binded to MS2-Aptamers
this figure show That the alignment plot scores high diagonal intensity which indicates the similarity between our structures and the experimental one .
MS2-MCP-NB1 binding stability
Alignment Plot
3D structure of MCP binded to MS2-Aptamers
this figure show That The alignment plot scores higher diagonal intensity than MCB-MS2 plot which indicates higher protein stability in the presence of NB1 .
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) .
Literature characterization
The study tested if MCP-ADAR activated the translation of cargo, specifically if the sensor contained MS2 RNA hairpins that encoded this cargo.
Off-state refers to mNeonGreen expression in the absence of iRFP720 trigger mRNA, while on-state refers to mNeonGreen expression in the presence of iRFP720 trigger mRNA. Orange points refer to the sensor with MS2, and blue points refer to the sensor without MS2. They found that MS2 increased the specificity of the switch.
To understand how various hammerhead ribozyme motifs affect gene activity, they incorporated eight different motifs into relevant mRNA sequences. To compare results across diverse genetic systems, they assessed reporter gene expression in human cells, baker's yeast (S. cerevisiae), and E. coli bacteria. Well-established plasmid-based gene expression constructs served as their reporter systems.
(A)The researchers placed the HHR motifs at the end of a reporter gene called Renilla luciferase (hRluc) within a vector named psi-CHECK2. The reporter gene is responsible for producing light. (B)They investigated how HHR motifs influence the production of a LacZ gene by inserting them into a specific region (3'-UTR) of a separate Gal4 gene on a plasmid. The LacZ gene, located on a chromosome, is controlled by a promoter that responds to Gal4. (C,D) It shows a comparison of how different ribozymes affect gene activity in living cells (in vivo analysis). Black bars represent a reporter gene controlled by a functional HHR motif, while gray bars show the same gene controlled by a non-functional HHR. The control group (Ctrl) lacks any ribozyme sequences. (C) of the figure displays results in human HeLa S3 cells after 18 hours of introducing the genetic material (transfection). (D) shows gene activity in baker's yeast (S. cerevisiae) grown for 18 hours in a special nutrient solution (synthetic complete medium) at room temperature (30°C).
Reference
Gayet, R. V., Ilia, K., Razavi, S., Tippens, N. D., Lalwani, M. A., Zhang, K., ... & Collins, J. J. (2023). Autocatalytic base editing for RNA-responsive translational control. Nature Communications, 14(1), 1339.
Wurmthaler, L. A., Klauser, B., & Hartig, J. S. (2018). Highly motif-and organism-dependent effects of naturally occurring hammerhead ribozyme sequences on gene expression. RNA biology, 15(2), 231-241.
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 NotI site found at 58
- 21INCOMPATIBLE WITH RFC[21]Illegal BamHI site found at 83
Illegal XhoI site found at 71 - 23COMPATIBLE WITH RFC[23]
- 25INCOMPATIBLE WITH RFC[25]Illegal NgoMIV site found at 34
Illegal AgeI site found at 96
Illegal AgeI site found at 314 - 1000COMPATIBLE WITH RFC[1000]
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