Part:BBa_K5036019

MS2(x24)-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 24 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

we have used 24 repeats of MS2 due to its high activation rate of our switch. 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 circularization and thus stopping unintended translation

this figure illustrates the structure of MS2 and HHR in our switch .

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).

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 nasal activity that maybe 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) .

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

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BamHI site found at 7
  Illegal XhoI site found at 1
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal AgeI site found at 20
  Illegal AgeI site found at 238
  Illegal AgeI site found at 456
  
• 1000
  COMPATIBLE WITH RFC[1000]