Revision as of 17:53, 20 September 2024

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 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).

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.‏

Sequence and Features