RNA

Part:BBa_K3504012

Designed by: Ahmed Wael   Group: iGEM20_AFCM-Egypt   (2020-10-12)


MiRNA FF4

NOTICE: Parts in our range for this season have been created as a part of our Phase I design of our project. These parts HAVE NOT been tested or characterized in the lab due to COVID-19-related precautionary measures. We have enriched our new parts pages with data from literature and results from our modeling and simulations. If you are intending on using this part or others in our range, please keep in mind these limitations and update these parts with data from your experimentation. Feel free to reach us at: igem.afcm@gmail.com for further inquiries.


Part Description

MiRNA FF4 is a micro RNA that has the ability to repress transcription. MiRNA FF4 is one of microRNAs that were constructed according to the pPRIME approach with stem-loops of functional sequence cloned into miR-30 backbone and fused into 3’-UTR of CMV-driven neomycin resistance gene. Their names are identical to their parent siRNAmolecules, i.e., FF3, FF4, FF5 and FF6

Usage

Genetically encoded miRNA sensors (which sense a solitary miRNA binding) and cell classifiers (which sense different miRNA inputs at the same time) can give data about infection state, activate reactions in cells explicitly communicating either a healthy or diseased miRNA profile. Through analyzing and studying multiple synthetic miRNAs and testing them, we found that miRNA FF4 shows high potency in translational silencing and significant cross-talk in the tumor micro-environment, accordingly we modified our OFF-switch to contain miRNA FF4 target site in our circuit in the 5’ UTR OFF position. Once miRNA FF4 binds to its target site on the mRNA in the 5’ UTR OFF position, this triggers a series of post-transciption modifications that regulate the gene expression and transcription of the used SFV replicon.(1), (2)

Characterization

We characterized this part by the RNA Fold structures of multiple MiRNAs comparing there stabilities by free energy thermodynamic and showing there ability to induce down regulation of the circuit and work as an off switch as an input for a NOR gate that could protect our replicon vaccine vectors from being attacked by innate immunity inside dendritic cells and act as a safety switch

Figure 2.This figure shows the sbol format of the off switch as well as way in which a nor gate works.
Figure 1.These figures illustrate the RNA Fold structure of multiple MiRNAs comparing there stabilities by free energy thermodynamic and showing there ability to induce down regulation of the circuit and work as an off switch.

References

1-Gam, J., Babb, J., & Weiss, R. (2018, June 22). A mixed antagonistic/synergistic miRNA repression model enables accurate predictions of multi-input miRNA sensor activity. Retrieved October 26, 2020, from https://www.nature.com/articles/s41467-018-04575-0

2-DiAndreth, B., Wauford, N., Hu, E., Palacios, S., & Weiss, R. (2019, January 01). PERSIST: A programmable RNA regulation platform using CRISPR endoRNases. Retrieved October 26, 2020, from https://www.biorxiv.org/content/10.1101/2019.12.15.867150v1.full


Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


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Categories
Parameters
None