Part:BBa_K3730013:Design

3UTR with miRNA combining sequence to induce degradation of the protein synthesis sequence

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 215
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 215
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 1
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 215
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 215
  
• 1000
  COMPATIBLE WITH RFC[1000]

Design Notes

The design of this part starts with the difference of miRNA between HCC cell (HEPG2 cell line) and normal cell (HEK293T cell line), and we designed a specific virus gene with a corresponding target sequence in its 3’UTR. Based on the virus gene with special 3’UTR, we added the design of a sponge to enlarge the difference of miRNA.

Therefore, we measured the difference of miRNA content in HepG2 cell line and HEK293T cell line, constructed eGFP-3’UTR and sponge plasmids, and tested their function with miRNA added or not.

To make the ViruGuard to distinguish cancer cells from normal cells, we chose three different miRNAs which was said to have different content in HCC cells and normal cells as shown in Table. 1 and the detailed distribution of these miRNAs in differnt cell typeswere further confirmed by atlas of human miRNA in Fig. 1.

Name	Sequence
has-miR-22-5p	AGUUCUUCAGUGGCAAGCUUUA
hsa-miR-195-5p	UAGCAGCACAGAAAUAUUGGC
hsa-miR-199a-5p	CCCAGUGUUCAGACUACCUGUUC

          Table 1 The miRNA chosen to be including in 3’UTR of E1A. 
These three kinds of miRNA were previously confirmed to have different content in HCC cells and normal cells.

           

 

          Fig. 1 The distribution of miRNA22 in normal human cells.

The miRNA195 and 199 were examined in the same way. We chose Hek293T cells as our comparison cell in later experiments because the kidney cells were predicted to have lower expression of these 3 kinds of miRNA compared with other normal cells.

After specific miRNAs were selected, how to insert each miRNA to 3’UTR of E1A became a problem. With the tool provided online, we obtained the reverse complementary of miRNA first and designed the spacer which separated each miRNA according to Saetrom’s study in Fig. 3-2, the binding affinity of miRNA to 3’UTR were also predicted.

            

           Fig. 2 The design of E1A-3’UTR. 
Each miRNA copy were seperated by specific spacers.

            

           Fig. 3 The affinity of each miRNA to its complementary sequence in E1A-3’UTR (named "sponge" in this figure).
 It indicated that miRNA were likely to bind to 3’UTR that we designed.

Source

NO

References

[1] Elhendawy, M., Abdul-Baki, E. A., Abd-Elsalam, S., Hagras, M. M., Zidan, A. A., Abdel-Naby, A. Y., ... & Abdou, S. (2020). MicroRNA signature in hepatocellular carcinoma patients: identification of potential markers. Molecular Biology Reports, 47(7), 4945-4953.

[2] Murakami, Y., Yasuda, T., Saigo, K., Urashima, T., Toyoda, H., Okanoue, T., & Shimotohno, K. (2006). Comprehensive analysis of microRNA expression patterns in hepatocellular carcinoma and non-tumorous tissues. Oncogene, 25(17), 2537-2545.

[3] De Rie, D., Abugessaisa, I., Alam, T., Arner, E., Arner, P., Ashoor, H., ... & De Hoon, M. J. (2017). An integrated expression atlas of miRNAs and their promoters in human and mouse. Nature biotechnology, 35(9), 872-878.

[4] Saetrom, P., Heale, B. S. E., Snøve, O., Aagaard, L., Alluin, J., & Rossi, J. J. (2007). Distance constraints between microRNA target sites dictate efficacy and cooperativity. Nucleic Acids Research, 35(7), 2333–2342.