Coding

Part:BBa_K5129000:Design

Designed by: Ansal' Diassova, Adil Ispambetov, Anastassiya Tyazhelova, Rauan Muratuly, Tomiris Kudassova, Makpal Akishova, Nuriya Nurlankyzy, Yernur Kenzhegazin, Artyom German, Abylaikhan Seraliyev, Temirlan Karat   Group: iGEM24_NU-Kazakhstan   (2024-09-27)


PNC-27


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]


Design Notes

As for the construction of the plasmid, the genetic sequence of PNC-27 was not available on open sources prior to our work. Therefore, the sequence had to be computed for the creation of the PNC-27 coding part. To do so, we utilized amino acid alignments of the peptide to p53 and CPP obtained for the visualization. Afterward, appropriate amino acid fragments of both components of PNC-27 were aligned to the mRNA sequences that code for these amino acids in native proteins from which the synthetic construct was made. BLAST alignment was used for this analysis as well. Then, reverse DNA sequences coding these mRNA fragments were manually computed, which was followed by codon optimization and insertion of the obtained parts into the plasmid designed for this project through SnapGene. Hence, we were able to compute a new part coding PNC-27 anticancer peptide having only its amino acid sequence.

Source

PNC-27 is a synthetic protein firstly prepared via solid-phase synthesis. The sequece contains HDM2 binding domain of p53 (residues 12–26)[1], native to human, and CPP leader sequence derived from a leader sequence of the antennapedia protein, which is native to Drosophila pseudoobscura pseudoobscura, or a fruit fly[2].

References

[1] Kanovsky, M., Raffo, A., Drew, L., Rosal, R., Do, T., Friedman, F. K., Rubinstein, P., Visser, J., Robinson, R., Brandt-Rauf, P. W., Michl, J., Fine, R. L., & Pincus, M. R. (2001b). Peptides from the amino terminal mdm-2-binding domain of p53, designed from conformational analysis, are selectively cytotoxic to transformed cells. Proceedings of the National Academy of Sciences, 98(22), 12438–12443. https://doi.org/10.1073/pnas.211280698


[2] Davitt, K., Babcock, B. D., Fenelus, M., Poon, C. K., Sarkar, A., Trivigno, V., Zolkind, P. A., Matthew, S. M., Grin'kina, N., Orynbayeva, Z., Shaikh, M. F., Adler, V., Michl, J., Sarafraz-Yazdi, E., Pincus, M. R., & Bowne, W. B. (2014). The anti-cancer peptide, PNC-27, induces tumor cell necrosis of a poorly differentiated non-solid tissue human leukemia cell line that depends on expression of HDM-2 in the plasma membrane of these cells. Annals of clinical and laboratory science, 44(3), 241–248.