Coding

Part:BBa_K4829010:Design

Designed by: Aditya Kamath Ammembal   Group: iGEM23_IISc-Bengaluru   (2023-10-05)


This sequence codes for an scFv against PD1


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BamHI site found at 412
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    INCOMPATIBLE WITH RFC[1000]
    Illegal BsaI site found at 127


Design Notes

This sequence has been derived from a sequence published in 'Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy. Nissim L, Wu MR, Pery E, Binder-Nissim A, Suzuki HI, Stupp D, Wehrspaun C, Tabach Y, Sharp PA, Lu TK. Cell. 2017 Nov 16;171(5):1138-1150.e15. doi: 10.1016/j.cell.2017.09.049. Epub 2017 Oct 19. 10.1016/j.cell.2017.09.049 PubMed 29056342' We had to make massive changes to the sequence, as the required amino acids were not conserved in the right places, and these conserved amino acids are essential for stability.

Here, we elaborate on a procedure to design a preliminary version of such a molecule from a full-size antibody:

This part has not been tested out by methods like BLI. However, it does give very good results in silico. We also provide here, a method to make multiple such amino acid sequences by following a simple pipeline to get a good enough starting point to make a good, well-functioning sequence. The things to be kept in mind are:

  • In each of the two variable domains of the scFv, there are three distinct regions known as complementary determining regions (CDRs) that are interconnected by framework regions (FRs).

The CDRs play a key role in binding to antigens, with their structure tailored to match the epitope. On the other hand, the FRs serve primarily as a support structure and show minimal variability compared to the CDRs. Notably, each CDR contributes differently to antigen binding. For example, the heavy chain's CDR3 is especially vital, contributing to 29% of the binding specificity, whereas the CDR2L's contribution is a mere 4%.

  • In the conventional VH region, the FR2 consist of four highly conserved hydrophobic amino acids (Val37, Gly44, Leu45, and Trp47)that in contribution with Gln39, Gly44, Tyr91, and Trp103 form a conserved hydrophobic interface of ~700 Å 2 to facilitate VL joining.

So, to make a preliminary version of such a molecule, do the following:

  • Take the VH and VL regions, and go through the sequences. Change the amino acid positions to the ones mentioned above, if it is not already this.
  • Join the 2 sequences with a linker: GGGGSGGGGSGGGGS

This simple change is all it takes to get a preliminary scFv!

Source

This sequence is not from any living organism to the best of our knowledge.

References

  • Asaadi, Y., Jouneghani, F.F., Janani, S. et al. A comprehensive comparison between camelid nanobodies and single chain variable fragments. Biomark Res 9, 87 (2021). https://doi.org/10.1186/s40364-021-00332-6
  • Synthetic RNA-Based Immunomodulatory Gene Circuits for Cancer Immunotherapy. Nissim L, Wu MR, Pery E, Binder-Nissim A, Suzuki HI, Stupp D, Wehrspaun C, Tabach Y, Sharp PA, Lu TK. Cell. 2017 Nov 16;171(5):1138-1150.e15. doi: 10.1016/j.cell.2017.09.049. Epub 2017 Oct 19. 10.1016/j.cell.2017.09.049 PubMed 29056342