DNA

Part:BBa_K4391003:Design

Designed by: Kalyandeep Ghosh, Udbhas Garai   Group: iGEM22_IISER_Mohali   (2022-10-02)


ToxR Aptamer


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

Des-Fig-1. Protein Homology search of ToxR
Des-Fig-2. Protein Homology modelling of cytoplasmic domain of ToxR (7NMB)
Des-Fig-3. Docked complex of cytoplasmic domain (7NMB) and periplasmic domain (7NN6) of ToxR

In-silico design of the aptamer involved the following steps:

  1. We first conducted a homology search of the target protein to help identify regions that could be targeted, to ensure the specificity and selectivity of the Aptamer. This was done using NCBI BLAST search followed by a Clustal W analysis of the proteins. Des-Fig-1 shows homology search of the periplasmic region of the protein (PDB ID 7NMB).
  2. The protein was common in Vibrio Species but the DNA binding regions showed several differences making it the perfect target.
  3. The majority of the differences were noted in the amino acid sequences in the w-THT region, suggesting a narrower search space for our Aptamer.
  4. The initial Aptamer generated was larger than what could be transported into the inner membrane. This was a problem as the the protein of interest was an internal membrane protein.
  5. We planned on adding an initial lysis step, to be able to target the region of interest in the protein
  6. A stability analysis was performed to note if the protein would survive the lysis step - we docked the individual chains of the protein and noted the binding energy.
  7. The protein-protein docking was conducted for all five conformations of 7NMB and seven conformations of 7NN6. This set of 35 dockings gave us the average binding energy for the complex.
  8. The final docked binding energy was found to be -640.34kJ/mol which is lower than the energy released on membrane dissociation and hence confirmed that the protein would not dissociate due to the lysis step.
  9. We then modelled the structure of the 7NMB again using a homology modeller, Jpred4, to obtain the results shown in Des-Fig-2
  10. This was followed by a homological alignment of all the conformers of 7NMB obtained from crystallographic data. This was done to ensure that the protein of interest accurately matched the average of the docked conformers.
  11. We selected the region of interest as the periplasmic domain (PDB ID 7NMB). The target showed a high affinity for AT-rich sequences when docked against a random list of nucleotide triplets.
  12. Using the DPP-PseAAC server, we identified the DNA binding propensity of 7NMB, using λ = 5-10. The sequence was hence verified to have good DNA binding propensity.
  13. A further inspection revealed that the 7NMB dimer forms a Wing Helix-Turn-Helix (w-HTH) domain that binds to the aptamer. The Turn domains form the internal linkage, while the helix domain is the targeted sequence for our Aptamer generation.
  14. A literature review and docking tests confirmed that the protein could bind to the following sequence motif.

References

[1] https://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearch&LINK_LOC=blasthome
[2] https://www.genome.jp/tools-bin/clustalw
[3] Drozdetskiy A, Cole C, Procter J & Barton GJ. Nucl. Acids Res. (first published online April 16, 2015) doi: 10.1093/nar/gkv332
[4] Rahman MS, Shatabda S, Saha S, Kaykobad M, Rahman MS. DPP-PseAAC: A DNA-binding protein prediction model using Chou's general PseAAC. J Theor Biol. 2018 Sep 7;452:22-34. doi: 10.1016/j.jtbi.2018.05.006. Epub 2018 May 16. PMID: 29753757.
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