Coding

Part:BBa_K5036015

Designed by: Emad hamdy Matter   Group: iGEM24_AFCM-Egypt   (2024-09-12)
Revision as of 05:48, 30 September 2024 by Em100 (Talk | contribs) (→‎Dry lab Characterization)


Nanobody1

Part Description

nanobodies are a unique type of antibody derived from camelids. These single-domain antibodies are significantly smaller and more stable, making them ideal for applications in diagnostics and therapeutics. Nanobodies can be used to develop highly sensitive and specific tests for a variety of diseases, including cancer and autoimmune disorders. Their compact size and stability also make them promising candidates for targeted drug delivery and other biomedical applications

Usage

we insert nanobodies to reach the conditioned circular RNA by mediating the attachment between NSP3 and the MCP of the aptamer

Dry lab Characterization

We Started by making a decision about which Nanobodies are candidates to be used in our project. We had to nominate 2 of 3 available nanobodies for the MMP-9 so we started by validating the nanobodies binding affinity to the MMP-9

MMP9-NB1 complex binding stability

This figure confirmed that The first nanobody-MMP9 interaction scored a binding stability (ΔG) of -12.3 kcal mol-1 which is considered a high binding stability .

Then we compared the binding stability between different nanobodies to MMP9

This figure shows that NB1 and NB3 have higher affinity over the NB2 which make them candidates to be used in our switch .


We measured the effect of the nanobodies on the NSP3-CAP binding stability, so we measured NSP3-CAP before and after binding to nanobody1 and nanobody3

NSP3-CAP

this figure illustrates The estimated binding stability (ΔG) between NSP3 and the Cap binding protein at the 5’ end of the mRNA equals -13.8 kcal mol-1 .


NSP3-Cap-NB1

this figure illustrates The estimated binding stability (ΔG) between NSP3 and the Cap binding protein, in the presence of nanobody1 , at the 5’ end of the mRNA equals -27.8 kcal mol-1. .


Then we compared between the previous three states of the 5’ prime end and we conclude that Nanobodies presence stabilized the proteins at the 5’ end.

this figure shows that adding the NB1 to the NSP3 and the Cap increased their binding stability (ΔG) from -13.8 kcal mol-1 to -27.8 kcal mol-1. While adding the NB3 intensified their binding stability (ΔG) from -13.8 kcal mol-1 to -34.9 kcal mol-1 Therefore, we used the NB3 at the 5’ end to increase its stability, and NB1 at the 3’ prime end putting its high binding stability with MMP9 in our consideration .


We measured the effect of the nanobodies on the MCP-MS2 binding stability, so we measured MCP-MS2 before and after binding to nanobody1

MCP-MS2 binding stability

Alignment Plot

3D structure of MCP binded to MS2-Aptamers

this figure show That the alignment plot scores high diagonal intensity which indicates the similarity between our structures and the experimental one .


MS2-MCP-NB1 binding stability

Alignment Plot

3D structure of MCP binded to MS2-Aptamers

this figure show That The alignment plot scores higher diagonal intensity than MCB-MS2 plot which indicates higher protein stability in the presence of NB1 .

Characterization by Mathematical Modeling

The model provides the interaction kinetics of MMP-9 to Nanobody-1 that can be connected to MCP or NSP3 to mediate our TID switch circulation. The result shows an increase in the binding complex upon MMP-9 interaction based on parametric values from literature

Graph(1). Illustrates the relation between decreasing free MMP-9 (Blue line) upon their binding to nanobody-1 (orange line) that results forming a binding complex ( Green line) .

Reference

Lee DW, Kochenderfer JN, Stetler-Stevenson M, Cui YK, Delbrook C, Feldman SA, Fry TJ, Orentas R, Sabatino M, Shah NN, Steinberg SM. T cells expressing CD19 chimeric antigen receptors for acute lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial. The Lancet. 2015 Feb 7;385(9967):517-28.

Shin YJ, Park SK, Jung YJ, Kim YN, Kim KS, Park OK, Kwon SH, Jeon SH, Trinh le A, Fraser SE, Kee Y, Hwang BJ. Nanobody-targeted E3-ubiquitin ligase complex degrades nuclear proteins. Sci Rep. 2015 Sep 16;5:14269. doi: 10.1038/srep14269. PMID: 26373678; PMCID: PMC4571616.


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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