Part:BBa_K5036015
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 inserted nanobodies in NSP3A and MCP to bind specifically to MMP9 so that when MMP9 level increases, Nanobodies will bind to it and mediate circularization and initiate translation of YAP-1
This figure illustrates the structure of MMP9 Nanobody in our TID switch .
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 performed molecular dynamics for MMP9-NB1 to measure its stability in normal physiological function
We used an amber notebook to simulate MMP-9 binded to nanobody-1 in normal biological circumstances. Despite significant fluctuation, the RMSD is revolves around a mean value of 3 Å. Then, RMSD increased to be approximately 4.5 Å. Overall, MMP-9-NB1 shows a relatively stable protein under physiological conditions .
We measured the effect of the nanobodies on the NSP3A-CAP binding stability, so we measured NSP3A-CAP before and after binding to nanobody1 and nanobody3
NSP3A-CAP
this figure illustrates The estimated binding stability (ΔG) between NSP3A and the Cap binding protein at the 5’ end of the mRNA equals -13.8 kcal mol-1 .
NSP3A-Cap-NB1
this figure illustrates The estimated binding stability (ΔG) between NSP3A 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 three states((NSP3A-CAP) -(NSP3A-Cap-NB1) -(NSP3A-Cap-NB3)) 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 NSP3A 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 and NB1
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 NSP3A 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) .
Literature Characterization
A two-stage method was investigated for creating variable heavy-chain fragments (VHHs). This approach involved transferring complementarity-determining regions (CDRs) from non-camel antibodies to VHH frameworks, followed by affinity maturation using a synthetic phage library. botulinum neurotoxin A (BoNT/A) Was selected and fluorescein as model antigens. antibodies and antibody fragments Was generated against both targets and used the variable heavy-chain domains of scFvs that recognized the light chain of BoNT/A (BoNT/A-LC) or fluorescein as CDR donors. The cAbBCII-10 nanobody and the enhancer nanobody were used as acceptor frameworks for the BoNT/A-LC and fluorescein-specific CDRs, respectively
the figure compares the sequences of the constructed proteins. Complementarity-determining regions (CDRs) and framework regions were identified using the Kabat numbering system. In addition to the CDRs, amino acids in the upper core of the variable domains, which influence CDR conformation and orientation, were aligned with the donor sequences. The resulting framework changes are highlighted in this figure .
The engineered VHHs were expressed in E. coli, purified using affinity chromatography and evaluated for their ability to bind to their target antigens. As shown in Figure A, the grafted BoNT/A-LC VHH exhibited slightly improved binding to its antigen compared to the negative control (fluorescein-conjugated BSA).
The figure shows that The grafted fluorescein-specific VHH also demonstrated enhanced binding to fluorescein-conjugated BSA compared to the non-conjugated form .
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.
Wagner HJ, Wehrle S, Weiss E, Cavallari M, Weber W. A Two-Step Approach for the Design and Generation of Nanobodies. Int J Mol Sci. 2018 Nov 2;19(11):3444. doi: 10.3390/ijms19113444. PMID: 30400198; PMCID: PMC6274671.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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