Difference between revisions of "Part:BBa K5036050"

Revision as of 15:56, 26 September 2024

MMP9 Nanobody3- NSP3A

Part Description

Our engineered switch contains a nanobody that recognizes MMP9, an enzyme elevated in injured cells. This nanobody is linked to NSP3 protein, which is attached to the cap from the other end.

Usage

Our TID device contains NSP3 protein linked to specific sensors (nanobodies) that detect MMP9, a protein elevated within cells after tissue injury. TID switches from off state to on state when MMP9 is present in the cell cytoplasm. This triggers the circularization of YAP mRNA, which is essential for protein production

this figure illustrates the structure of NSP3-MMP9 Nanobody which is attached to the cap of our switch .

Dry lab Characterization

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. .

NSP3-Cap-NB3

this figure illustrates The estimated binding stability (ΔG) between NSP3 and the Cap binding protein, in the presence of nanobody 3 , at the 5’ end of the mRNA equals -34.9 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 .

Characterization by Mathematical Modeling

The model provides the interaction kinetics of MMP-9 to both Nanobody-3 NSP3 from the cap side and Nanobody-3 MCP from MS2 aptamer side to form a binding complex to activate our TID switch. 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-3 NSP3 (orange line) and nanobody-3 MCP (Red line) at the same time, which results in 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