We evaluated the HER2 binding affinity of the aptamer quantitatively. Methods were based on a type of sandwich ELISA Kit, HER2 proteins were incubated with increasing concentrations of FAM-labeled aptamer and analyzed by spectrofluorometer. Using non-linear regression analysis, the Kd of the aptamer for binding with the HER2 protein was estimated to be 1.803 μM. (See more in
We evaluated the HER2 binding affinity of the aptamer quantitatively. Methods were based on a type of sandwich ELISA Kit, HER2 proteins were incubated with increasing concentrations of FAM-labeled aptamer and analyzed by spectrofluorometer. Using non-linear regression analysis, the Kd of the aptamer for binding with the HER2 protein was estimated to be 1.803 μM. (See more in
[[File:T--GreatBay_SCIE--ELONA_Quantitative.png|600px|thumb|center|<b>Figure 2 A further quantitative test for aptamer affinity.</b> The curve accounts for non-specific binding of aptamer on the bottom of the well plate, achieved through adding a paramater; the equation: Y=\frac{Bmax \times X}{(Kd +X)} + M \times X. The R<sup>2</sup> value for the curve is 0.9801 and Kd = 1.803 μM.]]
</p>
</p>
Revision as of 01:44, 9 October 2021
HR2 Aptamer, for targeting HER2 receptors HR2 Aptamer is an 86bp ssDNA strand that can be folded into 3D shapes to target Human Epidermal Growth Factor 2 (HER2)
Background
Human epidermal growth factor (HER2), belongs to the ErbB receptor tyrosine kinase family, involved in signal transduction pathways that regulate cell growth and differentiation. However, overexpression of HER2 can cause pathogenesis and progression of many carcinoma types. However, HER2 can also be used to treat these cancers. Current drugs like Herceptin bind HER2 through antibodies and inhibit its pathway, curing cancer. Some ADC drugs (Antibody-drug conjugates) also use HER2 as a target for binding, so drugs can be brought into proximity to the tumor cells. (e.g. T-DM1)
Aptamers are oligonucleotides that form unique 3D structures that can bind to practically anything, ranging from small molecules like ions, amanitin; to large proteins like HER2 in our case.
BBa_K3897000 is an aptamer, nicknamed HR2 aptamer, which specifically binds to HER2 ECD. HR2 aptamer is obtained from previous literature. It is synthesized through chemical synthesis, and its binding affinity for HER2 is tested through ELONA (An Enzyme-linked Oligonucleotide Assay).
Figure 1 2D(top) and 3D (bottom) simulations of HR2 aptamer, simulated by mFold and MATLAB, respecitively.
Experimental Data
We evaluated the HER2 binding affinity of the aptamer quantitatively. Methods were based on a type of sandwich ELISA Kit, HER2 proteins were incubated with increasing concentrations of FAM-labeled aptamer and analyzed by spectrofluorometer. Using non-linear regression analysis, the Kd of the aptamer for binding with the HER2 protein was estimated to be 1.803 μM. (See more in
<a href="https://2021.igem.org/Team:GreatBay_SCIE/Results">Results</a>).
Figure 2 A further quantitative test for aptamer affinity. The curve accounts for non-specific binding of aptamer on the bottom of the well plate, achieved through adding a paramater; the equation: Y=\frac{Bmax \times X}{(Kd +X)} + M \times X. The R2 value for the curve is 0.9801 and Kd = 1.803 μM.
Sequence and Features
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]
Reference
Liu, Z., Duan, J. H., Song, Y. M., Ma, J., Wang, F. D., Lu, X., & Yang, X. D. (2012). Novel HER2 aptamer selectively delivers cytotoxic drug to HER2-positive breast cancer cells in vitro. Journal of translational medicine, 10, 148.https://doi.org/10.1186/1479-5876-10-148
