Difference between revisions of "Part:BBa K5321002"
Line 7: Line 7:
 
In order to perform our proof of concept of our project, we choose thrombin as a model to mimic disease biomarkers, and its aptamers reported previously. thrombin_TBA_15mer is an aptamer originally characterized by Louis et al. in 1992. It specifically targets the heparin-binding site of thrombin.
 
In order to perform our proof of concept of our project, we choose thrombin as a model to mimic disease biomarkers, and its aptamers reported previously. thrombin_TBA_15mer is an aptamer originally characterized by Louis et al. in 1992. It specifically targets the heparin-binding site of thrombin.
  
−
'''Figure 1''' shows the interaction of thrombin_AYA1809004_40mer with human thrombin.
+
'''Figure 1''' shows the interaction of thrombin_TBA_15mer with human thrombin.
 
<html>
 
<html>
 
<div align="center">
 
<div align="center">
−
         <img src="https://static.igem.wiki/teams/5321/parts/400.png" width="700px" height="auto"/>
+
         <img src="https://static.igem.wiki/teams/5321/parts/15.png" width="700px" height="auto"/>
 
</div>
 
</div>
 
</html><br>
 
</html><br>
−
'''Figure 1 | Structure and affinity of thrombin_AYA1809004_40mer.''' (a) the secondary structure of the 40-mer aptamer. (b) ELISA-based competition assay to determine the affinity constant (Kd) for the aptamer AYA1809004. It was incubated with thrombin protein immobilized on a 96-well ELISA plate in the absence or presence of a 100-fold excess of non-biotinylated AYA1809004 respectively. <br>
+
'''Figure 1 | Binding affinity of thrombin_AYA1809004_40mer with thrombin.''' (a) Percent thrombin inhibition versus DNA concentration. Selection buffer containing human fibrinogen (2mg mi-1 final)and varying concentrations of DNA (~1 nM-150 nM) was incubated for 1 min at 37°C before adding thrombin. (b) Binding simulation predicted by Alphafold3. The thick yellow belt represents 15-mer aptamer. <br>
  
 
   
 
   

Revision as of 14:15, 21 September 2024

thrombin_TBA_15mer

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]

Usage and Biology

In order to perform our proof of concept of our project, we choose thrombin as a model to mimic disease biomarkers, and its aptamers reported previously. thrombin_TBA_15mer is an aptamer originally characterized by Louis et al. in 1992. It specifically targets the heparin-binding site of thrombin.

Figure 1 shows the interaction of thrombin_TBA_15mer with human thrombin.


Figure 1 | Binding affinity of thrombin_AYA1809004_40mer with thrombin. (a) Percent thrombin inhibition versus DNA concentration. Selection buffer containing human fibrinogen (2mg mi-1 final)and varying concentrations of DNA (~1 nM-150 nM) was incubated for 1 min at 37°C before adding thrombin. (b) Binding simulation predicted by Alphafold3. The thick yellow belt represents 15-mer aptamer.


Characterization

Electrophoretic mobility shift assay (EMSA)

An electrophoretic mobility shift assay (EMSA) is a common affinity electrophoresis technique used to study protein-DNA or protein-RNA interactions. This procedure can determine if a protein or mixture of proteins is capable of binding to a given DNA or RNA sequence. In the present study, EMSA was employed for affinity test of the aptamers.

After thrombin and aptamers were diluted with proper buffer, reaction systems were built with a gradient of aptamers. 15-mer, 29-mer and 40-mer aptamers were tested, and a gradient of concentration of thrombin were applied to reflect the binding affinity. After the aptamers were co-incubated with thrombin for 60 min, an 12% non-denaturing polyacrylamide gel electrophoresis was performed. The gel was then stained by fluorescent dye. GelRed was used as the DNA dye. Random extension was added to aptamer,