Part:BBa_K5321002
thrombin_TBA_15mer
Contents
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]
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,
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