Part:BBa_K4193000

RNA aptamer 1

RNA that can combine with GABA

Design

We first used the secondary structure of the aptamer and the specific binding with the ligand to de novo design the aptamer, and carried out molecular docking and dynamic simulation for testing. Then we got aptamer GABA-1.

Figure1.GABA-1 aptamer molecular docking with GABA

Characterization

Our team simulated RNA aptamer with high specificity for γ- aminobutyric acid(GABA) by modeling method. We then synthesized the target RNA aptamer sequence with fluorescence 3’ FAM. Therefore, the dissociation constant(Kd) of the aptamer was determined by fluorescence spectroscopy. We incubated the aptamer with a range of concentrations of GABA for two hours at room temperature, respectively. Next, we measured the fluorescence intensity at 525nm of each group and used the following formula to characterize the Kd value:

Here, F0 and F represent the fluorescence intensity of the fluorescent groups in the presence and absence of GABA. Experiments of each group was repeated three times to ensure the accuracy of the measurements and to reduce the chance of randomness.

Result

After statistical analysis, we found that GABA-1 had certain capacity of transporting, but it didn’t work well as we expected. That’s why we built GABA-2.

Figure2.Measurement of dissociation Constant of Aptamer GABA-1:Kd=6.143e+04 (-2.915e+08, 2.917e+08) 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]