Part:BBa_K4193001

RNA aptamer 2

Design

After building GABA-1, we had found that it didn’t work as we expected. That’s why we build the new RNA aptamer GABA-2. After obtaining experimental data of GABA-1, we learned experience and de novo designed an new aptamer with stronger binding ability on the basis of GABA-1.

Figure1.GABA-2 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 rough statistical analysis, we compared GABA-1 with GABA-2.Obviously GABA-2 has a stronger binding ability than GABA-1.So we adapted GABA-2 to our transport RNA and ribozyme riboswitch.

Figure2.Measurement of dissociation Constant of Aptamer GABA-2:Kd = 1.741e+05(-9.327e+10, 9.327e+10)

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]