Part:BBa_K4351021
Theophylline RNA aptamer + RNA Mango
Positive control for binding to small ligand. Theophylline should bind to the RNA aptamer and increase fluorescent output of RNA Mango biosensor.
Fluorescence experiments were completed to measure theophylline binding by the aptamer by combining the theophylline biosensor with RNA Mango part (BBa_K4351010). This biosensor was designed to hopefully act as a positive control as it has been shown previously to bind to ligand at nanomolar affinities.
RNA was unfolded by heating to 95°C and then let cool to room temperature in the presence of 20 µM ligand. The fluorophore TOI was only added to the RNA just prior to recording the fluorescence spectra and was excited at 510 nm. The emission spectra was recorded between 520 - 600 nm and two scans were completed.
Unfortunately, the fluorescence of the theophylline aptamer does not increase significantly in the presence of theophylline (Figure 4B). If the RNA Mango biosensor functions as we predicted, then this would indicate that the theophylline aptamer that we designed is not binding to the ligand under our experimental conditions. More analysis would be needed to confirm these results and testing of another theophylline aptamer could be attempted, possibly using parts BBa_J89000 or BBa_K784005.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000INCOMPATIBLE WITH RFC[1000]Illegal BsaI.rc site found at 92
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