Part:BBa_K1614014:Experience

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Applications of BBa_K1614014

Another biobrick, the ATP Aptamer JAWS1 Spinach2 (BBa_K1614014) was designed in this project as well. Similar to the biobrick BBa_K1614012, we performed in vitro transcriptions to sense ATP in real-time. The construct is a fusion of an ATP aptamer (Sassanfar 1993) and Spinach2 (Strack 2013), which we will call Spinach2-ATP-Aptamer system . To improve the binding of the ATP aptamer to ATP we apply our own implemented JAWS software (Fig. 1). Using our software, nucleotides which form the stem region of the ATP aptamer can be predicted, which will improve binding properties of this RNA to ATP advanced the stemming behavior of the ATP Aptamer which was then fused to the Spinach2. Measurements with the spectro fluorometer show that the ATP Aptamer JAWS1 Spinach2 (BBa_K1614014) has a lower fluorescence than ATP Aptamer JAWS2 Spinach2 (BBa_K1614015)(Fig.2) which is caused by a weaker steming behavior. Therefore ATP Aptamer JAWS1 Spinach2 is a better candidate for sensing ATP changes during biochemical reactions such as the in vitro transcription. For the in vitro transcription assay the RNA was renatured in 1x Renaturing buffer at 95 °C. 500 nM of the RNA was used for the in vitro transcription for measuring the ATP consumption during transcription. Experiments have shown that the detection range of this ATP sensor which correlates to the transcribed RNA is much more sensitive than traditional techniques that require UV-shadowing. The real time fluorescent readout system even allows the study of enzyme kinetics that depends on ATP (Fig.).

Fig.1 Fusion of Aptamers to Spinach to generate fluorescent small molecule sensors. (A) For the design of a new small molecule sensor, the second stem of the Spinach2 aptamer can be exchanged by an aptamer that binds specifically to a small molecule. We fused an ATP-binding Aptamer (yellow) to the Spinach. (B) We applied our JAWS software to predict the best ATP-Aptamers, that can form the best stem structure (blue and red highlighted) in presence of ATP. (C) The JAWS predicted stems can be fused to the Spinach Aptamer. The software can be validated by analyzing the fluorescence emission in presence of ATP and DFHBI (Fig.4B and C).

Figure 2.Establishment of a system to sense small molecule using the Spinach2 Aptamer. (A) Emission spectrum of the original Spinach2 Aptamer, which was applied as an internal control. (B) As another internal control, we reproduce the data for the c-di-GMP Spinach2 system, published by Kellenberger et al.. Indeed, highest fluorescence maximum for the c-di-GMP Spinach2 system was measured in presence of the ligand. (C) Analysis of the fluorescent properties of our ATP Aptamer Spinach2 constructs. The Spinach2 containing the Szostak ATP Aptamer shows the lowest fluorescence of all three ATP Aptamer Spinach2 variations. The JAWS-generated ATP AptamerJAWS1 Spinach2 and the ATP AptamerJAWS2 Spinach2 show higher fluorescence maxima in presence of ATP.

Figure 3 . Fig.3.Sensing of ATP using the ATP Aptamer Spinach in real time during in vitro transcription. (A) Assay design of the ATP-Aptamer Spinach2: ATP-AptamerJAWS1 Spinach2 RNA will be applied to a classical in vitro transcription. In presence of ATP, fluorescence emission can be determined. (B) As proof of principle, transcriptions were performed with different concentrations of T7 RNA polymerase. ATP consumption was monitored in real time by measuring the fluorescence of the ATP-AptamerJAWS1 Spinach RNA in regular intervals.(C) To confirm the results of the fluorescence measurements, the in vitro transcription reaction was analyzed using denaturing acrylamide gels.

References: 1.Sassanfar, M. and J.W. Szostak, An Rna Motif That Binds Atp. Nature, 1993. 364(6437): p. 550-553. 2.Strack, R.L. and S.R. Jaffrey, Live-cell imaging of mammalian RNAs with Spinach2. Methods Enzymol, 2015. 550: p. 129-46. 3. Strack, R.L., M.D. Disney, and S.R. Jaffrey, A superfolding Spinach2 reveals the dynamic nature of trinucleotide repeat-containing RNA. Nat Methods, 2013. 10(12): p. 1219-24.

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