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Discussion and Outlook

To sense specifically small molecules in biochemical reactions is problem that scientist tried to solve since many years.

In this project we developed an innovative method that can be applied as a tool box, enabling us to sense small molecules in real-time (real-time SMS) using a fluorescent readout at two different emission wavelengths. Using a spinach aptamer fused to an ATP aptamer we can sense ATP concentrations in real-time during a biochemical reaction. Thus we were able to analyze classical in vitro transcriptions in detail by monitoring simultaneously the ATP consumption as well as RNA strand synthesis.

A major achievement of our fluorescent tool-box was the validation of our own implemented JAWS software. ATP Aptamer-stems were successfully predicted which were causing a high turn-on effect of the fluorescence in presence of the small molecule. The ATP AptamerJAWS2 Spinach RNA shows a stronger interaction with ATP resulting in a higher fluorescence maximum than the ATP AptamerJAWS1 Spinach RNA. However, the tight binding to the small molecule ATP can result in a decreased bioavailability of this NTP for the polymerase. This results in decreased transcription efficiency. Due to this effect we decided to apply the ATP AptamerJAWS1 Spinach for our fluorescent read-out system. Nevertheless live-cell imaging might benefit from the good binding properties of ATP AptamerJAWS2 Spinach RNA. Imaging of ATP in cells might be also an interesting target to study.

Another important achievement was the monitoring of the change of ATP concentrations during the in vitro transcription. We can show that the concentration of used amount of polymerase changes the final RNA yield. We increased the polymerase concentration and observed low Spinach and high malachite green fluorescence levels. Thus ATP was consumed and RNA was successfully transcribed. In addition we were able to show the influence of different buffer systems, inhibitors and different polymerase. Using this huge amount of generated Data, we were able to model in vitro transcription reactions and to analyze the speed of nucleotide incorporation. By application of our tool-box, a in vitro transcription reaction can be optimized within a few hours. Thus difficult transcription targets can be easily optimized.

Furthermore, we were able to set-up a method to quantify the RNA yields in real-time. Similar to classical colorimetric assays like Bradford, we successfully applied instead of BSA, our Malachite Green Aptamer RNA to generate a calibration curve with a really good correlation coefficient. In addition, the JAWS generated ATP aptamer Spinach can be applied in several other biochemical reactions that depend on ATP. In interesting target would be the Poly(A) polymeraseBalbo2007, that attaches several adenosine moieties to the 3’-end of RNAs in eukaryotes.

Furthermore, by using the JAWS software we can generate new sensors that are specifically binding to new interesting small molecules for example rape drugs, cofactors like NAD(H) or FAD. The generated aptamers could be fused to the Spinach and binding can be monitored by an increase of the fluorescence in presence of the small molecule.

Those generated aptamers might by also applicable for live-cell imaging to sense small molecules. We have already demonstrated for ATP AptamerJAWS2 Spinach RNA a good binding efficiency, which might be a good candidate for imaging experiments as well.