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Methods

To describe in vitro transcription time-resolved in terms of NTP consumption, following conditions were applied: 500 nM renatured ATP Aptamer Spinach2 RNA, 10 µM Malachite Green Aptamer DNA template, 40 mM Tris pH 8.1, 1 mM spermidine, 20 mM MgCl2, 0.01% Triton X-100, 4 mM each NTP, 10 mM DTT, 5 % DMSO, 100 µM DFHBI, 1 mM malachite green, 0.1 mg/mL T7 RNA Polymerase, 0.2 U/µL Ribolock RNase and 0.1 U Pyrophosphatase (Thermo Scientific). The assay was performed in 384 well micro titer plates (black, flat round, transparent bottom [Corning, 3540]) on a 20 µL scale. Evaporation during transcription was prevented by using a sealing tape. Measurements on micro titer plates were performed in a microplate reader (Tecan Safire 2).

Results

By adding the ATP Aptamer Spinach construct to a setup containg T7 RNA Polymerase and ATP we achieved time resolved data that show the decrease of fluorescence connected to the depletion of ATP during in vitro transcription. Similar data could not be shown with setups not containing T7 RNA polymerase.Figure 1 shows the decrease of ATP using different T7 RNA Polymerase concentrations. The data shows that the ATP Aptamer Spinach is even sensitive to different ATP consumptions that are shown in different decreases of the curve.

Figure 1 . 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.

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